1. Biochemistry and Chemical Biology
  2. Cell Biology
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Plasmodium falciparum translational machinery condones polyadenosine repeats

  1. Slavica Pavlovic Djuranovic  Is a corresponding author
  2. Jessey Erath
  3. Ryan J Andrews
  4. Peter O Bayguinov
  5. Joyce J Chung
  6. Douglas L Chalker
  7. James AJ Fitzpatrick
  8. Walter N Moss
  9. Pawel Szczesny  Is a corresponding author
  10. Sergej Djuranovic  Is a corresponding author
  1. Department of Cell Biology and Physiology, Washington University School of Medicine, United States
  2. Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, United States
  3. Washington University Center for Cellular Imaging, Washington University School of Medicine, United States
  4. Department of Biology, Washington University, United States
  5. Department of Neuroscience, Washington University School of Medicine, United States
  6. Department of Biomedical Engineering, Washington University, United States
  7. Institute of Biochemistry and Biophysics Polish Academy of Sciences, Department of Bioinformatics, Poland
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Cite this article as: eLife 2020;9:e57799 doi: 10.7554/eLife.57799

Abstract

Plasmodium falciparum is a causative agent of human malaria. Sixty percent of mRNAs from its extremely AT-rich (81%) genome harbor long polyadenosine (polyA) runs within their ORFs, distinguishing the parasite from its hosts and other sequenced organisms. Recent studies indicate polyA runs cause ribosome stalling and frameshifting, triggering mRNA surveillance pathways and attenuating protein synthesis. Here, we show that P. falciparum is an exception to this rule. We demonstrate that both endogenous genes and reporter sequences containing long polyA runs are efficiently and accurately translated in P. falciparum cells. We show that polyA runs do not elicit any response from No Go Decay (NGD) or result in the production of frameshifted proteins. This is in stark contrast to what we observe in human cells or T. thermophila, an organism with similar AT-content. Finally, using stalling reporters we show that Plasmodium cells evolved not to have a fully functional NGD pathway.

Introduction

The complex life cycle of Plasmodium falciparum, responsible for 90% of all malaria-associated deaths, involves multiple stages in both the human and mosquito hosts. Asexual replication during the intraerythrocytic development cycle (IDC) is tightly regulated over a 48 hr period, involving the expression of the majority of its genes (Gerald et al., 2011; Lu et al., 2017; Caro et al., 2014). Progression through asexual stages (ring, trophozoite, schizont) of the IDC requires a strictly controlled panel of gene expression profiles for each stage. A range of 16–32 daughter cells results from the IDC. Thus, a single, originating merozoite must undergo several rounds of DNA synthesis, mitosis, and nuclear division in a relatively short period (Gerald et al., 2011; Lu et al., 2017; Caro et al., 2014). The apparent necessity for rapid and accurate DNA replication needs to be accompanied by large-scale RNA transcription and protein synthesis. These processes occur during the 24 hr of trophozoite stage before erythrocytic schizogeny and cytokinesis. Recent saturation-level mutagenesis of the P. falciparum genome further emphasized and demonstrated that genes associated with cell cycle control, translation, RNA metabolism, protein folding, and drug resistance are more likely to be essential for parasites fitness and survival (Zhang, 2018). However, a faithful execution of these fundamental processes is challenged by the extremely AT-rich P. falciparum genome: averaging ~81% in overall AT content. With a relatively small difference in AT-richness between the non-coding and coding regions; P. falciparum represents a unique case from other AT-rich organisms (Glöckner, 2000; Szafranski et al., 2005; Zilversmit et al., 2010; Erath et al., 2019). While the underlying reasons for such disproportionate representation of the four nucleotides in any given genome may be different, it is of vital importance that shifts towards extreme AT- or GC-richness must be accommodated by adaptation of the transcription and translation apparatuses; enabling the cell to transcribe and translate each gene appropriately.

Recently, it was demonstrated that the translation of genes with polyadenosine runs (polyA tracts), primarily coding for lysine residues, is attenuated in the majority of tested organisms presumably due to ribosomal stalling and frameshifting on such RNA motifs by action of ribosome-quality control complex (RQC) and mRNA surveillance mechanisms (Ito-Harashima et al., 2007; Arthur et al., 2015; Arthur et al., 2017; Arthur and Djuranovic, 2018; Koutmou et al., 2015; Garzia et al., 2017; Juszkiewicz and Hegde, 2017; Sundaramoorthy et al., 2017; Tournu et al., 2019; Szádeczky-Kardoss et al., 2018; Chandrasekaran et al., 2019; Tuck et al., 2020; Tesina et al., 2020). In human tissue cultures, the presence of just 12 adenosines in an mRNA-coding region was found to reduce the yield of protein synthesis by more than 40%, and runs of 30–60 adenosine nucleotides reduce protein synthesis by more than 90% (Arthur et al., 2015; Arthur et al., 2017; Sundaramoorthy et al., 2017). This effect on translation was observed in all tested organisms that include fruit flies (Drosophila melanogaster), yeasts (Saccharomyces cerevisiae and Candida albicans), plants (Arabidopsis thaliana, and Nicotiana benthamiana) and E. coli (Zilversmit et al., 2010; Arthur et al., 2015; Koutmou et al., 2015; Tournu et al., 2019; Szádeczky-Kardoss et al., 2018; Tuck et al., 2020); arguing for a universal response to coding polyA repeats. The consequence of translational arrest or slippage on polyA runs is the activation of RQC and one or more mRNA surveillance mechanisms, mainly No-Go (NGD) and Non-sense Mediated Decay (NMD) (Arthur and Djuranovic, 2018). High AU-content within transcript coding regions and an extreme AAA and AAU codon bias increase the propensity for polyA tracts in the P. falciparum transcriptome (Saul and Battistutta, 1988). Additionally, a ‘just-in-time’ transcriptional and translational model of gene expression during the relatively short trophozoite stage of the P. falciparum IDC (Lu et al., 2017; Coulson et al., 2004) make a rapid protein synthesis in an AU-rich transcriptome an appealing problem. While both, DNA and RNA polymerases must contend with high DNA AT-content, it is also puzzling what adaptations P. falciparum have made to its translational machinery to overcome the unusual AU-richness of mRNAs, which would affect the fidelity and efficacy of protein synthesis. With ‘just-in-time’ translation of numerous A-rich coding sequences (Szafranski et al., 2005; Erath et al., 2019) and poly-lysine proteins harboring an AAA codon bias, expressed at all stages in the parasite life cycle (Lu et al., 2017; Caro et al., 2014; Bunnik et al., 2013), the Plasmodium translation machinery would represent an exception in protein synthesis compared to other organisms.

Here we present data that indicate that the P. falciparum translational machinery and its NGD pathway have adapted to translate long runs of polyadenosine nucleotides into poly-lysine repeats. Using comparative bionformatic analyses, we show that malaria parasites contain an unusually high numbers of polyA-tract-containing genes compared to other eukaryotic organisms. We find this to be a common feature of all analyzed Plasmodium species regardless of their AT-content, arguing for evolutionary conservation of such sequences in Plasmodium genomes. Expression of endogenous genes and reporters with polyA tract motifs in P. falciparum cells results in efficient and accurate protein synthesis in direct contrast to what we observe for human-tissue cultures and T. thermophila cells. We find no evidence for either ribosomal stalling or frameshifting during the translation of long polyadenosine runs in P. falciparum cells. Interestingly, induction of the NGD pathway by either reporter with stable RNA structure or isoleucine starvation results in reduced reporter protein levels but without any detectable changes in mRNA levels, arguing for alterations in the NGD pathway. Finally, our analysis of P. falciparum ribosome structure suggests a model whereby multiple changes may have evolved to accommodate the unusual AU-richness and high percentage of poly–lysine runs of the P. falciparum transcriptome and proteome, respectively.

Results

Plasmodium species: A paradigm-breaking genus

Previous studies indicated that polyA runs in the coding sequences serve as hurdles to translation (Ito-Harashima et al., 2007; Arthur et al., 2015; Arthur et al., 2017; Arthur and Djuranovic, 2018; Koutmou et al., 2015; Garzia et al., 2017; Juszkiewicz and Hegde, 2017; Sundaramoorthy et al., 2017; Tournu et al., 2019; Szádeczky-Kardoss et al., 2018) that efficiently reduce protein yield and initiate NGD to degrade the mRNA. Given the very high AT-content in certain eukaryotic species (Glöckner, 2000) we sought to explore the association between coding region AT-content and transcript polyA-tract-motif abundance. In doing so, we analyzed 152 eukaryotic genomes (Figure 1A). We focused on stretches of polyadenosine nucleotides, or as defined previously 12A-1 motifs; sequences with a minimal length of 12 A’s allowing for one mismatched base (Arthur et al., 2015). We settled on 12A-1 sequence pattern since the presence of this motif reduces protein production by 40–60% in multiple human genes (Arthur et al., 2015; Arthur et al., 2017; Tuck et al., 2020). Subsequent analyses indicated that the reduction in protein amounts for genes with 12A-1 motif could be attributed to ribosome stalling and frameshifting (Arthur et al., 2017; Koutmou et al., 2015). Analyses of the selected set of eukaryotic genomes indicate that P. falciparum and other members of the Plasmodium genus have a much higher ratio of polyA tract genes when normalized to genomic AT-content (Figure 1A).

Figure 1 with 1 supplement see all
Analyses of polyA tracts in eukaryotic genomes.

(A) The plot of 152 species representing a comparison of the ratio of polyA-affected transcripts (over a total number of transcripts) to the AT content of the coding region for each organism. H. sapiens, T. thermophila, and P. falciparum, as organisms pertinent to this paper are in black. For reference, other model organisms of interest are displayed in gray, including a position of high (65% average) and low (35%) AT-content Plasmodium spp. (B) Transcript counts for genes with 6 to 36 consecutive adenosines for H. sapiens, T. thermophila, and P. falciparum. H. sapiens and T. thermophila are limited to a single transcript at length of ≤17 As. The longest P. falciparum 3D7 transcript reaches maximal 65As, with multiple transcripts of ≤36 As. (C) Violin plot of lysine codon usage distribution in tracts of four lysine residues for 152 organisms. 3AAG+1AAA, 2AAG+2AAA and 1AAG+3AAA indicate different ratios of AAG and AAA codons in runs of four consecutive lysine codons. 4AAG and 4AAA indicate poly-lysine runs with only AAG or AAA codons, respectively. H. sapiens (circle), T. thermophila (triangle), and P. falciparum (square) are specifically noted.

Figure 1—source data 1

Lysine codons distibution in 4xLys runs in eukaryotic genomes.

https://cdn.elifesciences.org/articles/57799/elife-57799-fig1-data1-v2.xlsx

Interestingly, this feature of Plasmodium species is conserved regardless of their genomic AT-content, resulting in two groups (low and high AT-content Plasmodium spp.) with unusually large portions of polyA tract genes ranging from 35 to over 65% of the total transcriptome (Figure 1A; Erath et al., 2019). These two groups, which also appear to be separated geographically, demonstrate that even with a reduction in genomic AT-content, selective pressure exists for Plasmodium spp. to maintain polyA tract motifs. However, regardless of a selective reduction of AT-content in some Plasmodium species, or an almost complete lack thereof in the case of avian Plasmodium counterparts (Videvall, 2018), the trend of harboring a high ratio of polyA-affected transcripts given a particular AT-richness, as well as their conservation within a significant number of genes across species, remains a paradigm-breaking hallmark of the genus.

To further emphasize the differences between P. falciparum and other organisms, we analyzed the number of genes that harbor polyA runs as well as the total length of consecutive adenosine nucleotides in transcripts (Figure 1—figure supplement 1 and Figure 1B, respectively). P. falciparum showed a significantly higher amount of genes with polyA runs of ≥12 A’s in a row when compared with its human and mosquito (Anopheles gambiae) hosts (Figure 1—figure supplement 1). Over 63% of genes in P. falciparum have polyA tract motifs, while only 0.7–2% of total genes in humans and mosquito genomes, respectively, contain these motifs. We used the most recently updated Ensembl database to look into coding sequences of lab strain of malaria P. falciparum 3D7, H. sapiens, and T. thermophila (Hunt et al., 2018). While there is already a significant difference in the number of genes with eleven consecutive adenosines (P. falciparum 3D7 – 1555, T. thermophila - 364 and H. sapiens - 77 genes), the most striking sequence difference is associated with 16 consecutive adenosines (16As). The P. falciparum 3D7 genome contains 188 genes with 16As, while the human host and AT-rich T. thermophila together have only three genes (T. thermophila two and H. sapiens 1) (Figure 1B). Additionally, T. thermophila a protozoan with an overall 73% AT-rich genome and 24,000 predicted genes, had almost nine times less genes with twelve consecutive adenosines than P. falciparum, 172 and 1526, respectively. Ensembl database reports 105 P. falciparum 3D7 genes with exactly 18As and more than 150 genes with more than 20 consecutive adenosines (20-65As, Figure 1B). Analyses of P. falciparum genomes in the PlasmoDB database (Beznosková et al., 2013), curated explicitly for malaria parasite species, report more than double that number with 329 genes having runs of ≥20 consecutive adenosines. Most of these genes appear to be annotated as Plasmodium specific. However, PlasmoDB also shows genes with up to 59 consecutive adenosines in conserved pathways, such as the phosphatidylinositol-4-phosphate 5-kinase gene (PfML01_010014200), which is involved in the inositol metabolism and signaling pathways (Beznosková et al., 2013). Finally, the maximum length of consecutive adenosine runs in coding sequences of Plasmodium genomes indicated 111 As in P. falciparum fch four strain with the human host or 132 As in P. reichenowi, strain causing chimpanzee malaria (Erath et al., 2019; Habich et al., 2016). As such, the length of polyA runs in coding sequences of malaria parasites exceeds the length of the average size of 3’UTR polyA tails found on the majority of transcripts in eukaryotic species (Subtelny et al., 2014; Brown and Sachs, 1998; Chang et al., 2014). Translation of 3’UTR located polyA tail in eukaryotic organism activates yet another mRNA surveillance pathway - non-stop decay (NSD) (Chandrasekaran et al., 2019; Tesina et al., 2020; Shoemaker and Green, 2012).

The disparity in the number of genes with polyA tracts could be due to previously observed codon biases in P. falciparum (Saul and Battistutta, 1988). However, it was already shown that codon bias and tRNA abundance do not correlate with codon selection in genes coding for lysine repeats (Arthur et al., 2015; Koutmou et al., 2015). The poly-lysine repeats in proteins of the analyzed model organisms are usually encoded by AAG codons. To investigate the distribution of AAA and AAG codons in poly-lysine tracts in more detail, we analyzed transcripts from P. falciparum and 152 other eukaryotic genomes (Figure 1C). We observed a complete reversal of the trend exhibited in other organisms, including humans and the AT-rich T. thermophila. P. falciparum had the highest abundance of transcripts hosting four consecutive AAA codons in runs of four lysine residues (Figure 1C). This divergence from other analyzed transcriptomes is preserved in other members of Plasmodium spp., with P. berghei being an extreme example using only AAA codons in 68% of transcripts coding for poly-lysine runs. Finally, we analyzed the biological function and essentiality of the polyA tract and poly-lysine containing genes in P. falciparum genome. Previous analysis indicated that 70–85% of orthologs of polyA tract carrying genes from P. falciparum have the same polyA motifs in genes from other Plasmodium species, regardless of their genomic AT content (Habich et al., 2016). The high degree of the conservation of polyA tracts has also been noted for other eukaryotic organisms (Arthur et al., 2015). A majority of Plasmodium polyA tracts genes and poly-lysine proteins fall into the group of essential genes based on the recent mutagenesis studies (Zhang, 2018). This outcome is expected given that gene ontology results indicate enrichment in gene products involved in crucial cellular processes such as protein synthesis, RNA biogenesis, and chromosome segregation. The same gene ontology groups were previously observed in poly-lysine repeats and polyA tract genes of the other organisms (Arthur et al., 2015). In addition to these groups, Plasmodium species also had enrichment of polyA tract motifs in a group of genes defined as the cellular and pathological cell adhesion ontology group (Supplementary file 1). As such, our overall bioinformatic analyses demonstrate that Plasmodium genomes represent a unique set of organisms that have an enrichment of polyA tracts in the coding sequences. The overall conservation of both polyA tracts in the transcriptome and poly-lysine repeats in the proteome of P. falciparum has been evolutionary selected and conserved due to possible benefits for the parasite.

Reporters with polyA tracts are not a problem for P. falciparum

As mentioned earlier, polyA tracts and poly-lysine repeats cause a reduction in mRNA stability and protein amounts, respectively, due to the ribosomal stalling and frameshifting on such motifs (Ito-Harashima et al., 2007; Arthur et al., 2015; Arthur et al., 2017; Arthur and Djuranovic, 2018; Koutmou et al., 2015; Garzia et al., 2017; Juszkiewicz and Hegde, 2017; Sundaramoorthy et al., 2017; Tournu et al., 2019; Szádeczky-Kardoss et al., 2018; Tesina et al., 2020). To investigate further how the AU-rich P. falciparum transcriptome with multiple polyA tracts gets effectively translated, we used double HA-tagged reporter constructs. The 36 adenosine nucleotide (36 As) insertion, coding for 12 lysine residues, was inserted between the sequence of double HA-tag and a fluorescent protein (+polyA36, Figure 2—figure supplement 1). As a control, we used a reporter that had only double HA-tag in front of the fluorescent-protein sequence (-polyA36, Figure 2—figure supplement 1). We expressed our reporter constructs from plasmid vectors in the P. falciparum Dd2 lab strain. In parallel, we expressed the same constructs in human dermal fibroblasts (HDFs) and T. thermophila cells (Figure 2A and B). We followed mRNA abundance of each construct by qRT-PCR (Figure 2A), and expression of the reporter protein was followed by western blot detection of the double HA-tag in all three organisms (Figure 2B). We observed robust changes in normalized mRNA levels (Figure 2A) and substantial losses in protein expression (Figure 2B) for reporters with polyA tracts (+polyA36) in both HDFs and AT-rich T. thermophila (Arthur et al., 2017; Arthur and Djuranovic, 2018). In comparison to HDFs and T. thermophila, we observed minimal, if any, effects from polyA tract insertion on reporter mRNA and protein expression in P. falciparum cells (Figure 2A and B). Further analysis of P. falciparum cells by live-fluorescence microscopy confirms the equivalent expression of mCherry reporter, judging by the intensity of fluorescence between constructs with and without polyA tract (+polyA36 and –polyA36, Figure 2C).

Figure 2 with 3 supplements see all
P. falciparum cells express reporters with long polyA tracts.

(A) mRNA abundance of reporter constructs (+polyA36) by qRT-PCR relative to their counterpart lacking polyA stretches (-polyA36) in H. sapiens, T. thermophila, and P. falciparum cells. Data represent three biological replicates with a standard deviation. (B) Expression of reporter constructs in H. sapiens, T. thermophila, and P. falciparum followed by western blot analysis with αHA or αGFP antisera. Samples from two integrated clones for the -polyA36 control (-A) and the +polyA36 reporter (+A) are shown for T. thermophila. αβ-actin, α-Histone H3 trimethyl-lysine (H3kM) and αhDHFR are used as loading controls for western blot analysis from H. sapiens, T. thermophila and P. falciparum cells, respectively. (C) Images from live fluorescence microscopy of P. falciparum expression of reporter constructs with (+polyA36) and without (-polyA36) polyA tracts as well as parent (non-transfected) line, 2.5 µm scale bar. (D) Quantification of protein amounts for Thioredoxin-2HA-NanoLuciferase (Trx-2HA-NLuc) reporter without (-polyA36) and with 36 adenosine stretch (+polyA36) expressed in P. falciparum cells. Western blot analysis of Trx-2HA-NLuc reporter (Figure 2—figure supplement 2) and luminescence measurements were normalized to hDHFR or cell number, respectively. Luminescence data represent the mean value of three biological replicates with standard deviation.

Figure 2—source data 1

Luminescence and western blot quantification data.

https://cdn.elifesciences.org/articles/57799/elife-57799-fig2-data1-v2.xlsx

To assess whether the efficiency of protein synthesis is altered when the polyA tract is located further downstream of the start codon, we designed a construct with thioredoxin (Trx) and nano-luciferase (nanoluc) proteins separated with a double HA-tag and 36As coding for a poly-lysine run (Figure 2—figure supplement 2). Measurement of nanoluc luminescence from the same number of drug-selected parasites indicates similar expression of a reporter with a polyA tract (+polyA36) compared with the control reporter (-polyA36, Figure 2D). We observed the same ratio when we analyzed the expression of reporters using western blot analysis (Figure 2D and Figure 2—figure supplement 2), arguing that the position of polyA tracts in coding sequence does not influence the efficiency of protein synthesis in P. falciparum cells.

Since polyA tracts cause ribosomal frameshifting (Arthur et al., 2015; Koutmou et al., 2015; Sundaramoorthy et al., 2017) we analyzed the expression of our nanoluc and fluorescent reporters for the possible presence of frameshifted protein products. Western blot analyses of nanoluc reporters did not indicate the HA-tagged protein band at 14kD that would represent frameshifted nanoluc (Figure 2—figure supplement 2). Moreover, the immunoprecipitation of mCherry reporters showed only equivalent amounts of the full-length product (Figure 2—figure supplement 3). There is still a possibility that ribosomal frameshifting occurred in polyA tract reporters, but the protein product was unstable in P. falciparum cells, and we failed to detect it. However, if such frameshifting events did happen, they did not reduce overall levels of the full-length protein. Finally, to check whether polyA tract constructs resulted in the synthesis of poly-lysine peptides, we analyzed all constructs using 2D PAGE electrophoresis (Figure 3). This analysis indicated a shift in isoelectric point (pI) of approximately one pH unit for the (+polyA36) construct compared to the wild-type one (-polyA36), 7.2–7.4 and 6.25–6.5, respectively. This shift on the 2D PAGE gels was the same for construct with 12xAAG lysine codons and is expected based on the calculated isoelectric points if all 12xAAA or 12xAAG codons were translated into lysine residues. Taken together, our analyses of different reporter expression data (Figures 2 and 3, Figure 2—figure supplements 2 and 3) indicates that polyA tracts are tolerated by the parasite translational machinery, without apparent effects on either stability of mRNA or quality of synthesized protein.

Poly-A tracts are correctly translated in P. falciparum cells.

(A) 2D gel analysis of HA-IP samples of wild type reporter (-polyA36). The western blots show isoelectric point (pI) at 6.25 and 6.5 (arrows). (B) 2D gel of reporter with polyA tract (+polyA36) sample indicates pI 7.25 and 7.4 (arrows).( C) 2D gel analyses of reporter with twelve lysines coded by AAG codons (12 x AAG) indicates the same effect on pI value, pI is 7.25 and 7.4 (arrows). Overlay of images of PVDF membranes stained with Coomassie Brilliant Blue, and images of western blots probed with HA-antibody. The scale with pH is on the bottom. Coomassie-stained membranes show markers (right side) and tropomyosin (33 kDa) and pI at 5.2 (arrow) as an internal standard for Isoelectric focusing (IEF).

Endogenous polyA tract genes are efficiently expressed in P. falciparum

Due to the potential for negative selection against polyA tracts (Guler et al., 2013), particularly in laboratory conditions, we also wanted to investigate how P. falciparum translates endogenous genes with polyA tracts. With as much as 60% of the parasite transcriptome harboring polyA tract motifs, we performed a comparative analysis of ribosome profiling data from P. falciparum (Caro et al., 2014) and aggregated data for human tissues conveniently harmonized at GWIPS database (Michel et al., 2014). We analyzed whether endogenous polyA tracts and poly-lysine sequences induce translational pausing in both sets of data. Ribosome stalling can be observed in the ribosome profiling data as an increase in the abundance of ribosome footprints on sequences that cause ribosomes to pause during translation (Ingolia et al., 2014). Cumulative data for all transcripts with polyA tracts from human cells indicate substantial translational pausing on these sequences (Figure 4A). The same effect was noted on poly-lysine sequences that contained more than four consecutive lysine residues in multiple other studies using S. cerevisiae ribosome profiling datasets without cycloheximide treatment or datasets from human tissue cultures regardless of cycloheximide treatment (Arthur et al., 2015; Guydosh and Green, 2017; Requião et al., 2016; Charneski and Hurst, 2013). However, analyses of previously published P. falciparum ribosome profiling dataset (Caro et al., 2014) indicated no evidence for ribosome stalling in P. falciparum transcripts containing polyA tracts. Normalized ribosome occupancy for P. falciparum transcripts with a length of less than or equal to 22 consecutive adenosine nucleotides (≤22As), that code for more than seven consecutive lysines, indicated more or less equal ribosome occupancy over polyA tract (Figure 4B). We limited our ribosome profiling analyses of P. falciparum transcripts to polyA tracts of ≤22As, since the cumulative transcript analyses become hindered by the low sequence complexity of the region, or reduced number of reads, for the long polyA tracts (Figure 4—figure supplement 1). As such, the dip in P. falciparum occupancy plot is a result of a reads mapping artifacts. The longer polyA segment, the harder it is to uniquely map a read - typically such reads are discarded. This artifact is clearly seen in the Figure 4—figure supplement 1, where the occupancy is shown per length of polyA segment - the larger polyA track, the larger symmetrical gap around position 0. The correlation holds true to about 30nt, which is the read length in the original experiment by Caro et al., 2014.

Figure 4 with 5 supplements see all
Occupancy of elongating ribosomes (mapped to A-site) around the start of polyA segments in human (A) and P. falciparum (B).

Occupancy is shown on the same scale. In both cases, to avoid the inclusion of sparsely mapped segments, regions with average occupancy below the mean for the whole dataset were excluded. In the case of P. falciparum gene segments with polyA tracts shorter than 22 adenosine nucleotides were taken into account. Centerlines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Dip in around position 0 in the P. falciparum plot is an artifact of the reads mapping procedure shown in Figure 4—figure supplement 1. It is not observed in human data, because of shorter polyA segments and lower occupancy overall around these segments.

Since there is a massive increase in protein synthesis in P. falciparum cells during the trophozoite and schizont stage of IDC (Caro et al., 2014; Bunnik et al., 2013) we analyzed whether there is a different distribution of ribosome profiling reads surrounding polyA tracts at different stages of P. falciparum development. We found that the translation of endogenous polyA tracts is independent of different stages of P. falciparum IDC, as we did not notice any significant difference in the distribution of ribosome protected fragments (Figure 4—figure supplement 2) and ostensibly irrespective of the length of polyA tracts or poly-lysine runs. While we observe a relatively small increase in the number of elongating ribosomes on polyA segments in the late trophozoite and schizont stages of IDC (Figure 4—figure supplement 2), it is unclear if these indicate higher protein expression or are just an artifact of a general increase in protein synthesis at this IDC stages.

In parallel with analyses of ribosome profiling data, we also selected a small subset of polyA tract-containing genes for independent expression analysis. We followed PfGAPDH (PF3D7_1462800) as a control gene without polyA tract, and PfZFP (PF3D7_1464200 (three polyA tracts and run of 20 consecutive adenosines), PfCRK5 - PF3D7_0615500 (four polyA tracts and run of 20As) and PfIWS1L - PF3D7_1108000 (three polyA tracts and run of 31As), to represent genes with polyA tracts (Figure 4—figure supplement 3). PfZFP is a zinc-binding protein with homology to the human ZC3H6 gene (Zinc Finger CCCH Domain-Containing Protein 6), PfCRK5 is cdc2-related protein kinase five and PfIWS1L is transcription elongation factor associated with RNA Polymerase II. We used a sorbitol based method to synchronize P. falciparum Dd2 strain at the ring stage of IDC and followed transcript profiles over the next 48 hr of the parasite life cycle. The mRNA profiles of three polyA tract genes were analyzed using qRT-PCR and normalized to the expression of PfGAPDH in P. falciparum Dd2 lab strain. A time-course study of synchronized parasite culture indicated that the selected polyA tract transcripts are efficiently transcribed at all-time points when compared to the control gene (Figure 4—figure supplement 4). Moreover, the increase in transcription of all three polyA tract genes followed previously described just-in-time transcription profile in trophozoite and schizont IDC stage of P. falciparum.

Finally, mRNA translation could also be affected by the ability of RNA to fold into unique functional structures; for example RNA secondary structures have been shown to coordinate ribosomal frameshifting (Kim et al., 2014; Mouzakis et al., 2013), slow ribosomal progression to allow protein folding (Faure et al., 2016) and to affect ribosomal density (Zur and Tuller, 2012; Andrews et al., 2017). To determine the RNA structural characteristics of the P. falciparum transcriptome, we analyzed the ability of all coding sequences to form thermodynamically stable RNA secondary structures using an in silico approach to characterize RNA folding landscapes (Andrews et al., 2017; Andrews et al., 2019). We compared the relative structural stability of the P. falciparum transcriptome to that of humans and the AU-rich T. thermophila. On average, the coding sequences from T. thermophila and P. falciparum yielded structures with higher predicted minimum free energy (MFE; Figure 4—figure supplement 5), than those from the coding sequences of human genes; a result which mostly correlates with the higher overall AU-content of P. falciparum and T. thermophila transcripts. In this analysis, each MFE is also characterized by a thermodynamic z-score (see Materials and methods). The thermodynamic z-score normalizes for nucleotide content and suggests whether sequences may form potentially functional structures (Clote et al., 2005). Specifically, the thermodynamic z-score indicates whether a nucleotide sequence has been ordered to adopt a more stable secondary structure than its nucleotide content would typically produce (Clote et al., 2005) (which is the case for many ncRNAs (Freyhult et al., 2005) as well as RNA regulatory structures embedded in mRNAs O'Leary et al., 2019; Andrews et al., 2018).

Interestingly transcriptome z-scores revealed no significant differences between P. falciparum and T. thermophila (Figure 4—figure supplement 5). This result indicates that while RNA folding stability varies between species, following from skews in nucleotide content, the potential occurrence of ordered, structured motifs does not. This suggests that globally, P. falciparum coding sequences do not appear ordered to adopt structures any more stable than would be expected for random sequences or with the same nucleotide content found in T. thermophila. However, expression of reporters with polyA-tracts in T. thermophila is strongly attenuated (Figure 2). This computational effort does not take into account the most recent work describing the m6A mRNA methylation dynamics within P. falciparum coding sequences (Baumgarten et al., 2019). The m6A modification may impact RNA structure and association of P. falciparum mRNAs with certain RNA binding proteins (Liu et al., 2015). However, previous work has demonstrated that m6A methylation does not affect the ability of the ribosome to decode mRNAs but instead has a modest impact on the rate of translation (Hudson and Zaher, 2015; Choi et al., 2016), which could in-turn reduce the rate of protein synthesis in P. falciparum. Taken together, our analyses of the P. falciparum transcriptome and translation of genes with endogenous polyA tracts, as well as in silico assessment of overall structure and stability of AU-rich transcripts indicates that the P. falciparum translation machinery adapted to long and numerous polyA tracts in coding sequences of the majority of genes.

NGD pathway is not connected to mRNA degradation in P. falciparum

PolyA tracts and poly-lysine repeats are highly efficient at causing ribosome stalling and frameshifting in bacteria and most eukaryotes (Ito-Harashima et al., 2007; Arthur et al., 2015; Arthur et al., 2017; Arthur and Djuranovic, 2018; Koutmou et al., 2015; Garzia et al., 2017; Juszkiewicz and Hegde, 2017; Sundaramoorthy et al., 2017; Tournu et al., 2019; Szádeczky-Kardoss et al., 2018; Chandrasekaran et al., 2019; Tuck et al., 2020; Tesina et al., 2020). However, our analyses of endogenous P. falciparum genes and reporters with long polyA runs, as well as immunoprecipitations did not indicate any changes in either mRNA stability, protein amounts, or protein quality (Figures 2 and 3) that was previously observed in E. coli, yeast, and human cells (Arthur et al., 2015; Arthur et al., 2017; Koutmou et al., 2015; Tuck et al., 2020). So, we turned towards analyses of mRNA surveillance pathways in P. falciparum cells and their contribution to the synthesis of poly-lysine peptides from long polyA tracts. The NMD pathway seems to be intact in Plasmodium cells (Sorber et al., 2011), while the existence of the NGD pathway has not been previously tested. We initially focused our analysis on two proteins that have been documented to be crucial for NGD pathways in eukaryotes, Hbs1 and Pelota (Shoemaker and Green, 2012). The Pelota protein in complex with Hbs1 recognizes and rescues stalled ribosomes with an empty A site on long polyA stretches, absence of tRNAs during starvation, damaged mRNAs, or stable RNA structures (Shoemaker and Green, 2012; Guydosh and Green, 2017; Tsuboi et al., 2012; Hilal et al., 2016; Becker et al., 2011). Our bioinformatics search of the P. falciparum genome did not identify an apparent homolog of Hbs1 nor the recently reported S. cerevisiae endonuclease Cue2 (NONU-1 in C. elegans) (D'Orazio et al., 2019; Glover, 2019; Navickas, 2019) in P. falciparum (Supplementary file 2). To explore whether changes in the NGD pathway are potential adaptations of P. falciparum to polyA tracts and poly-lysine repeats, we first used CRISPR/Cas9 technology (Ghorbal et al., 2014; Nasamu et al., 2017) to HA-tag the endogenous Pelota homolog (PfPelo, PF3D7_0722100, (Figure 5—figure supplement 1) in P. falciparum Dd2 cells.

Since polyA-containing reporters, as well as endogenous genes, were efficiently and correctly translated in P. falciparum cells, we decided to test the P. falciparum NGD pathway with a common NGD substrate (Shoemaker and Green, 2012; Doma and Parker, 2006). We inserted a 78 bp long RNA stem-loop into the fluorescent reporter sequence (StL, Figure 5—figure supplement 2). The same stem-loop was previously described to stall ribosomes and induce NGD response with subsequent endonucleolytic cleavage in various reporters and multiple organisms (Arthur et al., 2015; Doma and Parker, 2006; Simms et al., 2017; Passos et al., 2009; Dimitrova et al., 2009). We followed both protein synthesis and mRNA abundance from the StL construct in P. falciparum cells (Figure 5A and B). Insertion of the RNA stem-loop resulted in a severe reduction of reporter protein levels (Figure 5A, Figure 5—figure supplement 2), but moderate increase in mRNA levels (Figure 5B). While we could not detect any HA-tagged StL reporter using western blot analyses (Figure 5A), we did notice the small but detectable amount of fluorescent reporter in the P. falciparum cells during live imaging (Figure 5—figure supplement 2). To test whether stem-loop structure caused ribosomes to stall, we analyzed the distribution of StL transcript in polysome profile (Figure 5C). We used constructs with and without polyA tracts as controls. Polysome profile analyses indicated that mRNA of StL construct was associated mostly with polysome fractions 7–9 that correspond to the disome peak. Such mRNA distribution was in sharp contrast with the distribution of mRNAs for constructs with and without polyA tracts (±polyA36) and argues for the potential translational stall (Figure 5C). mRNAs for constructs with and without polyA tracts (±polyA36) and for PfGAPDH were distributed more evenly along the polysome fractions (fractions 9–16). These experiments argue that stable RNA structures in mRNAs stall P. falciparum ribosomes, reducing protein synthesis from such transcript, however without significant impact on mRNA stability.

Figure 5 with 2 supplements see all
mRNA stem-loop pauses P. falciparum ribosome without mRNA degradation.

(A) The western blot of –polyA36, +polyA36, and stem-loop (StL) tagged reporter gene. The blot was probed with anti-HA antibody HRP labeled (sc-7392HRP). The HA-tagged proteins were episomally expressed in HA-tagged pelota P. falciparum Dd2 strain. HA-tagged Pelota protein serves as normalization control. (*) denotes human hemoglobin as 15kD contaminant band appearing and causing cross-reactivity in western blot analyses. (B) qRT-PCR measured mRNA levels of of reporter constructs with polyA tract and stem-loop insertions (polyA36, +polyA36, Stl). In each case, data represent the mean value of three biological replicates with standard deviation. (C) Distributions of mRNA (GAPDH, -polyA36, +polyA36, Stl) and rRNA, in polysome gradients as determined by RT-qPCR. Error bars represent measurement variability as determined by two qPCR replicates. The position of monosome and disome peaks is indicated in each analyzed sample (gray shade).

To further test whether global arrest of translation leads to the activation of RQC and NGD mechanisms, we used isoleucine (Ile) starvation (Figure 6A) to induce widespread ribosome pausing on Ile-codons (Babbitt et al., 2012; Liu et al., 2006). Parasites rely on extensive proteolysis of human serum proteins to supplement Ile, which is found in 99% of all P. falciparum proteins (Liu et al., 2006). As such, the majority of ribosomes will have either a reduced rate of protein synthesis or will be stalled on runs of Ile-residues encoded in endogenous P. falciparum transcripts. Ile starvation in P. falciparum was previously reported to induce a state of hibernation through the arrest of protein synthesis with phosphorylation of eIF2α, but in a GCN2-independent fashion, and without existing TOR-nutrient sensing pathway nor activation of ATF4 homologue (Babbitt et al., 2012). Surprisingly, during the metabolically induced hibernation, parasites maintain their morphology while slowing down protein synthesis, and importantly more than 50% of parasites recover even after 4 days of starvation (Babbitt et al., 2012). By analyzing P. falciparum cells 72 hr into Ile-starvation, we found that expression of PfPelo and PfHsp70 chaperone were upregulated in starved cells (Figure 6B and C). The mRNA and protein levels of PfPelo were moderately increased (approximately two-fold), while levels of PfHsp70 were strongly induced (five-fold increase) in response to the Ile-starvation. This increase in PfPelo levels is reminiscent of the recently reported increased human Pelo expression during elevated ribosome stalling and the absence of recycling during the developmental transition from reticulocyte to erythrocyte (Mills et al., 2016). The substantial increase in Hsp70 levels upon starvation in human cells was associated with cell protective mechanisms against global protein misfolding (Wu et al., 1985; Rosenzweig et al., 2019).

Figure 6 with 1 supplement see all
Isoluecine starvation in Plasmodium cells induces HSP70 and Pelo genes.

(A) The schematic presentation of the starvation experiment design. The cells were incubated for 48 hr in medium with and without Ile (+ / - Ile). After 48 hr samples were collected and protein and total RNA was isolated. (B) Relative mRNA of P. falciparum GAPDH (PF3D7_1462800), HSP70 (PF3D7_0818900), Pelo (PF3D7_0722100) transcripts after 48 hr isoleucine (Ile) starvation of the P. falciparum cells. Levels of each transcript are normalized to total GAPDH levels and represented as a ratio of transcript levels under Ile starvation (-Ile) over the control conditions (+Ile). In each case, data represent the mean value of three biological replicates with standard deviation. (C) Levels of P. falciparum HSP70 and Pelo proteins after 48 hr of Ile starvation. Western blot analysis of Ile starved (-Ile), and control (+Ile) sample are normalized to PfHAD1 (PF3D7_1033400) levels. HA-tagged P. falciparum Pelo protein was CRISPR/Cas9 engineered and detected using mouse HA-antibody. The molecular weight of each protein is indicated. (D) qRT-PCR measured mRNA levels of genes containing 3–7 Ile. PF3D7_0322300, PF3D7_0512700, PF3D7_0110600, PF3D7_0903400, PF3D7_1450600, PF3D7_1364700, PF3D7_1121000, PF3D7_1341600, PF3D7_1321400, PF3D7_1138600. Levels of each transcript are normalized to total GAPDH level and represented as a ratio of transcript levels under Ile starvation (-Ile) over the control conditions (+Ile). In each case, data represent the mean value of three biological replicates with standard deviation.

To test whether Ile-rich transcripts were targeted specifically by the RQC and NGD pathway during Ile-starvation, we examined transcripts that encode Ile-rich proteins in the P. falciparum genome. Previous studies indicated that while protein levels did not drastically change throughout starvation, protein synthesis was significantly reduced (Babbitt et al., 2012). The microarray analyses of mRNAs in Ile-starvation samples also showed similar overall levels for the majority of mRNA transcripts when compared to non-starved controls. To specifically test transcripts with three and more consecutive Ile-codons, we analyzed mRNA abundance for nine P. falciparum genes during Ile-starvation (Figure 6—figure supplement 1). We did not observe the general reduction in mRNA levels for Ile-rich P. falciparum genes when we compared starved to non-starved control samples. We, instead found a slight increase in mRNA abundance for several Ile-rich transcripts during starvation (Figure 6C). This result is rather similar to Leu- and Arg-starvation in mammalian cells, where no overall change in mRNA stability is observed during amino acid starvation (Darnell et al., 2018). However, our result indicated that both increase in expression of NGD-associated factor, Pelota, and protein chaperone HSP70 may contribute to cell survival, ribosome recycling and mRNA stabilization during amino acid starvation in P. falciparum. This result is opposite to the recently reported role of human Pelo protein in the regulation of global mRNA decay on stalled mRNAs in platelets (Mills et al., 2017). Transgenic overexpression of human Pelo protein potentially releases unrecycled ribosomes and stimulates mRNA degradation during translational arrest in platelet differentiation. This is not the case in P. falciparum, where the production of the PfPelo protein is increased during the P. falciparum Ile-starvation; however, there was a lack of targeted mRNA degradation (Figure 6C). We observed, rather, stabilization of mRNAs that should cause ribosomal stalling, like in the case of StL construct (Figure 5B). These results may indicate that RQC and NGD pathway in P. falciparum cells are not connected, at least directly, to targeted mRNA degradation.

Plasmodium ribosome structure accommodates poly-lysine repeats

In the light of our data with different reporters and analyses of endogenous P. falciparum genes (Figures 26), we were still interested as to how the P. falciparum translation machinery deals with polyA tracts and poly-lysine sequences. Ribosomal stalling on long poly-lysine runs (Arthur et al., 2015; Juszkiewicz and Hegde, 2017; Sundaramoorthy et al., 2017; Dimitrova et al., 2009; Kuroha et al., 2010; Brandman et al., 2012; Lu and Deutsch, 2008) was classically explained by electrostatic interactions of the polybasic peptide and the exit tunnel of the ribosome (Lu and Deutsch, 2008). More recent studies revealed that polyA tracts and mRNA directly contribute to ribosomal stalling and frameshifting (Arthur et al., 2015; Koutmou et al., 2015; Chandrasekaran et al., 2019; Tesina et al., 2020). Consecutive adenosines are engaged by the ribosome decoding center nucleotides and are stabilized on both sides by rRNA base stacking interactions (Chandrasekaran et al., 2019; Tesina et al., 2020), and adopt a helical conformation typical for single-stranded polyA stretches (Tang et al., 2019). Based on these reports, we analyzed P. falciparum ribosomes as the principal components that could accommodate translation of long polyA stretches.

The retention and conservation of polyA tracts, as well as the stage-independent expression of polyA genes (Figure 1), reveals that all rRNAs within Plasmodium spp. must deal with them. The structural data on Plasmodium ribosomes are limited to two recent cryo-EM studies of P. falciparum ribosomes isolated during the schizont stage of the IDC (Wong et al., 2014; Sun et al., 2015). Both studies reported that P. falciparum ribosomes have different structural and dynamic features that distinguish them from other organisms. While the majority of P. falciparum 28S associated rRNA expansion segments (ESs) are shorter than in human rRNA, 18S ESs are generally larger (Figure 7—figure supplement 1). The ES6S/7S is located next to the binding pocket of eIF3 and might be necessary for translation initiation or termination (Wong et al., 2014; Beznosková et al., 2013; Hashem et al., 2013). ES9S/10S are positioned at the head of the 40S subunit and are probably important for the recruitment of additional translation factors as well as for mobility of the head region of 40S subunit (Wong et al., 2014; Sun et al., 2015). Previous studies had shown that the absence of ZNF598 and RACK1 would help in the resolution of putative stalling and subsequent read through of polyA sequences (Garzia et al., 2017; Juszkiewicz and Hegde, 2017; Sundaramoorthy et al., 2017). However, the P. falciparum homolog of ZNF598 (PF3D7_1450400), appears to be abundantly expressed according to PaxDB database (Wang et al., 2015).

On the other hand, while being essential and one of the most well-expressed proteins in P. falciparum cells (Wang et al., 2015; Blomqvist et al., 2017), the absence of RACK1 protein on Plasmodium ribosomes could be the most prominent features regarding the translation of polyA tracts and poly-lysine repeats (Wong et al., 2014; Sun et al., 2015; Figure 7—figure supplement 1). Asc1 (yeast RACK1 homolog) is required for endonucleolytic cleavage of stalled mRNAs in yeast cells (Matsuo et al., 2017; Ikeuchi and Inada, 2016). The deletion of RACK1/Asc1 in human or yeast cells was also shown to increase the production of proteins with polybasic peptides, however, at the cost of increased frameshifting (Sundaramoorthy et al., 2017; Kuroha et al., 2010; Wolf and Grayhack, 2015). The recent report has shown that the absence of Asc1 in yeast generally slows down elongation, promoting frameshifting on problematic CGA-CGA pairs (Tesina et al., 2020). However, the mechanisms proposed for the polyA-induced ribosome stalling involves the inhibitory conformation of polyA tract mRNA in the A site of the ribosome (Chandrasekaran et al., 2019; Tesina et al., 2020). This proposed mechanism for polyA stalling induction should not depend on species, because ribosome nucleotides interacting with π-stacked polyA sequences are universally conserved (A1756 and C1634, Figure 7—figure supplement 2). No significant differences in the mRNA tunnel have been observed in P. falciparum ribosome structures (Wong et al., 2014; Sun et al., 2015). However, differences in the resolution and absence of mRNA in P. falciparum ribosome cryo-EM structures do not allow for precise comparison of the conformation of rRNA between Plasmodium and either yeast or mammalian ribosomes (Wong et al., 2014; Sun et al., 2015). Altogether these observations do not explain why the mRNA tunnel and interactors of the P. falciparum ribosome could influence the efficient translation of polyA tracts.

Recently it was postulated that the stalling on the polyA tracts is an effect of a synergy between polyA-induced suboptimal geometry at PTC and poly-lysine interactions with ribosome exit tunnel (Chandrasekaran et al., 2019).To further investigate whether additional differences in the ribosome structure contributed to P. falciparum adaptation to long polyA tracts and poly-lysine repeats, we analyzed the ribosome peptide exit tunnel of P. falciparum as predicted by the MOLE 2.5 toolkit (Figure 7; Sehnal et al., 2013). While a recent study found no differences in charge pattern along the peptide tunnel in eukaryotes, notably between P. falciparum and human (Dao Duc et al., 2019), we observed changes in hydrophobicity across the tunnel (Figure 7 and Figure 7—figure supplement 3). Interactions between clusters of positive charges and hydrophobic environments are generally unfavorable even during protein translocation (Fujita et al., 2011). Along this line, the ribosome exit tunnel of P. falciparum seems to be generally more hydrophilic, including the constriction site. Given a substantial length of poly-lysine peptide required to induce stalling on downstream polyA tracts (6 to 11, depending on the experimental system) (Chandrasekaran et al., 2019) it is plausible that the upper and the central tunnel are contributing to the observed effects. This is what is observed in other organisms. The free energy profile of lysine across the H. marismortui ribosome exit tunnel, which also has a hydrophobic region between the PTC and constriction site (Figure 7—figure supplement 3), indicates the presence of a significant energy barrier after the constriction site, not before (Petrone et al., 2008). Indeed, in vitro translation of poly-lysine and poly-phenylalanine using E. coli ribosomes resulted in rather different paths and rates of the extension of nascent peptide due to postulated hydrophobic entry to the ribosome exit tunnel (Picking et al., 1991). Moreover, in a recent structure of the polyA stalled yeast ribosome, the unresolved density was also indicated in this part of the exit tunnel next to the PTC and above the constriction site (Tesina et al., 2020). Taken together, it seems that the ribosome peptide exit tunnel of parasites adapted to accommodate synthesis of long runs of poly-lysine residues by reducing electrostatic barriers below the PTC and at the constriction site. The resulting dynamics of translation of poly-lysine might be high enough to reduce the chances for unfavorable interactions between mRNA and ribosome.

Figure 7 with 3 supplements see all
Polypeptide exit tunnel as predicted by MOLE service from Plasmodium ribosome (PDB:3j79) (A) and human ribosome (PDB: 6d90) (B).

The constriction site flanked by L22 and L4 is marked in the orange clamp, and lining residues in both cases are marked with ball-and-stick visualization. Vertical plot outline hydrophobicity (left) and charge (right) profiles across the tunnel (Pravda et al., 2018). The width of the tunnel is indicated on the scale at the bottom of the plot. The total lengths of the tunnel are indicated at the top of the plots.

Discussion

Runs of lysine and arginine residues are underrepresented in the proteome of multiple organisms compared to the runs of other amino acids, suggesting a selective pressure against polybasic amino acid sequences (Karlin et al., 2002). One potential explanation for this evolutionary selection is electrostatic interactions of the polybasic peptide and the exit tunnel of the ribosome that would impact the rate of protein synthesis (Brandman et al., 2012; Lu and Deutsch, 2008). However, multiple biochemical analyses of poly-lysine stalling sequences, in both prokaryotic and eukaryotic systems, revealed that runs of lysine AAA codons exhibit a more significant delay in ribosome movement than an equivalent number of AAG lysine codons (Glöckner, 2000; Szafranski et al., 2005; Zilversmit et al., 2010; Arthur et al., 2015; Koutmou et al., 2015; Tournu et al., 2019; Szádeczky-Kardoss et al., 2018; Chandrasekaran et al., 2019; Tuck et al., 2020; Tesina et al., 2020). More recent study in mouse embryonic stem cells indicates that besides 12A-1 sequences endogenous sites with 8–9 As are sufficient to cause recruitment of mRNA surveillance pathways (Tuck et al., 2020). So as is the case of polybasic residues, analyses of codon usage within poly-lysine peptides revealed that the expected frequency of AAA codons compared to AAG codons in runs of four consecutive lysine residues is lower in the coding regions of genes for multiple organisms (Arthur et al., 2015; Koutmou et al., 2015; Habich et al., 2016; Figure 1). However, ribosome stalling and frameshifting induced by polyA runs depend on the sequence and conformation of the mRNA in the ribosome, with a questionable contribution by the nascent polypeptide chain (Arthur et al., 2015; Arthur and Djuranovic, 2018; Koutmou et al., 2015; Tesina et al., 2020). As such, both polyA tracts and poly-lysine sequences are unfavorable in mRNAs or proteins, respectively, of most tested organisms.

Data presented here indicate P. falciparum, the most studied malaria-causing human pathogen, is an exception to this rule. With an 80% AT-rich genome and more than 63% of transcripts containing polyA tracts, P. falciparum represents a paradigm breaking species for polyA tracts (Figure 1). The atypical enrichment in polyA tracts in the coding sequence of mRNAs, translated into long poly-lysine runs in proteins, is a feature that is conserved in most Plasmodium species (Figure 1; Caro et al., 2014; Habich et al., 2016; Wang et al., 2015; Aurrecoechea et al., 2009). Such species-specific evolutionary conservations usually result in traits that are beneficial for survival. The increased AT-richness and number of polyA tracts in P. falciparum, as such, could be a result of selective pressure on biosynthetic pathways (AT vs. GC biosynthesis) (Seward and Kelly, 2016; Dietel et al., 2019) and the oxidative intraerythrocytic environment (Becker et al., 2004). An increase in poly-lysine runs might have been of different etiology. Gene ontology analyses of polyA tract and poly-lysine genes in P. falciparum suggested cellular and pathological adhesion proteins as one of the enriched gene groups (Supplementary file 1). It is attractive to speculate that advantages for the parasites to synthesize proteins with poly-lysine repeats could be solely driven by benefits in the adhesion and invasion of host cells (Kobayashi et al., 2013; Leitgeb et al., 2011) or export of numerous parasite proteins to the surface of infected erythrocytes (Davies et al., 2016; Romero et al., 2004; Hancock et al., 1990).

Our data further indicate that the P. falciparum translational machinery permits the translation of polyA tracts and poly-lysine repeats without mRNA degradation, protein synthesis attenuation, nor obvious activation of translational surveillance pathways (Figures 25). The biochemical assays with reporter sequences (Figures 2, 3 and 5) as well as analyses of ribosome profiling data (Caro et al., 2014; Figure 4) suggest that parasite ribosomes do not stall or frameshift on long polyA tracts and poly-lysine runs. Even though RNA folding propensity in P. falciparum has been skewed due to the nucleotide content, the predicted stability of native P. falciparum transcripts did not show differences compared to human cells. However, the change in nucleotide composition and a number of long polyA tracts of P. falciparum (Figure 1) probably shaped NGD/NSD mRNA surveillance responses in parasites (seen in Figures 5 and 6). Insertion of the stable stem-loop structure (StL) in the reporter construct resulted in reduced protein synthesis and ribosome stalling but without noticeable mRNA degradation (Figure 5 and Figure 5—figure supplement 2). Our results with stem-loop construct and Ile-starvation are rather disparate to experiments in other eukaryotes, including the human host (Arthur et al., 2015; Doma and Parker, 2006; Simms et al., 2017; Passos et al., 2009; Dimitrova et al., 2009), and argues for significant differences between human and Plasmodium NGD mRNA surveillance pathways.

Similarly, while following the general cellular response to induced metabolic stress with Ile-starvation we noticed the upregulation of Pelo and Hsp70 levels (Mills et al., 2016; Wu et al., 1985; Rosenzweig et al., 2019), however again with the lack of targeted mRNA degradation of Ile-rich transcripts or reduction in global mRNA stability (Babbitt et al., 2012; Figure 6). It seems plausible that amino acid starvation could allow uncharged Ile-tRNAs to fill the A-site and block Pelo binding thus preventing NGD and RQC activation in this case. Similarly, Leu- or Arg-starvation experiments in HEK293 cells do not result in global change in mRNA stability but rather increase in translation termination and ribosome recycling (Darnell et al., 2018). While authors in Hek293 amino acid starvation studies did not follow changes in Pelota protein levels, it was shown recently that transgenic overexpression of Pelo protein in human platelets led to rapid degradation of most cytoplasmic platelet transcripts (Mills et al., 2017). Our studies indicate that increased P. falciparum Pelo levels, at least during the amino acid starvation, do not cause targeted mRNA degradation. However, future studies on the role PfPelo levels during amino acid starvation, its role in ribosome recycling as well as identification of missing P. falciparum Hbs1 protein will give more insight in NGD and RQC process in Plasmodium. Nonetheless, results with the Stl construct and amino acid starvation suggest a difference between human and Plasmodium NGD mRNA surveillance and mRNA degradation pathways in both signaling and resolution of ribosome stalling events (Joazeiro, 2017). One of these differences could be potentially attributed to the loss of recently reported NGD endonucleases (D'Orazio et al., 2019; Glover, 2019; Navickas, 2019) in P. falciparum cells. The bioinformatics analyses of the P. falciparum genome did not indicate a homolog of either Cue2 or Nonu-1 endonuclease (Supplementary file 2; D'Orazio et al., 2019; Glover, 2019; Navickas, 2019). However, the loss of Cue2/Nonu-1 in Plasmodium cells can not completely explain the lack of mRNA degradation on the stem-loop construct (Figure 5) or in Ile-starvation experiments (Figure 6). Given that the majority of observed NGD-induced mRNA decay is processed by canonical exonucleolytic decay by Xrn1 (D'Orazio et al., 2019; Navickas, 2019), stabilization of NGD substrates in P. falciparum remains puzzling.

It is apparent that the accommodation of polyA tracts and poly-lysine repeats in translation required multiple adaptations to be vital and typically highly conserved components of the translation and mRNA surveillance pathways. Unambiguously, P. falciparum ribosomes had to change to accommodate correct and efficient protein synthesis from the runs of coding polyA tracts that are longer than the average size of 3’-UTR polyA tails in other organisms (Subtelny et al., 2014; Brown and Sachs, 1998; Chang et al., 2014). The fact that recent cryo-EM structures indicated that the Plasmodium ribosome lacks interaction with PfRACK1 protein (Figure 7) could be beneficial for the translation of polyA tracts into poly-lysine runs (Wong et al., 2014; Sun et al., 2015). Albeit, it is not clear what would substitute the role of RACK1/Asc1 in the correct reading frame maintenance during translation of long polyA tract or other stalling sequences (Sundaramoorthy et al., 2017; Tesina et al., 2020; Kuroha et al., 2010; Wolf and Grayhack, 2015). The lack of Asc1/RACK1 association with P. falciparum ribosomes could affect its association with other RQC protein homologs, such as ZNF598, which can influence the activation of RQC pathway and endonucleolytic cleavage (Ikeuchi and Inada, 2016). It is possible to speculate that P. falciparum specific ribosome extension segments in the 40S subunit may have the role in recruiting other Plasmodium proteins that may help with translation of polyA tracts and poly-lysine repeats (Wong et al., 2014; Sun et al., 2015). However, it was recently indicated that an inhibitory conformation of mRNA in the A-site of the ribosome, as well as the polybasic character of the nascent polypeptide chain in the ribosome, peptide exit tunnel, are crucial for the poly(A)-mediated stalling mechanism (Chandrasekaran et al., 2019; Tesina et al., 2020). It is not clear from the conservation of the 18S rRNA sequence (Figure 7—figure supplement 2) and structure of the ribosome A-site in P. falciparum ribosomes (Wong et al., 2014; Sun et al., 2015) how parasite ribosomes would circumvent an inhibitory conformation of the polyA mRNA in the A site seen in yeast and mammalian ribosomes (Chandrasekaran et al., 2019; Tesina et al., 2020). It might be that changes in the peptide exit tunnel of P. falciparum ribosomes (Figure 7) have partially contributed to the adaptation to the synthesis of long poly-lysine runs by reducing the potential stalling effects associated with translocation of polybasic peptides through hydrophobic environment (Fujita et al., 2011). However, contributions of Plasmodium’s ribosome exit tunnel in overcoming poly-lysine induced ribosome stalling still need to be tested experimentally. It is also plausible that translation rate is a factor here. Chandrasekaran and coworkers (Chandrasekaran et al., 2019) in their polyA stalling study had observed a significant difference in the number of lysines required for stalling between in vitro system and cells. They have explained the observation by a 5-fold differences in translation rate.

In conclusion, to adapt to polyA tract translation for the production of the polybasic and homopolymeric lysine repeats, the malaria-causing parasite has altered its mRNA translation quality control pathways as well as its ribosomal proteins and ribosome structure. The additional diversity of Plasmodium spp. rRNAs (Walliker et al., 1987; McCutchan et al., 1988; Waters et al., 1989) and possible activity of yet unknown ribosome-associated factors promote the possibility of Plasmodium ‘specialized ribosomes,’ which allow for polyA tract translation into poly-lysine sequences of functional proteins. Further insights into differences between components of translational machinery and mRNA surveillance pathways present in P. falciparum and host organisms, as well as the physiological role(s) of conserved poly-lysine repeats, will provide answers to these questions and enable identification of new drug targets against malaria.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Plasmodium falciparum)Pelota geneThis paperPF3D7_0722100CRISPR/Cas9 engineered with C-terminal 3xHA tag
Cell line (Plasmodium falciparum)Dd2 strainGoldberg labhttp://neurolex.com/wiki/NCBITaxon:57267P. falciparum strain used for all experiments
Cell line (Homo sapiens)Dermal fibroblast (normal, Adult)ATCCPCS-201–012
Cell line (Homo sapiens)Adult erythrocytes (primary cell line)Goldberg lab and BJCIH Children HospitalDeidentified human blood. 50% haematocrit, washed in complete medium (MCM)
Cell line (Tetrahymena thermophila)strain B2086 (II)Chalker Labhttp://neurolex.com/wiki/NCBITaxon:5911
Recombinant DNA reagentpBSICY-gtw (plasmid)Chalker Lab• PMCID:PMC3232721
T. thermophila transfection plasmid
Recombinant DNA reagentpc-DNA-DEST40 (plasmid)Thermo Fisher ScientificRRID:Addgene_45597HDF transfection plasmid
Recombinant DNA reagentpHHT-TK (plasmid)Goldberg LabPlasmid for standard CRISPR/Cas9 knock out approach in P. falciparum cells.
Recombinant DNA reagentpyDHOD-2A-Cas9 (plasmid)Goldberg labAll-in-one Cas9+gRNA cassette vector
DSM-1 resistance
Antibodyanti-HA antibody (mouse monoclonal antibody)Santa Cruz BiotechnologyCat# sc-7392, RRID:AB_627809WB (1:5000), used also as HRP conjugated
Antibodyanti-mouse (horse unknown clonality)Cell Signalling TechnologyCat# 7076, RRID:AB_330924)WB (1:5000)
Antibodyanti-rabbit (goat polyclonal antibody)Cell Signalling TechnologyCat# 7074, RRID:AB_2099233WB (1:5000)
AntibodyAnti-HSP70 (rabbit polyclonal antibody)AgriSeraCat# AS08 371, RRID:AB_2248616WB (1:1000)
Antibodyanti-PfHAD1 rabbitOdom labWB (1:10000) PMID:25058848
Chemical compound, drugCycloheximideSigma AldrichCat# C1988200 uM
Chemical compound, drugWR99210Sigma AldrichCat# W177010 nM
Chemical compound, drugDSM1Sigma AldrichCat#533304DHODH Inhibitor
1.5 uM
Commercial assay or kitllustra triplePrep KitGE HealthcareCat#
28942544
DNA, RNA and protein isolation kit

Parasite culturing

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P. falciparum line Dd2 was cultured at 2–5% hematocrit in O+ erythrocytes in RPMI 1640 supplemented with 5 g/l Albumax II (Gibco), 0.12 mM hypoxanthine (1.2 ml 0.1M hypoxanthine in 1 M NaOH), 10 μg/mL gentamicin (Trager and Jensen, 2005). Cultures were grown statically in a candle jar atmosphere. As required, cultures were synchronized with 5% (wt/vol) sorbitol. Medium for Isoleucine starvation: 16.1 g RPMI medium 1640 per l (ME011232P1 Gibco), 2 g of sodium bicarbonate,0.12 mM hypoxanthine, 10 μg/mL gentamicin, and with and w/o 100 uM Isoleucine.

Generation of mCherry-GFP–Expressing plasmids with and without 12 lysines coded with 36 As in a row (polyA- and polyA+) mCherry with and without polyA was amplified from plasmids previously created (Arthur et al., 2015) pBttlysforXhoI 5′-gcgcctcgagatgggctacccatacga-3′ (XhoI site underlined) and mCherryrevAvrII 5′-gcgccctaggcttgtacagctcgtccatgccg3′ (AvrII site highlighted) digested with XhoI and AvrII, and ligated into the same sites of the episomal over-expression (EOE) a containing the promoter region for PfHsp86, a C-terminal GFP tag, and a human dihydrofolate reductase (hDHFR) drug-selection cassette (Russo et al., 2009). mCherry reporter in pc-DNA-DEST40 previously published Arthur et al., 2015; Arthur et al., 2017 used to transfect HDF cells.

HDF cells culturing and transfection

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HDF cells (ATCC PCS-201–012) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) and supplemented with 10% fetal bovine serum, 5% minimum essential medium nonessential amino acids (100×, Gibco), 5% penicillin and streptomycin (Gibco), and L-glutamine (Gibco). HDF cells were Mycoplasm-free based on PCR-tests using Venor GeM Mycoplasma Detection Kit (Sigma Aldrich).

mCherry reporter in pc-DNA-DEST40 plasmid (1 ug) was introduced to the cells by the Neon Transfection System (Invitrogen) with 100 μl tips according to cell-specific protocols (www.lifetechnologies.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system/neon-protocols-cell-line-data.html). Cells electroporated with DNA plasmids were harvested after 48 hr if not indicated differently (Arthur et al., 2015).

Parasite transfection

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Asynchronous Dd2 parasites were incubated with 100 μg of maxi-prep DNA of each of the EOE constructs described above encoding mCherry-GFP (with and w/o polyA), transfected in Bio-Rad Gene Pulser cuvette (0.2 cm), 0.31 kV, 950 up, infinity resistance. 10 nM WR99210 was added to parasite 48 hr after transfection and used to select resistant parasites (Fidock and Wellems, 1997).

Saponin lysis of infected red blood cells (iRBC)

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The cell iRBCs were resuspended in two volumes of PBS containing 0.15% saponin, and incubated on ice for 10 min, with vigorous mixing every 3 min. Afterward, the samples were centrifuged 7000 g, 5 min, 4°C, and the pellets were washed three times more with the same buffer.

Genomic DNA (gDNA) extraction

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The extraction of gDNA was done from samples pretreated with a saponin lysis buffer, and we followed the protocol of the DNeasy Qiagen kit.

RNA extraction and qRT-PCR

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Total RNA was extracted from iRBCs using the RiboZol RNA extraction reagent (Amresco) with some changes (protocol from Jabos-Lorrrena Laboratory at Johns Hopkins School of Public Health), or we used Illustra triplePrep Kit GE Health Care. In 1 ml of pelleted iRBCs (500 g, 5 min, room temperature), parasitemia 5–7%, 5 ml of RiboZol was added. It was mixed thoroughly that all cells lysed. At this step, the sample can be snap-frozen and stored at −80°C or proceed further. Add 1 ml of chloroform and mix for 20 s. Transfer the aqueous phase to a new tube, and add 1/10th of the volume of 3 M sodium acetate, mix well and add 10 µl GlycoBlue. Add 2.5 ml of cold iso-propanol and leave overnight at −80°C. After overnight precipitation, the samples were centrifuged at 16000xg, 1 hr, 4°C. The supernatant was discarded and washed twice with 5 and 1 ml of cold ethanol. The pellets were dried and resuspended in pure water. RNA concentration was measured by NanoDrop (OD260/280). For total RNA extraction using Illustra TriplePrep Kit (item# 28942544 GE Health Care), we used 200 µl of iRBCs, parasitemia 5–7%. The cell pellet was resuspended in 600 µl PBS buffer containing 0.15% saponin (item# 47036–50 G-F Sigma), cOmplete Protease Inhibitor Cocktail without EDTA (item# 11873580001 Roche), RNase OUT 1 µl/ml (item# 10777019 Invitrogen), and incubated at RT for 10 min on ice. Afterward, it was centrifuged 7000xg, 5 min, 4°C, and the pellets were washed three times more with the same buffer. After this pretreatment to get rid of hemoglobin pellets were used for total RNA isolation following Illustra TriplePrep Kit protocol. iScript Adv cDNAkit for RT-qPCR (item# 172–5037, Bio-Rad) or SuperScript IV VILO Master Mix(item# 11756050 Life Technologies Corporation), was used with 100–200 ng of total RNA following the manufacturer’s protocol. iQ SYBR Green Supermix (item# 1708886, Bio-Rad) protocol was used for qRT-PCR on the CFX96 Real-Time system with Bio-Rad CFX Manager 3.0 software (Arthur et al., 2015). Cycle threshold (Ct) values were normalized to the hDHFR resistance gene expressed from the same plasmid or HSP86, GAPDH genes.

qRT-PCR primers

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  • PF3D7_14628002GAPDHqf

  • 5′-ACCAAAGGATGACACCCCAA

  • 2GAPDHqf 5′-ACCACCCTTTGATGGACCAT

  • PF3D7_1464200

  • 610gf 5′-CGACAAGGCCATTTTAGAGAA-3′

  • 610gR 5′TTTCGTTTTATCTCCGCTTACA-3′

  • PF3D7_0615500

  • 0615500for 5′-CCACAATTGGAGTCGTCGTA-3′

  • 0615500rev 5′-TCAAATCGAATTCTGTGACTCCT-3’

  • UniProtKB - P00374

  • qPCR_ hDHFR_F_5′-TCCTCCTGGACATCAGAGAGA-3′ qPCR_ hDHFR_R_5′-CTCAAGGAACCTCCACAAGG-3′

  • PF3D7_0818900

  • Hsp70qf 5′-GAATCGGTTTGTGCTCCAAT-3′

  • Hsp70qr 5′-CAACTGTTGGTCCACTTCCA-3′

  • PF3D7_1108000

  • qPCRrevIWS1w 5′-TGGTTGAAGAGGATGAGGAGA-3′ qPCRrevIWS1w 5′-ACCTTGTGCATATCATCATTTTCC-3′ mCherry mCherry qF 5′-TGACGTACCGGATTATGCAA-3′ mCherry qR 5′-ATATGAACTGAGGGGACAGG-3′

  • Ile rich genes

  • PF3D7_0322300_F 5′-TGGATGATCTGAGCAACAAAA-3′

  • PF3D7_0322300_R 5′-GGGTGGATCTTTATGCAAGC-3′

  • PF3D7_0512700_F 5′-TGGAACAGCATTAACGGAAA-3′

  • PF3D7_0512700_R 5′-GAGGTATTCCTACCCTTTTCTCAA-3′

  • PF3D7_0110600_F 5′-AGCATCACGACCTTTCCATC-3′

  • PF3D7_0110600_R 5′-TTGCATAAGCATTGGGATGA-3′

  • PF3D7_0903400_F 5′-TTCCATTATTGCATGCTCTCC-3′

  • PF3D7_0903400_R 5′-TCACACATGGATGTTGCTCA-3′

  • PF3D7_1450600_F 5′-ACGGATTACATGCAGCACAA-3′

  • PF3D7_1450600_R 5′-GATGACGTGTCGTCAAAAA-3′

  • PF3D7_1364700_F 5′-AAGGAAGCTCGGTTTTATTTGA-3′

  • PF3D7_1364700_R 5′-AAACCCTTCTTTTGTTTTGACA-3′

  • PF3D7_1121000_F 5′-CAAAAACAAATCCCGTAGATCC-3′

  • PF3D7_1121000_R 5′-CGATACAATTGTTGACCCACAT-3′

  • PF3D7_1341600_F 5′-GGGAATGGGAACCTTGTGTA-3′

  • PF3D7_1341600_R 5′-TCTTCATTTATCCATGCGTCA-3′

  • PF3D7_1321400_F 5′-TCCTTTCCATCCTCCCTTTT-3′

  • PF3D7_1321400_R 5′-TGGATTTTATCCACGGGTGT-3′

  • PF3D7_1138600_F 5′-ACAAGCGGAAAATATCGAATG-3′

  • PF3D7_1138600_R 5′-TCGTCTAAGTCCACTTCACTGC-3′

Immunoblotting and antibodies

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Samples pretreated with lysis buffer supplemented with cOmplete Protease Inhibitor Cocktail without EDTA were prepared with passive lysis buffer (Promega), BioRad sample buffer and BioRad reducing buffer. For immunoblotting, the PVDF membranes were blocked in 5% milk in PBS. The membranes were probed with anti-HA, or Anti-HA HRP (sc-7392, sc-7392HRP, Santa Cruz) or anti-HA mouse or rabbit antibody (7076 s, 7074 s respectively, Cell Signaling) diluted in 2.5% milk in PBS-Tween20 0.1% PBST) 1: 5000, an anti-hDHFR mouse antibody (sc-377091, Santa Cruz) diluted 1:5000 in 2.5% milk in PBST, anti-HSP70 mouse(AS08 371, Agrisera, generous gift of Goldberg lab) 1:1000 in 2.5% milk in PBST, anti-PfHAD1 rabbit antibody (generous gift of Odom lab) 1:10000 in 2.5% milk PBST. Secondary HRP-labeled anti-mouse or anti-rabbit antibodies are diluted 1:5000 in 2.5% milk in PBST and incubated for an hour. After incubation with the primary antibody, the PVDF membranes were washed three times for 5 min in PBST, Prepare Working Solution by mixing equal parts of the Stable Peroxide Solution and the Luminol/Enhancer Solution (34577 SUPERSIGNAL WEST PICO PLUS, 34096 SUPERSIGNAL WEST FEMTO MAXIMUM SENSITIVITY SUBSTRATE respectively). We incubate the blot in Working Solution for 5 min. Remove the blot from Working Solution and drain excess reagent. Afterward we tookimages were generated by BioRad Molecular Imager CHemiDoc XRS System with Image Lab software.

Starvation assay

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Asynchronous P. falciparum Dd2 parasites clones, with HA-tagged Pelota gene cultured in complete RPMI at 5% hematocrit and grown to ∼3–5% parasitemia. The parasites were washed twice in PBS, equally partitioned, and washed in complete, isoleucine-free, and then were replated in their respective medium. Parasite cultures were incubated at 37°C, in a candle jar atmosphere for 48 hr. After incubation, parasites were harvested. After harvesting, infected RBCs were lysed for total RNA/protein isolation or polysome profiling (Babbitt et al., 2012).

Polysome-associated RNA isolation

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For polysome/RNA isolation we did according to published protocols (Bunnik et al., 2013; Lacsina et al., 2011). Shortly, cycloheximide (100 mM) was added to parasite-infected red blood cell cultures to a final concentration of 200 μM. The culture was incubated for 10 min at 37°C following with pelleting erythrocytes (5 min at 500 x g at room temperature) and washed twice in PBS containing 200 μM cycloheximide. After the last wash, pellets were kept on ice and were subsequently lysed by adding 2.2 volumes of lysis buffer (1% (v/v) Nonident P-40% and 0.5% (w/v) sodium deoxycholate in polysome buffer (400 mM potassium acetate, 25 mM potassium HEPES pH 7.2, 15 mM magnesium acetate, 200 μM cycloheximide, 1 mM dithiothreitol (DTT), and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride HCl (AEBSF))) or cOmplete Protease Inhibitor Cocktail without EDTA (Roche), RNase OUT 1 µl/ml (Invitrogen). After 10 min incubation on ice, lysates were centrifuged for 15 min at 20,000xg at 4°C, at this point, the pellets were flash-freeze and stored at −80°C. The clarified lysates were then loaded on top of a sucrose cushion (35% sucrose in polysome buffer) to concentrate the ribosomes (4 ml polycarbonate ultracentrifuge tubes and then centrifuged for two h at 150000xg at 4°C in a Type 100.3 Ti rotor (Beckman Coulter, Brea, CA, USA). Ribosome pellets were resuspended in polysome buffer. Afterward, the ribosome suspension was layered on top of a 15 ml continuous linear 15% to 60% sucrose (w/v) 2 hr 40 min 260343xg (Beckman Optima XPN-90 and the swinging bucket rotor SW41 Ti). Fractions of 500 μl were collected using a UA-5 UV detector and model 185 gradient fractionator (ISCO, Lincoln, NE, USA). RNA was extracted with acid-phenol: chloroform pH 4.5 (Life Technologies), extracted twice with chloroform, and then precipitated using isopropanol.

Plasmodium falciparum sucrose cushion for polysome profiling

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This prep is used to isolate crude ribosome pellet, removing hemoglobin, which can then be later used for polysome profiling. We used 2 ml 100% hematocrit erythrocytes 8% parasitemia. The pellets were washed with 10 ml PBS and flash freeze in liquid nitrogen. Samples were stored at −80C before further use. Reagents: To make 10 ml Plasmodium falciparum Polysome Lysis Buffer (2.2 V/sample), we used 25 mM K-HEPES (1 M stock) 250 μL, 400 mM K-OAc (4 M stock) 1000 μL, 15 mM Mg-OAc (1 M stock) 150 μL, 1% Igepal CA-360 (100% stock), 100 μL 0.5% Na Deoxycholate (10% stock) 500 μL, 200 μM cycloheximide (100 mM stock) 20 μL, 1 mM AEBSF (200 mM stock) 50 μL, 1 mM DTT (1 M stock) 10 μL, RNase Inhibitory (40 U/μL stock) 10 μL, Molecular Grade Water 7 mL, 910 μL. Plasmodium falciparum Sucrose Cushion for Polysome Profiling (15 ml), 25 mM K-HEPES (1 M stock) 375 μL, 400 mM K-OAc (4 M stock) 1500 μL, 15 mM Mg-OAc (1 M stock) 225 μL, 200 μM cycloheximide (100 mM stock) 30 μL, 1 mM AEBSF (200 mM stock) 75 μL, 1 mM DTT (1 M stock) 15 μL, 40 U/mL RNase Inhibitory (40 U/μL stock) 15 μL, Ultrapure sucrose 5.135 g, Molecular Grade Water (to start, complete to 15 mL after dissolved and all components added) 7.5 mL. Plasmodium falciparum Polysome Wash Buffer (1.5 mL/sample) 25 mL. 25 mM K-HEPES (1 M stock) 625 μL, 400 mM K-OAc (4 M stock) 2500 μL, 15 mM Mg-OAc (1 M stock) 375 μL, 200 μM cycloheximide (100 mM stock) 50 μL, 0.1 mM AEBSF (200 mM stock) 12.5 μL, 1 mM DTT (1 M stock) 25 μL, 10 U/mL RNase Inhibitory (40 U/μL stock) 6.25 μL, Molecular Grade Water 21 mL. 406.25 μL Plasmodium falciparum Polysome Lysis Buffer (500 μL/sample) 2.5 mL 25 mM K-HEPES (1 M stock) 62.5 μL 400 mM K-OAc (4 M stock) 250 μL, Plasmodium falciparum Sucrose Cushion for Polysome Profiling - 15 mM Mg-OAc (1 M stock) 37.5 μL, 1% Igepal CA-360 (100% stock) 25 μL, 200 μM cycloheximide (100 mM stock) 5 μL, 1 mM AEBSF (200 mM stock) 12.5 μL, 1 mM DTT (1 M stock) 2.5 μL, 40 U/mL RNase Inhibitory (40 U/μL stock) 2.5 μL, Molecular Grade Water 2 mL and 102.5 μL. All steps were performed on ice. Pellets, 2 ml, were lysed with 2.2 V of lysis buffer, vortex to mix well. Incubate at 4 °C for 10 mins while rocking or rotating. Centrifuge 11,800 x g, move lysate to the fresh tube (~6.2 mL) For Sucrose Cushion Setup, we needed three cushions per sample. We were using 1 mL cushion per ~2 mL lysate. Add 1 mL sucrose to each tube. Layer sample lysates over sucrose cushion. Add 2 mL to each, then split the remainder (usually a few hundred microliters) over the three tubes. We used TLA 100.3 rotor (kept cold in the refrigerator) for ultracentrifugation. Speed: 100,000 x g, Time: 1 hr 30 mins 15 c. Temp: 4 °C When the centrifugation is finished, carefully aspirate off supernatant without touching sides so as not to disrupt pellets. Wash pellet 3X with 500 μL wash buffer, gently pipetting on the side opposite the pellet near the bottom of the tube. Resuspend and combine pellets for each sample in a total of 500 μL ribosome resuspension buffer. Pipet to disperse pellet. Use 200 μL tips to further disperse. Move to 1.5 mL microcentrifuge tube Incubate at 4 °C, rotating end-over-end, for at least 10 min. To remove remaining hemoglobin, set up another 1 mL sucrose cushion for each sample as previously. 23. A quick spin to collect samples to the bottom of the tube. Layer ribosome suspension over sucrose cushion, balance using ribosome resuspension buffer. Centrifuge as previously. Wash 3X with 500 μL wash buffer as previously. Resuspend in 500 μL ribosome resuspension buffer as previously. If not, proceed to the next steps immediately, flash-freeze in liquid nitrogen and store at −80 °C.

Parasite live imaging

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To image P. falciparum Dd2 strain episomally expressing polyA- and polyA+ constructs, we used 50 µl of infected erythrocytes, washed two times in PBS. The nucleus was stained with 1:1000 dilution of Hoechst 33342 for 10 min at room temperature. The cells were washed two times with PBS (500xg, 5 min, room temperature). After the washing step, the cells were resuspended in PBS (500 µl), 5 µl of cell resuspension was put on positively charged slides and put Zeiss cover glasses (item number:474030-9000-000). The cover glasses were sealed with nail polish, and subsequently, microscopy was performed.

Samples were visualized using an upright Zeiss Examiner.Z1-based 880 LSM with a 100x/1.46 oil-immersion objective. DAPI was excited using a 405 nm diode laser, mCherry was excited with a 561 nm DPSS laser, and GFP was excited with an Argon laser tuned to 488 nm. Optical sections (0.3 µm) were acquired with an Airyscan super-resolution detector and were processed using ZEN Blue v. 2.3.

Generating Tetrahymena thermophila expressing YFP plasmids without (polyA-) and with (polyA+) coding for 12 lysines

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Tetrahymena thermophila strain B2086 (II) was used for all experiments reported. Similar results were obtained with strain CU428 [(VII) mpr1-1/mpr1-1]. To assess the effect of LysAAA codons on protein accumulation, we modified a fluorescent protein tagging vector, pBSICY-gtw (Motl and Chalker, 2011) so as to fuse YFP to the carboxyl-terminus of a macronucleus-localized protein of unknown function (TTHERM_00384860), separated by a Gateway recombination cassette (Invitrogen/Life Technologies, Inc), and expressed from the cadmium inducible MTT1 promoter (Shang et al., 2002). The TTHERM_00384860 coding region was amplified with oligonucleotides 5’ ALM Bsi’ 5’ - CAC CCG TAC GAA TAA AAT GAG CAT TAA TAA AGA AGA AGT-3’ and 3’ ALM RV 5’- GAT ATC TTC AAT TTT AAT TTT TCT TCG AAG TTG C 3’ and cloned into pENTR-D in a topoisomerase mediated reaction prior to digesting with BsiWI and EcoRV and inserting into BsiWI/PmeI digested pBSICY-gtw. Subsequently, LR Clonase II was used to insert a linker containing the sequence coding for an HA epitope tag alone (N) or the tag plus 36 adenosines (K12) in place of the Gateway cassette. The expression cassette is located within the 5’ flanking region of a cycloheximide resistant allele of the rpL29 gene to direct its integration into this genomic locus. These constructs were linearized with PvuI and SacI in the region flanking the Tetrahymena rpl29 sequences and introduced into starved Tetrahymena cells by biolistic transformation (Bruns and Cassidy-Hanley, 2000; Cassidy-Hanley, 1997). Transformants were selected in 1x SPP medium containing 12.5 μg/ml cycloheximide. To control for copy number, PCR assays with primers MTT2386 5’- tc tta gct acg tga ttc acg −3’and Chx-117, 5’- ATG TGT TAT TAA TCG ATT GAT −3’ and Chx85r, 5’- TCT CTT TCA TGC ATG CTA GC – 3’ verified that all rpL29 loci contained the integrated expression construct. Transgene expression was induced by addition of 0.4 μg/ml CdCl2 and cells were grown 12–16 hr before monitoring protein accumulation. YFP accumulation was visualized by epifluorescence microscopy as previously described (Matsuda et al., 2010). Whole cells extracts were generated by boiling concentrated cell pellets in 1x Laemmli sample buffer, followed by were fractionation on 10% SDS polyacrylamide gels and transferred to nitrocellulose. YFP accumulation was a monitored with mouse anti-GFP antisera (G28R anti-GFP (OAEA00007) antibody, Aviva Systems Biology) and normalized to acetylated Rabbit anti-Histone H3 trimethyl-lysine (Upstate Biotechnologies/Millipore, NY, 07–473). Accumulation of transcripts was assessed on Northern blots as previously described using a 32P-random-primer-labeled YFP probe (Chalker and Yao, 2001).

2D-electrophoresis

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2 ml (100% hct) of P. falciparum infected erytrocyes (HA-pelo Dd2 strain, parasitemia 7–9%) were treated with PBS saponin 0.15%, and protease inhibitor w/o EDTA. The pellets were washed in PBS-saponin bufer three times. Afterwards, the samples were used to do immunoprecipitation (IP). We used Pierce HA-magnetic beads 25 µl and further proceeded with cell lysis:Lysis Buffer:150 mM NaCl, 50 mM Tris pH 7.5, 1% IGPAL-CA-630 (Sigma, #I8896), 5% Glycerol, Protease (1 mM PMSF) and phosphatase inhibitors. Sample wash: Wash Buffer: 150 mM NaCl, 50 mM Tris pH 7.5, 5% Glycerol. After the washing step the samples were snap frozen in liquid nitrogen for 2D gel analysis.

Two-dimensional electrophoresis was performed according to the carrier ampholine method of isoelectric focusing (O'Farrell, 1975; Burgess-Cassler et al., 1989) by Kendrick Labs, Inc (Madison, WI) as follows: Isoelectric focusing was carried out in a glass tube of inner diameter 2.0 mm using 2% pH 3–10 isodalt servalytes (Serva, Heidelberg, Germany) for 9600 volt-hrs. One μg of an IEF internal standard, tropomyosin, was added to the sample. This protein migrates as a doublet with lower polypeptide spot of MW 33,000 and pI 5.2. The enclosed tube gel pH gradient plot for this set of ampholines was determined with a surface pH electrode.

After equilibration for 10 min in Buffer 'O' (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M tris, pH 6.8), each tube gel was sealed to the top of a stacking gel that overlaid a 10% acrylamide slab gel (0.75 mm thick). SDS slab gel electrophoresis was carried out for about 4 hr at 15 mA/gel. After slab gel electrophoresis, the duplicate gel for blotting was placed in transfer buffer (10mMCaps, pH 11.0, 10% MeOH) and transblotted onto a PVDF membrane overnight at 200 mA and approximately 100 volts/two gels. The following proteins (Millipore Sigma) were used as molecular weight standards: myosin (220,000), phosphorylase A (94,000), catalase (60,000), actin (43,000) carbonic anhydrase (29,000) and lysozyme (14,000). These standards appear as bands at the basic edge of the Coomassie Brilliant Blue R-250-stained membrane.

Coding sequence metrics

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Coding sequences for each organism (Plasmodium falciparum 3D7 - EPr 1, Tetrahymena thermophila – JCVI-TTA1-2.2, and human - GRCh38.p7) were downloaded from their respective Ensembl BioMart pages (accessed on March 19th 2018). Each set of genes was filtered to include only the longest coding sequence variant. The resulting sequences were each analyzed using a folding window approach; a window of 120 nt was scanned across each sequence with a single nucleotide step size. Each window sequence was folded (using RNAfold) to determine its minimum free energy structure/value (MFEnative) (Lorenz et al., 2011). At the same time, MFE values were calculated for 30 randomized versions of the native window sequence (MFErandom). The mean of MFErandom values were compared to that of the native in a method adapted from Clote et al., 2005 and illustrated in the following equation:

zscore= MFEnativeMFErandom¯Std DevMFE

The mean window MFE and z-score values of each gene were calculated and compiled in separate spreadsheets. Box and whisker plots (generated using BoxPlotR Spitzer et al., 2014) are shown, depicting the distribution of these means for each organism.

Bioinformatics analyses

Ribosome profiling

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GWIPS database was used as the source of ribosome profiling data. We downloaded Caro et al., 2014 dataset for Plasmodium falciparum (the only dataset available for that species), while for humans, we used aggregate for all deposited studies. In both cases, we took a dataset for elongating ribosomes mapped to A-site.

The definition of polyA-tracts was taken from Arthur et al., 2015, which are twelve consecutive adenines allowing for one mismatch. Genomic coordinates of such segments were downloaded from PATACSDB (Habich et al., 2016). We took 50 residues upstream and downstream from the beginning of the polyA segment, preserving the proper strand orientation. Occupancy plot was generated with two conditions:

  1. discarding fragments that had less than five occupancy values in the given window of 101 nucleotides

  2. taking into account fragments that had the average occupancy equal or higher than the mean for the dataset (this was to remove the influence of poorly mapped segments from the plots)

To make occupancy plots between different life stages comparable (Figure 4—figure supplement 2), we have introduced a normalization mechanism, where occupancy of polyA region was divided by a mean from similarly sized distribution of random fragments of the same length that had the 0 position within the coding region. Randomization was preserving chromosome distribution of the original polyA-carrying genes dataset. All the analyses were done using R language.

Gene ontology analysis

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Gene Ontology analysis was done using the Gene Ontology Enrichment tool at PlasmoDB website (Aurrecoechea et al., 2009), using default options. For Supplementary file 1 we took only those terms that had Bonferroni-adjusted p-value better than 0.05.

Structural analyses

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Structural analyses were done using MOLEonline service version 2.5 (Pravda et al., 2018) using default parameters, with manual selection of the proper tunnel among all predicted by this service. Due to limits of the method, it was not possible to enforce the same length of the tunnel in all structures, yet the hydrophobic patches formed by interaction with rRNA and L4/L22 proteins were easy to observe in all structures.

Data availability

All data generated or analysed during this study are included in the manuscript, supporting files or referenced. Source data files have been referenced for Figures 1, 3 and 5, as well as for figure supplements.

The following previously published data sets were used
    1. Caro F
    2. Ahyong V
    3. Betegon M
    4. DeRisi JL
    (2014) NCBI Gene Expression Omnibus
    ID GSE58402. Ribosome Profiling in P. falciparum asexual blood stages.

References

    1. Cassidy-Hanley D
    (1997)
    Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment
    Genetics 146:135–147.
    1. O'Farrell PH
    (1975)
    High resolution two-dimensional electrophoresis of proteins
    The Journal of Biological Chemistry 250:4007–4021.
    1. Picking WD
    2. Odom OW
    3. Tsalkova T
    4. Serdyuk I
    5. Hardesty B
    (1991)
    The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes
    The Journal of Biological Chemistry 266:1534–1542.

Decision letter

  1. Nahum Sonenberg
    Reviewing Editor; McGill University, Canada
  2. James L Manley
    Senior Editor; Columbia University, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Plasmodium falciparum genome is extremely AT-rich (81% of the genome). PolyA tracts in open reading frames in eukaryotes and prokaryotes trigger ribosome quality control mechanisms. The authors show that in contrast to other organisms, in Plasmodium falciparum, mRNAs containing long polyA sequences are efficiently and accurately translated. Also, polyA tracts fail to engender No Go Decay (NGD) process or production of frameshift-protein fragments.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Plasmodium falciparum translational machinery condones polyadenosine repeats" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work cannot be considered further for publication in eLife, at least in its current form.

In general, the reviewers agree that the main observation of the absence of mRNA decay of RQC substrates in Plasmodium is very interesting and that the field would greatly benefit from knowing it. However, in the present form, the ribosome profiling data are unreliable due to the pre-treatment with cycloheximide. The analysis in Figures 3-4 is significantly lacking. Evidence should be obtained to determine whether ribosomes are actually stalling or not. The eLife rules require resubmission of a revised version of the paper in 2 months. This would appear to be hard to accomplish. We hope that you can respond to the reviewers' criticisms given extra time. As the reviewers' comments are extensive but highly constructive, they are detailed below. A revised version of your manuscript that addresses all these issues would likely be suitable for reconsideration.

Reviewer #1:

Ribosome stalling and the RQC pathway, which resolves the stalled ribosome and degrade the mRNA and nascent peptide, have drawn considerable attention in recent years. However, there are significant gaps in our understanding of the various aspect of these phenomena, particularly their importance in physiology, considering that their substrate mRNAs with runs of multiple AAA codons, are quite rare in eukaryotic genomes. Therefore, it has been previously proposed that RQC on mRNAs with polyA sequence within ORFs may have anti-parasitic significance. In this manuscript, Djuranovic and colleagues demonstrate a very interesting observation that the malaria-causing parasite P. falciparum, circumvent the ribosome stalling process at polyA sites, despite having a remarkable number of mRNAs with long polyA tracks. Thus, this manuscript clearly advances our understanding of this process and extend the field further and should be of interest to investigator in various fields. However, despite its strengths in providing convincing data on the unusual nature of the P. falciparum transcriptome and the apparent deficiency of the mRNA decay pathway in this species, the manuscripts does not properly address the other possible aspect of the RQC-induced silencing; mRNA translation. In addition, the robustness of the data produced by ribosome profiling assay maybe compromised due to technical issues as described below. Therefore, the manuscript may benefit from additional experimentation/analyses to verify this aspect.

1) The contribution of translational repression in this process is not properly addressed. This is an important issue in light of the emphasize that the authors placed on the main role of the mRNA sequence, rather than amino acid sequence of the nascent peptide, in stalling. Nevertheless, while the authors quite clearly demonstrate the lack of efficient mRNA decay pathway, their explanation for the absence of stalling at polyA sequences in P. falciparum seems to rely solely on the differences in the exit channel of the ribosome. This comes across as changing the goal post. Rather than explaining why the mRNA sequence does not induce stalling, the authors in fact try to address why the peptide doesn't induce stalling. On this note, it is unclear why the authors focused only on measuring the mRNA levels of a few of the candidate genes with more than 22As (Figure 4). It is quite possible that due to deficiency in the mRNA decay pathway, the target mRNAs are only translationally repressed, but it does not rule out the potential translational repression. As it stands, it is unclear whether the ribosome stalling and RQC pathway in P. falciparum is deficient, or the observed phenomenon is merely due to the deficient mRNA decay. Therefore, performing a polysome profiling assay and western blot for the candidate proteins would be suitable additions to confirm the lack of stalling on mRNAs with long polyA tracts in P. falciparum.

2) It has been previously shown that pre-treatment of the samples with translation inhibitors such as cycloheximide and anisomycin could complicate the analysis of ribosome stalling by ribosome profiling assay and result in false negative outcome, when investigating ribosome pausing/stalling on poly-basic sites (Requião, et al., 2016). From what could be deduced in the Materials and methods section, the samples seem to have been pre-treated with cycloheximide for 10 minutes before collection. Therefore, this may in fact take away from the reliability of the observation that there is no ribosome pausing at polA tracts in P. falciparum based on the ribosome profiling assay, as it may be a false negative observation due to artefacts caused by cycloheximide.

Reviewer #2:

In this manuscript, Pavlovic-Djuranovic et al. show that poly(A) tracks in Plasmodium falciparum are efficiently translated and their translation does not trigger No Go Decay (NGD). This is very interesting since translation of poly(A) sequence elicits NGD in human cell or in Tetrahymenathermophila, which is an organism with an AT-content similar to P. falciparum. The authors further show that isoleucine starvation causes upregulation of Pelo but suggest this Pelo is not involved with NGD. Inserting a stem loop in their reporter construct disrupts protein synthesis but still does not trigger NGD. The authors hypothesize that the ribosome rescue pathway is evolved in this parasite differently than other organisms and NGD is not triggered upon ribosome stalling. They speculate that loss of RACK1's association with the ribosome and other structural differences could underlie these trends, though no data are offered to test these hypotheses. Overall, this paper grapples with an intriguing observation and improves our understanding of ribosome quality control system in different organisms. However, the paper seems to have been quickly written and lacks clarity. While much of the data are negative, it is worth sharing if further experiments could be performed in some cases to more clearly explain what models are being tested and directly show the results in Plasmodium are unlike those in other organisms.

List of major concerns:

Is it possible that during poly(A) translation, P. falciparum ribosomes miscode and incorporates amino acids other than Lys? A mass spectrometry analysis of the reporter peptide could address this possibility and show that the ribosome faithfully translates Lys codons without miscoding and frameshifting.

It is difficult to see the difference between human and P. falciparum ribosome occupancy in Figures 3A and 3B. It should be shown by distinct colors and the color code should be included in the figure legends. What do the error bars signify? Is the large central dip in 3B significant? Also, the authors should explain how many genes are going into the analysis and how they are controlling for the possibility that a few strongly-expressed genes are dominating these effects (how was normalization done?).

The authors refer to previous studies where frameshifting was attributed to translation of poly(A) sequences. They do not see frameshifting when poly(A) reporters were examined here in Plasmodium. However, they do not show that their reporters do cause frameshifting in human cells. Is there a positive control to show these reporters can cause it? (Figure 2, Figure 2—figure supplement 2, Figure 2—figure supplement 3). If not, perhaps the language should be adjusted to not limit the effects to frameshifting.

The authors do not show or cite evidence that Ile-starvation leads to substantial ribosome stalling in their cells. They also do not cite cases where Pelo has been observed to target ribosomes that are stalled awaiting an aa-tRNA. Gcn1 and Gcn20 have been thought to directly sense stalled ribosomes under these conditions. Are those factors present here? Is eIF2α phosphorylated and could Pelo be downstream of ATF4?

The argument that overexpression of Pelo can stimulate mRNA degradation has not been previously shown (i.e. a study of mRNA stability in an overexpression strain). The DHH8 mRNA level could be increased during Ile starvation for any number of reasons and the authors have not shown ribosome stalling on this transcript is occurring. The authors should provide results from other Ile codon containing transcripts or correlate mRNA levels with Ile content globally by RNA-seq. It's not clear this is the best test for NGD.

Figure 4C: the stem loop construct, HA-pelo protein level seems to be increased – why is this occurring? Is this the natural variation between experiments? If so, how do we know the difference in 4B is significant? Were replicates done? Also, it is concerning that +polyA36 containing peptide migrates to the same exact place in the gel as -polyA36 peptide despite additional 12 positively charged amino acids. Is this just a resolution issue in this particular gel? Also, what is the band migrating at ~15 kD and why could it be changing with different constructs? Finally, there is a faint band that is just a bit heavier than the HA-mCherry product – is this the reporter with stem loop present?

The section on RNA structure and, particularly, z-scores for transcript stability was not very comprehensible. It appears the authors were asking whether poly(A) regions ("native" regions?) were more or less stable than randomly selected regions, but I could be wrong. Overall, this section did not seem to make any compelling arguments toward explaining the lack of no-go decay in this organism.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Plasmodium falciparum translational machinery condones polyadenosine repeats" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.

Revisions:

The two original reviewers recommend publication of your new version of the manuscript based on the additional conclusive evidence that you provided. The second reviewer provides comments on the text and offers excellent suggestions for improvements, vis-a-vis the explanation for the rationale for experiments. Also, some of the later figure panels might be better added to supplemental materials. The comments are noted below. We hope you find these suggestions helpful.

Reviewer #1:

The authors have mainly answered my major concerns in the first version of the manuscript, particularly regarding the potential artifacts caused by cycloheximide pretreatment.

Reviewer #2:

The revised manuscript addressed the major concerns by performing new experiments (polysome profiling, 2D gels) and by including additional controls. The text and figures are improved, and the story is now more compelling. The remaining issues are fixable and the most significant one is the writing, which still does not offer clear rationales for why some experiments were performed and tends to overinterpret results. In addition, the writing suffers from many typographic errors (some noted below) and some figures can benefit from a better presentation.

1) Figure 4: While the explanation of methodology is improved, I'm not sure what information Figures 4C and 4D add to the story. The text implies that the AT-rich genome could affect structure of mRNAs (less stable). The rationale for this analysis was not clearly stated. It is implied that if evolution has compensated for structure, there would be an effect in the z-scores. But how would this be related to assays of stalling? What is the predicted observation? Since the assay is indirect (structure is not measured) and the purpose is not clearly articulated, I suggest either placing these figures into supplementary or removing them from the manuscript.

2) Figure 6: The authors state that previous microarray analysis in Plasmodium showed that Ile starvation does not globally change mRNA levels. Therefore, the rationale to choose this experiment to study NGD in P. falciparum is unclear. Why did the authors expect changes on Ile-rich mRNAs given the prior study already showed "similar overall levels for the majority of mRNA transcripts"? Second, is it known that in mammalian cells that aa starvation induces NGD? If so, that should be clearly cited and explained. Alternatively, amino acid starvation could allow uncharged tRNAs to fill the A-site and block Pelota binding, so positive control data in mammalian cells establishing this expectation would add confidence. Third, in yeast, the mRNA stabilizing effects of loss of Dom34 is not that strong and I'm not sure anyone has observed increased mRNA degradation in a Dom34/Pelota overexpression experiment. If such data exists, it should be cited, as it would add confidence. Without offering a stronger rationale for these experiments, the conclusions from this figure are highly speculative and the authors should soften the claims and possibly move it to supplementary data.

3) It is widely accepted that NGD is part of RQC and the authors should clarify that, as previously requested, in the Introduction.

4) To make Figures 4A and 4B clearer, I recommend directly adding labels for H. sapiens and P. falciparum on top of the graphs and improving the figure legends. It's not clear that the first sentence of the legend refers to both A and B. It's not clear what "taken into account" means without context of the main text. Showing full, uncut error bar in Figure 4B and then providing an inset with the y-axis occupancy of 200 may help readers to better grasp this figure since the boxes extending off the plot is misleading. Also, the technical artifact leading to the dip should be acknowledged in the legend. It's not clear why this artifact is not also present in A and therefore whether this artifact is masking the pause in B. To make this more convincing, the authors could create a control dataset where pauses were computationally introduced and then successfully detected (i.e. a positive control). Alternatively, some explanation should be offered in the text as to why this dip does not void this analysis.

https://doi.org/10.7554/eLife.57799.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

In general, the reviewers agree that the main observation of the absence of mRNA decay of RQC substrates in Plasmodium is very interesting and that the field would greatly benefit from knowing it. However, in the present form, the ribosome profiling data are unreliable due to the pre-treatment with cycloheximide. The analysis in Figures3-4 is significantly lacking. Evidence should be obtained to determine whether ribosomes are actually stalling or not.

In our revised manuscript, we did multiple experiments to address reviewer's comments. Two major arguments of reviewer’s were, lacking of evidence for ribosomal stalling and the use of cycloheximide in the ribosome profiling dataset from Plasmodium falciparum (previously published in Caro et al., 2014). We should point out that there are articles that indicate that cycloheximide artifacts (described in Requião et al., 2016 or Santos et al., NAR, 2019) might be restricted to S. cerevisiae cells, ie: Sharma et al., bioRxiv 746255; doi: https://doi.org/10.1101/746255; Duncan and Mata, Sci. Rep., 2017; https://doi.org/10.1038/s41598-017-10650-1.

As seen from the above articles cycloheximide artifacts are not present in the ribosome profiling data from humans and other species and are somewhat challenging to infer to P. falciparum. However, we have decided to address this problem experimentally. We have now included experiments to show that P. falciparum ribosomes can be stalled without inducing targeted mRNA degradation. We did polysome profiles without pre-incubation of parasites with cycloheximide and inspected the distribution of wild type (-polyA36), polyA tract (+polyA36) and stem loop (StL) transcripts. We found that stem loop mRNA distribution correlates mostly with disomes, while polyA and will type constructs are found in higher polysome fractions. These results are now included in Figure 5. These results strengthens our data that polyA tracts are not stalling Plasmodium ribosomes. The data on stem-loop construct further indicate that the stalling of ribosomes, and reduced protein production, in Plasmodium cells does not lead to targeted mRNA degradation (as shown in our original data). This answers also general comment to our original submission, whether the Plasmodium ribosome are able to stall or not.

Reviewer #1:

[…]

1) The contribution of translational repression in this process is not properly addressed. This is an important issue in light of the emphasize that the authors placed on the main role of the mRNA sequence, rather than amino acid sequence of the nascent peptide, in stalling. Nevertheless, while the authors quite clearly demonstrate the lack of efficient mRNA decay pathway, their explanation for the absence of stalling at polyA sequences in P. falciparum seems to rely solely on the differences in the exit channel of the ribosome. This comes across as changing the goal post. Rather than explaining why the mRNA sequence does not induce stalling, the authors in fact try to address why the peptide doesn't induce stalling. On this note, it is unclear why the authors focused only on measuring the mRNA levels of a few of the candidate genes with more than 22As (Figure 4). It is quite possible that due to deficiency in the mRNA decay pathway, the target mRNAs are only translationally repressed, but it does not rule out the potential translational repression. As it stands, it is unclear whether the ribosome stalling and RQC pathway in P. falciparum is deficient, or the observed phenomenon is merely due to the deficient mRNA decay. Therefore, performing a polysome profiling assay and western blot for the candidate proteins would be suitable additions to confirm the lack of stalling on mRNAs with long polyA tracts in P. falciparum.

2) It has been previously shown that pre-treatment of the samples with translation inhibitors such as cycloheximide and anisomycin could complicate the analysis of ribosome stalling by ribosome profiling assay and result in false negative outcome, when investigating ribosome pausing/stalling on poly-basic sites (Requião, et al., 2016). From what could be deduced in the Materials and methods section, the samples seem to have been pre-treated with cycloheximide for 10 minutes before collection. Therefore, this may in fact take away from the reliability of the observation that there is no ribosome pausing at polA tracts in P. falciparum based on the ribosome profiling assay, as it may be a false negative observation due to artefacts caused by cycloheximide.

In response to reviewer #1 major comments, we have now included polysome profiles without cycloheximide pre-treatment and look for the distribution of wild type (-polyA36), polyA (+polyA36) and stem-loop (StL) constructs. We followed the distribution of all transcripts, control transcript (GAPDH) and Plasmodium ribosomes by qRT-PCR. We found that stem loop construct distribution correlates mostly with disomes, while polyA and wild type constructs are found evenly distributed in polysomes. This strengthens our data that polyA tracts are not stalling Plasmodium ribosomes. This also further indicates that the stalling of ribosomes with stem loop in Plasmodium cells does not lead to targeted degradation (our original data). We are puzzled with reviewer’s comment:

“It is quite possible that due to deficiency in the mRNA decay pathway, the target mRNAs are only translationally repressed, but it does not rule out the potential translational repression”.

If the target polyA tract mRNAs are indeed translationally repressed, we should see much less protein being produced from our reporters (shown in Figure 2 and 3, and Figure 2—figure supplement 2 and 3), as well as in ribosome profiling data shown in Figure 4. The western blot analyses of reporters show opposite, protein synthesis from polyA tract transcripts is comparable, if not enhanced, compared to synthesis from transcripts without polyA tracts. The same is through in ribosome profiling data where polyA tract constructs from human tissue cultures indicate ribosome stalling (Figure 4A, regardless of cycloheximide treatment) while P. falciparum transcripts do not (Figure 4B). This analysis is plotted for 1167 transcripts from the P. falciparum cells with polyA tracts shorter than 22 adenosine nucleotides (22A-1 sequences). While we would be happy to test the expression of some of these endogenous constructs by western blot analyses, there are only a few available antibodies for P. falciparum proteins. PfHAD1 and PfHSP70 are used in our western blots, but unfortunately, these proteins do not host polyA tracts. However, our western blot analyses are done on reporters with and without polyA tract with 36 consecutive adenosines. We have commented on cycloheximide treatment in our general response.

Reviewer #2:

[…] However, the paper seems to have been quickly written and lacks clarity. While much of the data are negative, it is worth sharing if further experiments could be performed in some cases to more clearly explain what models are being tested and directly show the results in Plasmodium are unlike those in other organisms.

We are thankful for reviewer #2 for comments on our manuscript. We have now added several additional experiments that further support our original data and clarify our major points: 1. Lack of ribosomal stalling on polyA tracts and poly-lysine repeats in P. falciparum cells; 2. Lack of targeted mRNA degradation of ribosome stalled transcripts. We have also increased the clarity of our manuscript by further changing original text.

List of major concerns:

Is it possible that during poly(A) translation, P. falciparum ribosomes miscode and incorporates amino acids other than Lys? A mass spectrometry analysis of the reporter peptide could address this possibility and show that the ribosome faithfully translates Lys codons without miscoding and frameshifting.

In response to the reviewer’s comment, we have analyzed our reporter constructs using 2D gels to show whether fidelity and quality of protein synthesis stay intact in Plasmodium cells. We show now in the new Figure 3 that regardless of the lysine codons (AAA or AAG) both polylysine containing proteins shift their position from the wild-type (-polyA36) control by +1 overall charge in isoelectric point (pI for wild-type 6.2, pI for 12xLys 7.2). The position of protein products from 12xAAA and 12xAAG is identical on those gels arguing for no significant miscoding and frameshifting on either of these constructs. If such events do occur, they do not contribute significantly to overall quality or change in protein expression of polyA tract constructs.

It is difficult to see the difference between human and P. falciparum ribosome occupancy in Figures 3A and 3B. It should be shown by distinct colors and the color code should be included in the figure legends. What do the error bars signify? Is the large central dip in 3B significant? Also, the authors should explain how many genes are going into the analysis and how they are controlling for the possibility that a few strongly-expressed genes are dominating these effects (how was normalization done?).

Both figures are symmetrical, with the center of the plot being the beginning of the polyA segment, left side being upstream of polyA segment. Both plots intentionally use the same y-axis scale to enable comparison. Barplots at each position denote first and third quantile (box ranges), the median is denoted with the horizontal line inside a box. Vertical lines denote 1.5*IQR range from the box (standard box plot representation). There was no normalization done (other than applied by GWIPS pipeline), only removing of entries without occupancy or with low average occupancy, to improve signal to noise ratio (as described in the Materials and methods). For a human this resulted in the plot based on 32 polyA fragments (32 transcripts), out of 102, that had occupancy at the start if the polyA segment more significant than 0. This is due to the fact that polyA tract genes are generally not expressed well in most of the organisms and, as such, have reduced amount of reads over polyA segments in ribosome profiling data sets. P. falciparum analyses is done on 1167 genes. That is why outer whiskers (1.5*IQR lines) extend beyond the plot because there are many more data points with good occupancy. Plasmodium plot has a central dip due to the inability of bioinformatics software to correctly map very long polyA fragments (as shown in Figure 4—figure supplement 1). A similar procedure of removing poorly mapped fragments was applied here as well. There was no statistical assessment done in these plots – they simply show the difference between occupancy distributions in these two species.

The authors refer to previous studies where frameshifting was attributed to translation of poly(A) sequences. They do not see frameshifting when poly(A) reporters were examined here in Plasmodium. However, they do not show that their reporters do cause frameshifting in human cells. Is there a positive control to show these reporters can cause it? (Figure 2, Figure 2—figure supplement 2, Figure 2—figure supplement 3). If not, perhaps the language should be adjusted to not limit the effects to frameshifting.

Ribosomal frameshifting on polyA tracts was previously shown in E. coli, yeast, Drosophila and human cells with the identical or similar constructs, as well as endogenous human genes (ZCRB1). These experiments are described in Arthur et al., 2015; Koutmou et al., 2015; as well as Sundaramoorthy et al., 2017. We should also point out that ribosomal frameshifting was seen on sequences shorter than 36 consecutive adenosine constructs starting with 3 or 4 consecutive lysine AAA codons (9 or 12A polyA tracts).

The authors do not show or cite evidence that Ile-starvation leads to substantial ribosome stalling in their cells. They also do not cite cases where Pelo has been observed to target ribosomes that are stalled awaiting an aa-tRNA. Gcn1 and Gcn20 have been thought to directly sense stalled ribosomes under these conditions. Are those factors present here? Is eIF2α phosphorylated and could Pelo be downstream of ATF4?

Dan Goldberg’s lab originally reported Ile-induced starvation in P. falciparum (cited as Babbitt et al., 2012). This manuscript argues that Ile-starvation of P. falciparum cells is able to induce a state of hibernation through the arrest of protein synthesis with phosphorylation of eIF2α. This phosphorylation of eIF2α in Plasmodium is happening in a GCN2-independent fashion, and without existing TOR-nutrient sensing pathway nor activation of ATF4 homolog. The same manuscript gives micro-array analyses of transcript levels that also indicate no significant change in the mRNA levels of the majority of P. falciparum genes during the 48 hours starvation period (our focused qRT-PCR analysis in Figure 5C).

The argument that overexpression of Pelo can stimulate mRNA degradation has not been previously shown (i.e. a study of mRNA stability in an overexpression strain). The DHH8 mRNA level could be increased during Ile starvation for any number of reasons and the authors have not shown ribosome stalling on this transcript is occurring. The authors should provide results from other Ile codon containing transcripts or correlate mRNA levels with Ile content globally by RNA-seq. It's not clear this is the best test for NGD.

We have now included qRT-PCR analyses for additional eight P. falciparum genes with multiple runs of Ile-residues (3-7 consecutive Ile). As noted from the new Figure 5C, the general trend is rather no-change or stabilization of Ile-rich transcripts during the Ile-starvation experiment. We have also expanded our analyses of stem loop constructs and included new Figure 4C, which indicates the stalling of ribosomes on the stem loop transcript in P. falciparum cells.

Figure 4C: the stem loop construct, HA-pelo protein level seems to be increased – why is this occurring? Is this the natural variation between experiments? If so, how do we know the difference in 4B is significant? Were replicates done? Also, it is concerning that +polyA36 containing peptide migrates to the same exact place in the gel as -polyA36 peptide despite additional 12 positively charged amino acids. Is this just a resolution issue in this particular gel? Also, what is the band migrating at ~15 kD and why could it be changing with different constructs? Finally, there is a faint band that is just a bit heavier than the HA-mCherry product – is this the reporter with stem loop present?

We have not noticed an increase in the Pelo transcript or protein in parasites transfected with stem-loopp construct. HA-pelo was supposed to be normalization control in Figure 4C (now Figure 5A) and difference for Pelo protein is due to the differences in loading amounts of samples. The difference in HA-pelo amounts and transcript levels in Figure 6A and 6B (or previous Figure 4A and 4B) is a consequence of 48-hour Ile-starvation of parasites. As noted in figure legends, these experiments were done in 3 biological replicates. We think that no obvious change between -polyA36 and +polyA36 in this gel is due to the resolution issue. We have now also included 2D gels (Figure 3) of -polyA36, +polyA36 constructs and 12xAAG Lys construct to indicate a shift in isoelectric point of polylysine protein. 15kD band appearing in gels and causing cross-reactivity in western blots is contaminant of human hemoglobin. The Plasmodium parasite cultures are grown in human erythrocytes (98% of total protein is hemoglobin) and while we tried to lyse erythrocytes and get rid of most of hemoglobin prior to parasite lysis the amount of hemoglobin present in lysed samples is very hard to control. This is why we used PfHAD1 and endogenously tagged HA-pelo as normalization controls for western blot analyses. Finally, we would like to think that the faint band in Figure 5A is indeed HA-mCherry protein from stem-loop construct (StL). We just did not want to speculate whether this band indeed represents protein product from StL construct. We have also tried HA-immunoprecipitations with lysates expressing this particular construct (StL) without clear enrichment of protein corresponding to this band.

The section on RNA structure and, particularly, z-scores for transcript stability was not very comprehensible. It appears the authors were asking whether poly(A) regions ("native" regions?) were more or less stable than randomly selected regions, but I could be wrong. Overall, this section did not seem to make any compelling arguments toward explaining the lack of no-go decay in this organism.

Since mRNA translation could be affected by the ability of RNA to fold into unique functional structures, we wanted to test whether mRNA transcripts between three tested species (human, Tetrahymena, and Plasmodium) have a different propensity in RNA folding. This is because RNA secondary structures, especially A-rich sequences, have been shown to coordinate ribosomal frameshifting, impact RNA stability, or slow ribosomal progression to allow protein folding.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Reviewer #2:

The revised manuscript addressed the major concerns by performing new experiments (polysome profiling, 2D gels) and by including additional controls. The text and figures are improved, and the story is now more compelling. The remaining issues are fixable and the most significant one is the writing, which still does not offer clear rationales for why some experiments were performed and tends to overinterpret results. In addition, the writing suffers from many typographic errors (some noted below) and some figures can benefit from a better presentation.

We are thankful reviewer #2 for their comments. In the revised manuscript, we have followed majority of the suggestions on improving the text as well as presentation of the data.

1) Figure 4: While the explanation of methodology is improved, I'm not sure what information Figures 4C and 4D add to the story. The text implies that the AT-rich genome could affect structure of mRNAs (less stable). The rationale for this analysis was not clearly stated. It is implied that if evolution has compensated for structure, there would be an effect in the z-scores. But how would this be related to assays of stalling? What is the predicted observation? Since the assay is indirect (structure is not measured) and the purpose is not clearly articulated, I suggest either placing these figures into supplementary or removing them from the manuscript.

4) To make Figures 4A and 4B clearer, I recommend directly adding labels for H. sapiens and P. falciparum on top of the graphs and improving the figure legends. It's not clear that the first sentence of the legend refers to both A and B. It's not clear what "taken into account" means without context of the main text. Showing full, uncut error bar in Figure 4B and then providing an inset with the y-axis occupancy of 200 may help readers to better grasp this figure since the boxes extending off the plot is misleading. Also, the technical artifact leading to the dip should be acknowledged in the legend. It's not clear why this artifact is not also present in A and therefore whether this artifact is masking the pause in B. To make this more convincing, the authors could create a control dataset where pauses were computationally introduced and then successfully detected (i.e. a positive control). Alternatively, some explanation should be offered in the text as to why this dip does not void this analysis.

In the revised manuscript, we have moved Figure 4C and 4D to supplementary material as a new Figure 4—figure supplement 5. In order to improve presentation of the data we have included new Figures 4A and 4B as well as further explained data presented in Figure 4. and new Figure 4—figure supplement 5. Based on the reviewer suggestion we have also included explanation for the technical artifact in the figure legend.

2) Figure 6: The authors state that previous microarray analysis in Plasmodium showed that Ile starvation does not globally change mRNA levels. Therefore, the rationale to choose this experiment to study NGD in P. falciparum is unclear. Why did the authors expect changes on Ile-rich mRNAs given the prior study already showed "similar overall levels for the majority of mRNA transcripts"? Second, is it known that in mammalian cells that aa starvation induces NGD? If so, that should be clearly cited and explained. Alternatively, amino acid starvation could allow uncharged tRNAs to fill the A-site and block Pelota binding, so positive control data in mammalian cells establishing this expectation would add confidence. Third, in yeast, the mRNA stabilizing effects of loss of Dom34 is not that strong and I'm not sure anyone has observed increased mRNA degradation in a Dom34/Pelota overexpression experiment. If such data exists, it should be cited, as it would add confidence. Without offering a stronger rationale for these experiments, the conclusions from this figure are highly speculative and the authors should soften the claims and possibly move it to supplementary data.

The rationale for the Ile-starvation experiment was taken from the previous studies with histidine-induced stalling (using 3-AT, Guydosh and Green, Cell 2014), protein synthesis and subsequent translational arrest during erythroid and platelet maturation (Mills et al., 2016 and Mills et al., 2017), Leu- and Arg-starvation experiments (Darnell et al., Mol Cell 2018) as well as cycloheximide-induced stalling (Simms et al., 2017). We wanted to check whether Plasmodium cells react differently during global translational arrest looking more specifically to Ile-rich transcripts as well as levels of NGD factor Pelota and HSP70 chaperone. Based on the reviewer comments and results from Darnell et al., 2018, we have softened our claims and included other explanations for Ile-starvation experiment. We have previously cited Mills et al., 2017, for Pelota overexpression experiments in human cells. This study clearly states that transgenic overexpression of the Pelota in human platelets results in more rapid degradation of most cytoplasmic platelet transcripts (average half-life, 3.7 vs 5.7 hours in control).

3) It is widely accepted that NGD is part of RQC and the authors should clarify that, as previously requested, in the Introduction.

We have now included clarification of RQC and NGD in the Introduction.

https://doi.org/10.7554/eLife.57799.sa2

Article and author information

Author details

  1. Slavica Pavlovic Djuranovic

    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Jessey Erath
    For correspondence
    spavlov@wustl.edu
    Competing interests
    No competing interests declared
  2. Jessey Erath

    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Slavica Pavlovic Djuranovic
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8802-635X
  3. Ryan J Andrews

    Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, United States
    Contribution
    Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0275-0019
  4. Peter O Bayguinov

    Washington University Center for Cellular Imaging, Washington University School of Medicine, St. Louis, United States
    Contribution
    Formal analysis, Investigation, Visualization, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Joyce J Chung

    Department of Biology, Washington University, St Louis, United States
    Contribution
    Formal analysis, Investigation, Writing - original draft
    Competing interests
    No competing interests declared
  6. Douglas L Chalker

    Department of Biology, Washington University, St Louis, United States
    Contribution
    Formal analysis, Validation, Investigation, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0285-3344
  7. James AJ Fitzpatrick

    1. Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, United States
    2. Washington University Center for Cellular Imaging, Washington University School of Medicine, St. Louis, United States
    3. Department of Neuroscience, Washington University School of Medicine, St. Louis, United States
    4. Department of Biomedical Engineering, Washington University, St Louis, United States
    Contribution
    Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Writing - review and editing
    Competing interests
    No competing interests declared
  8. Walter N Moss

    Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, United States
    Contribution
    Data curation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6419-5570
  9. Pawel Szczesny

    Institute of Biochemistry and Biophysics Polish Academy of Sciences, Department of Bioinformatics, Warsaw, Poland
    Contribution
    Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    For correspondence
    szczesny.pawel@gmail.com
    Competing interests
    No competing interests declared
  10. Sergej Djuranovic

    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    sergej.djuranovic@wustl.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9417-0822

Funding

National Institute of General Medical Sciences (R01 GM112824)

  • Sergej Djuranovic

National Science Foundation (MCB 1412336)

  • Douglas L Chalker

National Institute of General Medical Sciences (R00 GM112877)

  • Walter N Moss

National Institute of General Medical Sciences (T32 GM007067)

  • Jessey Erath

St. Louis Children’s Hospital (CDI-CORE-2015-505)

  • Peter O Bayguinov
  • James AJ Fitzpatrick

St. Louis Children’s Hospital (CDI-CORE-2019-813)

  • Peter O Bayguinov
  • James AJ Fitzpatrick

Washington University School of Medicine in St. Louis

  • Peter O Bayguinov
  • James AJ Fitzpatrick

Foundation for Barnes-Jewish Hospital (3770)

  • Peter O Bayguinov
  • James AJ Fitzpatrick

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Hani Zaher as well as members of Daniel Goldberg’s and Sergej Djuranovic’s lab for helpful comments. This work is supported by NIH R01 GM112824 to SD, NIH R00 R00GM112877 to WM, NSF MCB 1412336 to DLC, and NIH T32 GM: 007067 to JE. POB and JAJF are supported by the Washington University Center for Cellular Imaging (WUCCI), which is funded in part by Washington University School of Medicine, The Children’s Discovery Institute of Washington University, and St. Louis Children’s Hospital (CDI-CORE-2015–505 and CDI-CORE-2019–813) and the Foundation for Barnes-Jewish Hospital (3770). The authors declare that they have no competing interests.

Senior Editor

  1. James L Manley, Columbia University, United States

Reviewing Editor

  1. Nahum Sonenberg, McGill University, Canada

Publication history

  1. Received: April 16, 2020
  2. Accepted: May 28, 2020
  3. Accepted Manuscript published: May 29, 2020 (version 1)
  4. Version of Record published: June 15, 2020 (version 2)

Copyright

© 2020, Pavlovic Djuranovic et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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