Genome-wide regulatory dynamics of translation in the Plasmodium falciparum asexual blood stages
- University of California, San Francisco, United States
- Howard Hughes Medical Institute, University of California, San Francisco, United States
Figures
Figure 1 with 3 supplements
Ribosome profiling of the P. falciparum asexual blood stages, experimental outline.
(A) Synchronized parasite cultures were maintained in hyperflasks at 5% hematocrit and maximum 15% parasitemia. Cycloheximide-treated cultures containing ∼1010 parasites were harvested at ring, …
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Figure 1—source data 1
Illumina sequencing mapping statistics against P. facliparum W2 SNP-corrected genome.
- https://doi.org/10.7554/eLife.04106.004
Figure 1—figure supplement 1
Polysome profiles of the P. falciparum asexual blood stages.
Sucrose gradient A260 absorbance profiles of polysome extracts treated with micrococcal nuclease (green, +MNase) and untreated controls (gray, No treatment). Red dotted line indicates monosome peak …
Figure 1—figure supplement 2
Read size influence on mappability.
Single nucleotide sliding windows ranging from 10 to 50 nt were used to generate in silico libraries of the P. falciparum W2 SNP corrected genome. These were uniquely aligned, allowing no …
Figure 1—figure supplement 3
Reproducibility and coverage threshold determination using two fully independent biological replicates.
mRNA abundance measurements (A) and ribosome footprint densities (B) in terms of rpkM in two fully independent biological replicates of the late trophozoite timepoint. Genes with at least ≥32 total …
Figure 2
Ribosome profiling through the P. falciparum IDC.
(A) Reproducibility among biological replicates. Two fully independent biological replicas of each stage were sampled for RNA-seq (left panels, blue) and ribosome profiling (right panels, green). …
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Figure 2—source data 1
P. falciparum ribosome profiling data set.
- https://doi.org/10.7554/eLife.04106.009
Figure 3 with 1 supplement
Transcription and translation are highly correlated.
(A) Ribosome footprint (green) and mRNA (blue) coverage profiles of two neighbor genes, the eIF2 gamma subunit (PF14_0104) and the conserved protein PF14_0105 (CDS, white boxes; HMM-defined UTRs, …
Figure 3—figure supplement 1
mRNA abundance and ribosome footprint density are highly correlated for the majority of genes expressed during the IDC.
Pearson correlation of mRNA abundance and ribosome footprint density of every gene expressed in at least three stages (2412 genes).
Figure 4
Genome-wide measurements of translation.
(A) Translational efficiency distributions in each stage. Rings and merozoites have most extreme TE values; ± 2 SD above (yellow bars) and below (blue bars) the mean. TE values of translationally …
Figure 5 with 2 supplements
Translationally down-regulated genes have decreased CDS ribosome density.
(A) Ribosome footprint (green) and mRNA (blue) profiles of the PfRh3 pseudogene (PFL2520w) in merozoites (M). In the detail the bars above the gene model indicate AUG, stop, and any other codon, in …
Figure 5—figure supplement 1
Translation of a truncated form of PfRh3 during the IDC.
Ribosome footprint (green) and mRNA (blue) profiles of the PfRh3 pseudogene (PFL2520w) in rings (R), early trophozoites (ET), late trophozoites (LT), schizonts (S), and merozoites (M). (A) …
Figure 5—figure supplement 2
Translationally down-regulated genes have decreased CDS ribosome density.
(A) Ribosome footprint (green) and mRNA (blue) profiles of the ring-infected erythrocyte surface antigen 2, RESA2 pseudogene (PF11_0512) in rings (R) and merozoites (M). Both, the annotated isoform …
Figure 6 with 1 supplement
Example of extended transcript annotations using the HMM.
5′ leaders and 3′ UTRs of the gene pair Myb2 (PF10_0327) and bromodomain protein (PF10_0328) were defined using the HMM designed (see ‘Materials and methods’). The sizes of 5′ leaders and 3′ UTRs of …
Figure 6—figure supplement 1
HMM-defined 5′ leader and 3′ UTR characteristics.
5′ leader (A) and 3′ UTR (B) length distribution and their statistics (C) per stage.
Figure 7 with 6 supplements
Transcripts accumulate ribosome density within the 5′ leader.
(A) Proportion of mRNA or ribosome footprint reads mapping to CDS, to HMM-defined 5′ leaders and 3′ UTRs, antisense to annotated coding genes or to other regions of the genome such as mitochondria, …
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Figure 7—source data 1
Predicted uORFs.
- https://doi.org/10.7554/eLife.04106.019
Figure 7—figure supplement 1
5′ leader footprints are derived from ribosomes.
Ribosome footprint read length distributions for reads mapping either to CDSs, 5′ leaders, 3′ UTRs, antisense, rRNAs or tRNAs are plotted. Read lengths of rRNA and tRNA mapping footprints are …
Figure 7—figure supplement 2
Distribution of uORF coverage on 5′ leaders of genes expressed during the IDC.
The proportion of ribosome footprints mapping inside predicted uORFs was calculated for each gene expressed in each stage. The median of each of these distributions is ∼0.5.
Figure 7—figure supplement 3
uORFs present on 5′ leaders have no effect on TE.
Translational efficiency (log2TE) for all genes expressed in each stage is plotted against the proportion of reads mapping within uORFs, the number of predicted uORFs, or the length of predicted …
Figure 7—figure supplement 4
Detection of ribosome density on uORFs.
(A) Ring stage mRNA (blue) and ribosome footprint (green) profiles of VAR2CSA (PFL0030c) are shown. There is virtually no ribosome density on transcript CDS (log2TERings = −4.2). Ribosomes do …
Figure 7—figure supplement 5
uORFs present on 5′ leaders have no effect on TE.
Ribosome density on the 5′ leader (log25′RD) for all genes expressed in each stage is plotted against the proportion of reads mapping within uORFs, the number of predicted uORFs, or the length of …
Figure 7—figure supplement 6
5′ ribosome density can be found on 5′ leaders devoid of AUGs.
Ring stage mRNA (blue) and ribosome footprint (green) profiles of (A) aquaglyceroporin (PF11_0338) and (B) PFC0486c (unknown function) are shown. Both genes display high ribosome density on their 5’ …
Figure 8
5′ ribosome density is commonly found on genes expressed during the IDC.
(A) 5′ RD distributions in each stage. Transcripts in rings and merozoites have on average higher 5′ RD values; ± 2 SD values lie outside gray shade. μ = mean log25′RD, n = total number of genes. (B)…
Figure 9 with 1 supplement
3′ UTR ribosome density.
(A) Late trophozoite stage mRNA (blue) and ribosome footprint (green) profiles of the conserved plasmodium protein, PF13_0160. Ribosomes can be detected up to ∼130 nt beyond the stop codon on the 3′ …
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Figure 9—source data 1
Genes with 3′ UTR ribosome occupancy.
- https://doi.org/10.7554/eLife.04106.028
Figure 9—figure supplement 1
3′ UTR ribosome footprint size distribution.
Cummulative read length distributions of all reads mapping to the 3′ UTR of the 19 genes with 3′ ribosome density identified compared to the read length distributions of reads mapping to all CDSs in …
Figure 10 with 1 supplement
Strand-specific libraries can distinguish antisense from sense gene transcription.
(A) Schizont stage mRNA (blue) and ribosome footprint (green) profiles of the nucleoside transporter pfNT4 (PFA0160c). The antisense transcript covers the full extent of the sense transcript and …
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Figure 10—source data 1
Antisense transcripts.
- https://doi.org/10.7554/eLife.04106.031
Figure 10—figure supplement 1
Strand-specific RT-PCR detection of the antisense to Pfs16.
Read coverage on the plus and minus strand of the stage-specific protein precursor Pfs16 (PFD0310w) locus. The gene is encoded on the plus strand and the antisense transcript covers and extends …
Author response image 1
Author response image 2
Additional files
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Supplementary file 1
MochiView genome browser data tracks.
- https://doi.org/10.7554/eLife.04106.033
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Supplementary file 2
RESA2 mapping reads.
- https://doi.org/10.7554/eLife.04106.034
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Supplementary file 3
Strand-specific RT-PCR primers.
- https://doi.org/10.7554/eLife.04106.035
