Figures and data
RNA Pol III-transcribed genes are downregulated in asymptomatic individuals during the dry season.
Steady state RNA levels as quantified by RT-qPCR using primers to tRNA Asparagine (Pf3D7_0714700) (A) and tRNA Valine (Pf3D7_0312600) (B) as well as RNA Pol III-transcribed RUF6 ncRNA (C). DBLalpha primers were used to detect RNA Pol II-transcribed var genes (D). Normalized expression is shown using fructose-bisphosphate aldolase (FBA Pf3D7_1444800) as the reference gene in symptomatic individuals during the wet season (n = 14+) and asymptomatic individuals during the dry season (n = 17+). + with the exception of Asparagine tRNA wet symptomatic (n=11) and dry asymptomatic (n=12), and DBLalpha wet symptomatic (n = 12) and dry asymptomatic (n = 16). Box plots indicate the mean with standard deviation. Wilcoxon matched-pairs signed rank test was done to determine significance (* indicates P < 0.05 and ** indicates P < 0.005).
External factors modulate RNA Pol III-transcribed genes.
(A) Plasma magnesium levels are significantly increased in asymptomatic individuals during the dry season compared to symptomatic individuals during the wet season. Concentration is shown in nmol/μL (mM) (B) Schematic showing underlying molecular mechanism summary using in vitro cultured P. falciparum. (C, D) Volcano plot showing log2 (fold change, FC) against −log10 (p-value) of transcripts identified by RNA-seq analysis of 3D7 control and addition of MgCl2 harvested during ring (C) and trophozoite stages (D). Expressed transcripts from three replicates between control and addition of MgCl2 that are significantly up-regulated are highlighted in red while significantly down- regulated RNA Pol III genes are highlighted in blue (FDR corrected p-value of <0.05) and a FCL≥±L1.95) with examples listed as text. Black dots indicate non-significant transcripts with a FCL≤L2.0.
Nuclear PfMaf1 is essential to regulate RNA Pol III.
(A) Illustration of the TORC1-dependent cellular localization of Maf1 protein in unfavorable conditions in typical eukaryotic organisms compared P. falciparum that has no TORC1 pathway. (B, top) Illustration of recombinant PfMaf1 with a 3HA tag followed by a ddFKBP domain to allow for knockdown studies. (B, bottom) Western blot analysis for PfMaf1 in total extracts in pSLI-Maf1-FKBP transfected parasites at 24hpi after 1, 2, and 3 cycles without addition of Shield-1 (-) and control, with addition of Shield-1 (+). Aldolase levels are also shown. Representative of 3 replicates. (C) Transcript levels as quantified by RT-qPCR using the same primers in (Figure 2B) in parasites harvested at 18hpi in control group and without Shield for 2 cycles. Error bars are displayed from 3 biological replicates. Statistical significance was determined by two-tailed Student’s t-test (** p<0.005). (D) Growth curve over 5 days of clonal pSLI-Maf1-FKBP parasites for 2 conditions: in the presence or absence of Shield-1. Uninfected red blood cells (“blood” in red) serve as reference of background. Error bars indicate standard deviation of three technical replicates in different blood from two different clones (n = 6). (E) Data is represented as box-whisker plot of mean merozoite number per schizont ±SD (Mann-Whitney), with the with the median represented at the center line. Boxplots show the data of 100 segmented schizonts counted per condition (n = 100). Statistical significance was determined by two-tailed Student’s t-test (*** p<0.001). Representative giemsa images are shown to the right for + and – Shield. (F) Visual representation of Co-IP MS analysis of cytoplasmic and nuclear PfMaf1. Labeled proteins represent important significant and unique proteins in cytoplasmic and nuclear fractions not found in either of the controls.
External factors modulate virulence through PfMaf1-regulated RNA Pol III inhibition
(A) Immunoprecipitation western blot analysis for cytoplasmic and nuclear extracts for PfMaf1 expression in PfMaf1-FKBP transfected parasites with addition of MgCl2 ([3mM] total) and control parasites harvested at 18hpi. Controls are shown in Figure S4B. (B) Transcript levels as quantified by RT-qPCR using primers to tRNA Valine (Pf3D7_0312600), RUF6 ncRNA, var DBLalpha, and normalized to FBA (Pf3D7_1444800) for 4 conditions: control (black), addition of MgCl2 (green), KD of PfMaf1 (blue), and KD of PfMaf1 and addition of MgCl2 (orange). Error bars are displayed from 3 biological replicates. Statistical significance was determined by two-tailed Student’s t-test (** p<0.005). (C) Cytoadhesion binding assay data is represented as box-whisker plot of mean number of bound iRBC ±SD (Mann-Whitney) mm2, with the median represented at the center line. Boxplots show the data of 3 biological replicates (n = 3). Statistical significance was determined by two-tailed Student’s t-test (**** p<0.001). Representative images are shown below for 3D7 and 3D7 +MgCl2. (D) Western blot analysis for extracts for ATS-PfEMP1 expression in 3D7 control parasites and with addition of MgCl2 ([3mM] total) harvested after plasmion. NapL control levels are also shown. Representative of 3 replicates. (E) Schematic showing summary of study linking decreased cytoadherence, associated with disease severity, with increased RNA Pol III-inhibition, triggered in response to external factors.
(A) Multiple sequence alignment of highly conserved genes encoding GC-rich ncRNA elements. 15 members of RUF6 gene family aligned by Clustal Omega (http://www.ebi.ac.uk) and presented in Jalview (http://www.jalview.org). Degree of conservation per base and consensus sequence are displayed below. Black lines show position of potential A- and B-box consensus motifs (as assessed in (Guizetti et al, 2016)). (B) RT- qPCR shows transcript levels for tRNA Asparagine, tRNA Valine, RUF6 ncRNA and a housekeeping gene encoding ubiquitin-conjugating enzyme in synchronized wildtype parasites at 24 hpi that were untreated or treated with RNA Pol III inhibitor or DMSO. Transcript levels are normalized to fructose-bisphosphate aldolase (PF3D7_1444800) transcript levels. MeanLSEM of two independent experiments are shown. Statistical significance was determined by two-tailed Student’s t-test (* p<0.05).
(A) tRNA Leucine (Pf3D7_API05400) and tRNA Asparagine (Pf3D7_API05500) transcripts identified by RNA-Seq analysis from ((Andrade et al, 2020)) for wet symptomatic (n = 12) and dry asymptomatic (n = 12) individuals. Statistical significance was determined by two-tailed Student’s t-test (* p<0.05). (B) Transcript levels as quantified by RT-qPCR on 3D7 parasites harvested during late ring stage for control parasites, parasites in the absence of isoleucine (w/o Ile), at 40 degrees Celsius, at low-glucose levels (0.5mg/mL), and presence of additional MgCl2 ([3mM] total). Primers were used for Pol II-transcribed ubiquitin- conjugating enzyme (UCE Pf3D7_0812600), Pol I-transcribed rRNA A1, Pol III-transcribed RUF6 ncRNA, Pol III-transcribed tRNA Valine (Pf3D7_0312600), and Pol II-transcribed active var gene (Pf3D7_1240900). Results are normalized to an RNA Pol II-transcribed reference gene FBA (Pf3D7_1444800) and presented as fold change/control. Error bars are displayed from 3 biological replicates. Statistical significance was determined by two-tailed Student’s t-test (* p<0.05).
(A) Transcript levels as quantified by RT-qPCR on 3D7 parasites harvested during late ring stage for control parasites and parasites in the presence of additional MgCl2 levels ([1mM], [1.5mM], [2mM], and [3mM] total). Primers were used for Pol II-transcribed ubiquitin-conjugating enzyme (UCE Pf3D7_0812600), Pol III- transcribed RUF6 ncRNA, Pol III-transcribed tRNA Valine (Pf3D7_0312600) and Alanine (PF3D7_0411500), and Pol II-transcribed var DBLalpha. Results are normalized to an RNA Pol II-transcribed reference gene FBA (Pf3D7_1444800) and presented as fold change/control. Error bars are displayed from 3 biological replicates. (B) Cell cycle progression estimation of a wildtype 3D7 clone in the absence (‘C’) or presence (‘Mg’) of MgCl2 supplementation. RNA-seq data from synchronized parasites harvested at 12 and 24 hpi were compared to microarray data (from (Bozdech et al, 2003) as in (Lemieux et al, 2009). Replicates are represented with circles.
PfMaf1 cytoplasmic and nuclear interactome.
Co-IP MS volcano plot of enrichment for all 5 replicates for cytoplasmic (A) and nuclear (B) PfMaf1 vs control proteins are indicated and labeled. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. x-axis = log2(fold-change), y-axis = −log10(p-value), horizontal red line indicates adjusted p- value = 0.05, and vertical green lines indicate absolute fold-change = 2.0. Side panels indicate proteins uniquely identified in either sample (y-axis = number of peptides per 100 amino acids) with a minimum of 2 distinct peptides in 3 replicates of a same state. (C) Table showing values for significantly and uniquely enriched proteins from both extracts as labeled in (Figure 3F). (D) Volcano plot showing the distribution of significant and unique proteins in cytoplasmic and nuclear fractions not found in either of the controls. Each dot represents a protein, and its size corresponds to the sum of peptides from both conditions used to quantify the ratio of enrichment. x-axis = log2(fold-change), y-axis = −log10(p-value), horizontal red line indicates adjusted p- value = 0.05, and vertical green lines indicate absolute fold-change = 2.0. Side panels indicate proteins uniquely identified in either sample (y-axis = number of peptides per 100 amino acids) with a minimum of 2 distinct peptides in 3 replicates of a same state.
(A) Immunoprecipitation western blot analysis for cytoplasmic and nuclear extracts for PfMaf1 expression in PfMaf1-FKBP transfected parasites with addition of MgCl2 ([3mM] total) and control parasites harvested at 18hpi from Figure 4A. Anti-HA PfMaf1, aldolase and histone H3 are shown from input and flow- through (FT). (B) Representative immunofluorescence images show brightfield, Dapi, anti-HA PfMaf1, and Dapi-HA merge for PfMaf1 in control and addition of MgCl2 ([3mM] total) in parasites harvested and fixed at late ring stage.
