Abstract
BackgroundTranscriptome-wide ribosome occupancy studies have suggested that during the intra-erythrocytic lifecycle of Plasmodium falciparum, select mRNAs are post-transcriptionally regulated. A subset of these encodes parasite virulence factors required for invading host erythrocytes, and are currently being developed as vaccine candidates. However, the molecular mechanisms that govern post-transcriptional regulation are currently unknown.ResultsWe explore the previously identified DNA/RNA-binding protein PfAlba1, which localizes to multiple foci in the cytoplasm of P. falciparum trophozoites. We establish that PfAlba1 is essential for asexual proliferation, and subsequently investigate parasites overexpressing epitope-tagged PfAlba1 to identify its RNA targets and effects on mRNA homeostasis and translational regulation. Using deep sequencing of affinity-purified PfAlba1-associated RNAs, we identify 1193 transcripts that directly bind to PfAlba1 in trophozoites. For 105 such transcripts, 43 % of which are uncharacterized and 13 % of which encode erythrocyte invasion components, the steady state levels significantly change at this stage, evidencing a role for PfAlba1 in maintaining mRNA homeostasis. Additionally, we discover that binding of PfAlba1 to four erythrocyte invasion mRNAs, Rap1, RhopH3, CDPK1, and AMA1, is linked to translation repression in trophozoites whereas release of these mRNAs from a PfAlba1 complex in mature stages correlates with protein synthesis.ConclusionsWe show that PfAlba1 binds to a sub-population of asexual stage mRNAs and fine-tunes the timing of translation. This mode of post-transcriptional regulation may be especially important for P. falciparum erythrocyte invasion components that have to be assembled into apical secretory organelles in a highly time-dependent manner towards the end of the parasite’s asexual lifecycle.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0771-5) contains supplementary material, which is available to authorized users.
Highlights
Transcriptome-wide ribosome occupancy studies have suggested that during the intra-erythrocytic lifecycle of Plasmodium falciparum, select mRNAs are post-transcriptionally regulated
When ALBA1-Escherichia coli DHFR destabilization domain (eDHFR)-HA transgenic parasites were grown in the absence of TMP, we did not observe a significant reduction in the levels of PfAlba1eDHFR-HA, even after 10 days (Figure S1c in Additional file 1, day 7 and day 10 panels), indicating that the tagged protein is refractory to the stabilizing drug
Excess levels of PfAlba1 inhibit intra-erythrocytic growth of P. falciparum Before functionally characterizing PfAlba1-Ty1, we explored if we could modulate the expression levels of the tagged protein during the P. falciparum intra-erythrocytic developmental cycle (IDC), and if so, Fig. 1 C-terminally tagged PfAlba1-Ty1 was successfully expressed from an episome in P. falciparum asexual blood stages. a The transfection and maintenance of the pPfAlba1-Ty1C plasmid was confirmed by PCR of genomic DNA
Summary
Transcriptome-wide ribosome occupancy studies have suggested that during the intra-erythrocytic lifecycle of Plasmodium falciparum, select mRNAs are post-transcriptionally regulated. A subset of these encodes parasite virulence factors required for invading host erythrocytes, and are currently being developed as vaccine candidates. Recent high throughput sequencing studies have shown that >80 % of the parasite genome is pervasively transcribed in a monocistronic manner during the IDC [11, 12], hinting at robust post-transcriptional regulation to fine-tune stage-specific IDC expression. Such a mode of regulation is further supported by an enrichment of genes predicted to encode RNA-binding proteins (RBPs) in the P. falciparum genome [13]. We recently found that in ring stages the chromatin-associated exoribonuclease PfRNaseII controls the post-transcriptional silencing of nascent RNA synthesized from a subset of genes, including virulence genes encoding surface adhesion molecules linked to severe malaria [14]
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