Vembar Shruthi Sridhar, Macpherson Cameron Ross, Sismeiro Odile, Coppée Jean-Yves, Scherf Artur
Unité Biologie des Interactions Hôte-Parasite, Département de Parasites et Insectes Vecteurs, Institut Pasteur, Paris, 75015, France.
CNRS, ERL 9195, Paris, 75015, France.
Genome Biol. 2015 Sep 28;16:212. doi: 10.1186/s13059-015-0771-5.
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. However, the molecular mechanisms that govern post-transcriptional regulation are currently unknown.
We 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.
We 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.
全转录组核糖体占有率研究表明，在恶性疟原虫红细胞内生命周期中，某些mRNA在转录后受到调控。其中一部分编码入侵宿主红细胞所需的寄生虫毒力因子，目前正被开发为候选疫苗。然而，目前尚不清楚转录后调控的分子机制。
我们研究了先前鉴定的DNA/RNA结合蛋白PfAlba1，它定位于恶性疟原虫滋养体细胞质中的多个位点。我们确定PfAlba1对无性增殖至关重要，随后研究过表达表位标签PfAlba1的寄生虫，以确定其RNA靶标以及对mRNA稳态和翻译调控的影响。通过对亲和纯化的PfAlba1相关RNA进行深度测序，我们鉴定出1193个在滋养体中直接与PfAlba1结合的转录本。对于其中105个这样的转录本，43%为未表征的，13%编码红细胞入侵成分，其稳态水平在这一阶段发生显著变化，证明PfAlba1在维持mRNA稳态中发挥作用。此外，我们发现PfAlba1与四种红细胞入侵mRNA（Rap1、RhopH3、CDPK1和AMA1）的结合与滋养体中的翻译抑制有关，而在成熟阶段这些mRNA从PfAlba1复合物中的释放与蛋白质合成相关。
我们表明PfAlba1与无性阶段mRNA的一个亚群结合，并微调翻译时机。这种转录后调控模式对于恶性疟原虫红细胞入侵成分可能尤为重要，这些成分必须在寄生虫无性生命周期接近尾声时以高度时间依赖的方式组装到顶端分泌细胞器中。
