TRIBE uncovers the role of Dis3 in shaping the dynamic transcriptome in malaria parasites [RIP-seq]. TRIBE uncovers the role of Dis3 in shaping the dynamic transcriptome in malaria parasites [RIP-seq]

Identification of RNA targets of RNA-binding proteins (RBPs) is essential for complete understanding of their biological functions. However, it is still a challenge to identify the biologically relevant targets of RBPs through in vitro strategies of RIP-seq, HITS-CLIP, or GoldCLIP due to the potentially high background and complicated manipulation. In malaria parasites, RIP-seq and gene disruption are the few tools available currently for identification of RBP targets. Here, we have adopted the TRIBE (Targets of RNA binding proteins identified by editing) system to in vivo identify the RNA targets of PfDis3, a key exoribonuclease subunit of RNA exosome in Plasmodium falciparum. We generated a transgenic parasite line of Pfdis3-ADARcd, which catalyzes an adenosine (A)-to-inosine (I) conversion at the potential interacting sites of PfDis3-targeting RNAs. Most of PfDis3 target genes contain one edit site. The majority of the edit sites detected by PfDis3-TRIBE locate in exons and spread across the entire coding regions. The nucleotides adjacent to the edit sites contain ~ 75% of A+T. PfDis3-TRIBE target genes are biases toward higher RIP enrichment, suggesting that PfDis3-TRIBE preferentially detects stronger PfDis3 RIP targets. Collectively, PfDis3-TRIBE is a favorable tool to identify in vivo target genes of RBP with high efficiency and reproducibility. Additionally, the PfDis3-targeting genes are involved in stage-related biological processes during the blood-stage development. PfDis3 appears to shape the dynamic transcriptional transcriptome of malaria parasites through post-transcriptional degradation of a variety of unwanted transcripts from both strands in the asexual blood stage Overall design: RIP-seq data of Pfdis3 targets throughout the IDC

鉴定RNA结合蛋白（RNA-binding proteins, RBPs）的RNA靶标，对于全面解析其生物学功能至关重要。然而，通过RIP-seq、HITS-CLIP及GoldCLIP等体外策略鉴定RBPs的生物学相关靶标，仍面临较高背景与操作复杂的挑战。在疟原虫中，目前可用于鉴定RBP靶标的工具仅有RIP-seq与基因敲除等寥寥数种。本研究采用TRIBE（Targets of RNA binding proteins identified by editing，即通过编辑鉴定RNA结合蛋白靶标）系统，在体内鉴定恶性疟原虫（Plasmodium falciparum）RNA外切体的关键核糖核酸外切酶亚基PfDis3的RNA靶标。我们构建了Pfdis3-ADARcd转基因疟原虫株，该株系可在PfDis3靶向RNA的潜在互作位点催化腺苷（A）向肌苷（I）的转化。大多数PfDis3靶基因仅含一个编辑位点。PfDis3-TRIBE检测到的绝大多数编辑位点位于外显子中，并遍布整个编码区。编辑位点邻近的核苷酸中，A+T占比约为75%。PfDis3-TRIBE的靶基因倾向于具有更高的RIP富集水平，这表明PfDis3-TRIBE可优先检测到富集度更高的PfDis3 RIP靶标。综上，PfDis3-TRIBE是一种高效且可重复的体内鉴定RBP靶基因的优良工具。此外，PfDis3靶向基因参与疟原虫红内期发育过程中的阶段特异性生物学过程。PfDis3似乎通过在无性红内期降解来自两条基因组链的多种多余转录本，从而调控疟原虫的动态转录组。整体实验设计：全红细胞内发育周期（intraerythrocytic developmental cycle, IDC）中Pfdis3靶标的RIP-seq数据。