TRIBE uncovers the role of Dis3 in shaping the dynamic transcriptome in malaria parasites [TRIBE]

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. TRIBE result of Pfdis3 targets throughout the IDC

RNA结合蛋白（RNA-binding proteins, RBPs）的RNA靶标鉴定，是全面阐明其生物学功能的必要前提。然而，借助RIP-seq、HITS-CLIP及GoldCLIP等体外策略鉴定RBPs的生物学相关靶标，仍存在背景信号偏高、操作流程复杂等难点。在疟原虫中，目前可用于鉴定RBP靶标的工具十分有限，仅RIP-seq与基因敲除技术可供使用。本研究采用TRIBE（Targets of RNA binding proteins identified by editing，即通过编辑鉴定RNA结合蛋白靶标的系统），在体内鉴定恶性疟原虫RNA外泌体的关键核糖外切酶亚基PfDis3的RNA靶标。我们构建了Pfdis3-ADARcd转基因疟原虫株系，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）的碱基转换。多数PfDis3靶基因仅含单个编辑位点；PfDis3-TRIBE检测到的绝大多数编辑位点位于外显子区域，且覆盖完整的编码区。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富集度，提示该系统可优先检测到富集程度更高的PfDis3 RIP靶标。综上，PfDis3-TRIBE是一种高效且可重复的RBP体内靶基因鉴定工具。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进程。PfDis3似乎可通过在无性红内期降解双向转录链上的多种冗余转录本，重塑疟原虫的动态转录组。Pfdis3靶标的TRIBE检测结果覆盖整个红细胞内发育周期（Intraerythrocytic Development Cycle, IDC）。