TRIBE Uncovers the Role of Dis3 in Shaping the Dynamic Transcriptome in Malaria Parasites
TRIBE Uncovers the Role of Dis3 in Shaping the Dynamic Transcriptome in Malaria Parasites
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TRIBE 揭示了 Dis3 在塑造疟疾寄生虫动态转录组中的作用
DOI：
10.3389/fcell.2019.00264
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发表时间：
2019-11-01
影响因子：
5.5
通讯作者：
Zhang, Qingfeng
中科院分区：
文献类型：
--
作者：
Liu, Meng;Lu, Binbin;Zhang, Qingfeng
识别RNA结合蛋白（RBP）的RNA靶标对于全面了解其生物学功能至关重要。然而，由于潜在的高背景和复杂的操作，通过RIP-seq、HITS-CLIP或GoldCLIP的体外策略来鉴定RBP的生物学相关靶标仍然是一个挑战。在疟疾寄生虫中，RIP-seq和基因破坏是目前可用于识别RBP靶标的少数工具。在这里，我们采用TRIBE（通过编辑识别的RNA结合蛋白的目标）系统在体内鉴定恶性疟原虫RNA外泌体的关键核糖核酸外切酶亚基PfDis3的RNA靶标。我们产生了PfDis3-ADARcd的转基因寄生虫系，其催化在PfDis3靶向RNA的潜在相互作用位点处的腺苷（A）至肌苷（I）的转化。大多数PfDis3靶基因含有一个编辑位点。PfDis3-TRIBE检测到的大多数编辑位点位于外显子中并分布在整个编码区。与编辑位点相邻的核苷酸含有约75%的A + T。PfDis3-TRIBE靶基因偏向于更高的RIP富集，表明PfDis3-TRIBE优先检测更强的PfDis3 RIP靶。PfDis3-TRIBE是一种高效、重复性好的RBP体内靶基因鉴定工具。此外，PfDis3靶向基因参与血液阶段发育期间的阶段相关生物学过程。因此，PfDis3似乎通过转录后降解来自无性血液阶段的两条链的各种不需要的转录物来塑造疟原虫的动态转录转录组。
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. Thus 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.