Genomics and Bioinformatics Core, University at Buffalo School of Medicine and Biomedical Sciences, Buffalo, NY 14203.
Department of Biochemistry, University at Buffalo School of Medicine and Biomedical Sciences, Buffalo, NY 14203.
Proc Natl Acad Sci U S A. 2024 Jul 16;121(29):e2403188121. doi: 10.1073/pnas.2403188121. Epub 2024 Jul 11.
The kinetoplastid parasite, , undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in developmental control and identifies a critical RBP in this process.
锥虫寄生虫 在哺乳动物中经历了一个复杂的生命周期，包括细长和粗短的血液形式，以及在采采蝇宿主中的前环和后环形式 (MFs)。在宿主之间以及每个宿主中的活跃和静止阶段之间分化的许多基因调控事件是在转录控制几乎不存在的情况下发生的。相反，分化是由与 mRNA 3'非翻译区 (3'UTR) 结合的 RNA 结合蛋白 (RBPs) 控制的，这些蛋白影响 RNA 的稳定性和翻译效率。DRBD18 是一种多功能的 RBP，已被证明可以影响 mRNA 的稳定性、翻译、输出和加工。在这里，我们使用单细胞 RNAseq 来描述 DRBD18 耗竭后细胞群体中出现的转录组变化，并可视化全转录组 3'UTR 长度的改变。我们表明，在前环昆虫阶段，DRBD18 抑制了粗短血液形式和 MF 转录本的表达。此外，DRBD18 调节了超过 1500 个转录本的 3'UTR 长度，通常促进使用远端多聚腺苷酸化位点，从而包含 3'UTR 调节元件。值得注意的是，将 DRBD18 敲低的多聚腺苷酸化模式与粗短血液形式的多聚腺苷酸化模式进行比较，显示出许多相似之处，揭示了多聚腺苷酸化位点选择在发育基因调控中的作用，并表明 DRBD18 控制了一组转录本的这一过程。RNA 免疫沉淀支持 DRBD18 在多聚腺苷酸化位点选择中的直接作用。本报告强调了可变多聚腺苷酸化在发育控制中的重要性，并确定了这一过程中的一个关键 RBP。
