Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
PLoS Genet. 2024 May 14;20(5):e1011284. doi: 10.1371/journal.pgen.1011284. eCollection 2024 May.
The Integrator is a multi-subunit protein complex that catalyzes the maturation of snRNA transcripts via 3' cleavage, a step required for snRNA incorporation with snRNP for spliceosome biogenesis. Here we developed a GFP based in vivo snRNA misprocessing reporter as a readout of Integrator function and performed a genome-wide RNAi screen for Integrator regulators. We found that loss of the Argonaute encoding csr-1 gene resulted in widespread 3' misprocessing of snRNA transcripts that is accompanied by a significant increase in alternative splicing. Loss of the csr-1 gene down-regulates the germline expression of Integrator subunits 4 and 6 and is accompanied by a reduced protein translation efficiency of multiple Integrator catalytic and non-catalytic subunits. Through isoform and motif mutant analysis, we determined that CSR-1's effect on snRNA processing is dependent on its catalytic slicer activity but does not involve the CSR-1a isoform. Moreover, mRNA-sequencing revealed high similarity in the transcriptome profile between csr-1 and Integrator subunit knockdown via RNAi. Together, our findings reveal CSR-1 as a new regulator of the Integrator complex and implicate a novel role of this Argonaute protein in snRNA 3' processing.
整合作者是一种多亚基蛋白复合物,通过 3' 切割催化 snRNA 转录本的成熟,这是 snRNA 与 snRNP 结合用于剪接体生物发生所必需的步骤。在这里,我们开发了一种基于 GFP 的体内 snRNA 错误处理报告器,作为整合作者功能的读出器,并进行了全基因组 RNAi 筛选以寻找整合作者调节剂。我们发现 Argonaute 编码基因 csr-1 的缺失导致 snRNA 转录本的广泛 3' 错误处理,同时伴随着剪接的显著增加。csr-1 基因的缺失下调了 Integrator 亚基 4 和 6 的生殖系表达,并伴随着多个 Integrator 催化和非催化亚基的翻译效率降低。通过同工型和基序突变分析,我们确定 CSR-1 对 snRNA 加工的影响依赖于其催化切割活性,但不涉及 CSR-1a 同工型。此外,mRNA 测序显示 csr-1 和 Integrator 亚基通过 RNAi 敲低之间的转录组图谱高度相似。总之,我们的发现揭示了 CSR-1 是整合作者复合物的一个新调节剂,并暗示了这种 Argonaute 蛋白在 snRNA 3' 加工中的新作用。