Center for Molecular Biology of RNA, Department of Molecular Cell Developmental Biology, University of California, Santa Cruz, California, United States of America.
UCSC Genomics Institute, University of California, Santa Cruz, Santa Cruz, California, United States of America.
PLoS Genet. 2022 Feb 10;18(2):e1010028. doi: 10.1371/journal.pgen.1010028. eCollection 2022 Feb.
Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5' GU and ending with 3' AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and "custody" of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5' splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5' splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5' splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5' splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.
前体 mRNA 剪接是真核生物基因表达的一个基本步骤,由一系列动态的大分子蛋白/RNA 复合物完成,这些复合物统称为剪接体。这一系列剪接体复合物定义、组装并催化内含子的切除。该过程的中间分子模型快照是根据 cryo-EM 数据创建的,然而,剪接体中发生的许多动态变化的方面尚未完全了解。秀丽隐杆线虫遵循 GU-AG 剪接规则,几乎所有内含子都以 5'GU 开头,以 3'AG 结尾。这些剪接位点在剪接循环的早期被识别,但随着循环的进行,随着催化位点的构建,前体 mRNA 剪接位点的“监管”从一个因子传递到另一个因子,剪接位点身份通过这些动态变化得以维持或重新建立的机制尚不清楚。我们在秀丽隐杆线虫中进行了一个遗传筛选,以寻找能够改变 5'剪接位点选择的因子。我们报告说,KIN17 和 PRCC 参与剪接位点选择,这是这两种蛋白中第一个被提出的功能剪接作用。以前鉴定的隐性 5'剪接抑制因子促进远端隐性 GU 剪接位点,然而,KIN17 和 PRCC 的突变反而促进了不寻常的近端 5'剪接位点的使用,该位点定义了一个以 UU 开头的内含子,与 GU 供体相隔 1nt。我们进行了高通量 mRNA 测序分析,发现 PRCC 的突变,在较小程度上还有 KIN17 的突变,改变了基因组范围内的天然位置的替代 5'剪接位点的使用,通常促进了附近非保守位点的使用。我们的工作揭示了剪接体在复杂组装过程中维持剪接位点身份的精细和粗略机制。