Dissecting the role of U5 snRNA isoforms as drivers of U5 snRNP diversity and spliceosome plasticity during development and homeostasis

前体mRNA剪接是后生动物基因表达调控的关键步骤，对组织和器官发育、稳态和生物体健康至关重要。由剪接体催化的“切割和连接”过程必须是高效和精确的，但也必须是灵活的，以根据需要产生特定的转录物。相反，剪接体成分的突变和失调是许多人类疾病和生物衰老的基础。产生不同细胞类型特异性剪接模式的能力与剪接体的动态组成有关。剪接体由五个小的核核糖核蛋白颗粒（snRNP）组成，每个颗粒由特定的富含尿苷的小核RNA（U snRNA）和一组核心蛋白和辅助蛋白组成。有趣的是，高等真核生物，不像酵母，编码几种亚型的个别U snRNAs，显示特定的时空表达。它们的作用和贡献的动态剪接体结构和功能可塑性仍然在很大程度上未知。我们的工作，使用遗传上易处理的果蝇模型，提供了对剪接体生物发生、功能和调节机制的新见解（Claudius et al.，2015年，埃尔克伦茨等人，2021年）。此外，我们已经证明了其与由核心剪接因子Prp8突变引起的退行性眼病视网膜色素变性的病因学和病理学的机制研究的相关性（Stankovic et al.，2020年）。在这里，我们建议联合收割机功能遗传学，基因组学和蛋白质组学的方法在果蝇模型中解开的组织和发育阶段的特定要求的7个U5 snRNA亚型编码的苍蝇基因组。我们将确定单个U5 snRNA亚型在形成U5 snRNP组合物中的作用，以及它们对建立发育阶段和组织特异性剪接程序的贡献。最后，我们将定义U5 snRNA· RNA碱基配对相互作用的体内功能，作为剪接位点选择和维持转录组完整性的晚期检查点机制的一部分。我们相信，我们的工作将是基础和生物医学研究的普遍重要性，推进的机制，控制典型的和可选的前mRNA剪接后生动物的理解。

Pre-mRNA splicing represents a crucial step in the regulation of gene expression in metazoans pivotal to tissue and organ development, homeostasis, and organismal healthspan. The “cut and join” process catalyzed by a spliceosome must be efficient and precise but also flexible to generate specific transcripts on demand. In contrast, mutations in, and deregulation of, spliceosome components underlie numerous human diseases and organismal aging. The capacity to generate diverse cell-type-specific splicing patterns has been linked to the dynamic composition of the spliceosome. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs) each comprising of specific uridine-rich small nuclear RNA (U snRNA), and a set of core and accessory proteins. Intriguingly, higher eukaryotes, unlike yeast, encode several isoforms of individual U snRNAs, which show specific spatiotemporal expression. Their roles and contribution to the dynamic spliceosome architecture and functional plasticity remain largely unknown. Our work, using the genetically tractable Drosophila model, has provided novel insights into mechanisms governing spliceosome biogenesis, function, and regulation (Claudius et al., 2015, Erkelenz et al., 2021). Moreover, we have demonstrated its relevance for mechanistic studies of etiology and pathology of a degenerative eye disorder retinitis pigmentosa caused by mutations in the core splicing factor Prp8 (Stankovic et al., 2020). Here we propose to combine functional genetics, genomic and proteomic approaches in the Drosophila model to unravel the tissue- and developmental-stage specific requirements for the seven U5 snRNA isoforms encoded in the fly genome. We will determine the role of the individual U5 snRNA isoforms in shaping the U5 snRNP composition and their contribution to establishing the developmental stage- and tissue-specific splicing programs. Finally, we will define the in vivo function of the U5 snRNA• RNA base-pairing interactions as a part of the late checkpoint mechanism for splice site selection and the maintenance of transcriptome integrity. We believe that our work will be of general importance for basic and biomedical research, advancing the understanding of mechanisms that control canonical and alternative pre-mRNA splicing in metazoans.