Nuclear quality control of pre-mRNA splicing

pre - mrna在转运到细胞质进行翻译之前在细胞核中产生和加工。在mRNA的生命周期中，许多蛋白质与具有各种功能的核糖核粒子（RNP）结合或分离。富含穿梭丝氨酸/精氨酸（SR）的蛋白Gbp2和Hrb1在剪接后期被募集到pre-mRNA中，并作为输出受体异二聚体Mex67-Mtr2的适配蛋白，允许mRNP通过核孔复合物（NPC）进入细胞质。重要的是，我们的工作表明sr蛋白作为质量控制因子起作用，并防止错误或过早转录本泄漏到细胞质中。然而，它们在RNA监视中的确切功能尚不清楚，需要进一步研究。在本项目中，我们将通过遗传学和生化研究相结合的方法，研究质量控制的机制，确定Gbp2和Hrb1在这一过程中的个体功能。通过高通量方法（如质谱），我们将鉴定出主要存在Gbp2和Hrb1的蛋白复合物。此外，我们将使用mRNA降解因子的突变体，例如标记假mRNA降解的trump复合物的组分或核外泌体突变体和mRNA输出突变体来丰富这些复合物中的sr蛋白，不仅研究它们的相互作用，而且研究它们的潜在修饰，这可能为降解或输出提供开关。在降解过程中，突变体还将通过rip实验和随后的RNA-Seq分析分析mrna Gbp2和Hrb1与哪些mrna结合。通过这种方式，我们将描述它们与缺陷mrna的结合。除此之外，我们将确定负责rna结合的蛋白质序列。最后，我们将研究由Mlp1和Mlp2介导的NPC的最后一个控制步骤。这些研究将确定Gbp2和Hrb1在核质量控制中的单独功能，并阐明其潜在机制。

Pre-mRNAs are generated and processed in the nucleus before they are transported to the cytoplasm for translation. During the life cycle of an mRNA many proteins associate and dissociate from the ribonucleoparticle (RNP) that have various functions. The shuttling serine/arginine (SR)-rich proteins Gbp2 and Hrb1 are recruited to the pre-mRNA during late phases of splicing and function as adapter proteins for the export receptor heterodimer Mex67-Mtr2, that allows transit of the mRNP through the nuclear pore complex (NPC) into the cytoplasm. Importantly, our work has shown that the SR-proteins function as quality control factors and prevent leakage of false or premature transcripts to the cytoplasm. Their exact function in RNA surveillance, however, is unclear and requires further studies. In this project we will study the mechanisms of quality control and determine the individual functions of Gbp2 and Hrb1 in this process through a combination of genetic and biochemical studies. With high throughput methods (e.g. mass spectrometry) we will identify the protein complexes in which Gbp2 in comparison to Hrb1 are mainly found. Additionally, we will use mutants of mRNA degradation factors, e.g. components of the TRAMP-complex that marks false mRNAs for degradation or nuclear exosome mutants and mRNA export mutants to enrich the SR-proteins in these complexes not only to study their interactions but also their potential modifications, which might provide switches for degradation or export. In the degradation mutants will also analyze to which mRNAs Gbp2 and Hrb1 bind by RIP-experiments and subsequent RNA-Seq analyses. In this way we will characterize their binding to defective mRNAs. In addition to that we will determine the responsible protein sequence for RNA-binding. Finally, we will investigate the last control step at the NPC that is mediated by Mlp1 and Mlp2. These studies will identify individual functions of Gbp2 and Hrb1 in nuclear quality control and illuminate the underlying mechanisms.