Nuclear quality control of pre-mRNA splicing

mRNA 前体剪接的核质量控制

• 批准号: 394490813
• 负责人: Professorin Dr. Heike Krebber
• 金额: --
• 依托单位: Abteilung Molekulare Genetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/394490813?language=en
• 关键词: Nuclear; quality; control; pre; mRNA

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.

前mRNA在细胞核中产生和加工，然后被转运到细胞质进行翻译。在mRNA的生命周期中，许多蛋白质与具有各种功能的核糖核颗粒（RNP）结合和分离。富含丝氨酸/精氨酸（SR）的穿梭蛋白Gbp 2和Hrb 1在剪接的后期被募集到前体mRNA中，并作为输出受体异源二聚体Mex 67-Mtr 2的衔接蛋白发挥作用，允许mRNP通过核孔复合物（NPC）转运到细胞质中。重要的是，我们的工作已经表明，SR蛋白作为质量控制因子发挥作用，并防止错误或过早的转录物泄漏到细胞质中。然而，它们在RNA监测中的确切功能尚不清楚，需要进一步研究。在这个项目中，我们将研究质量控制的机制，并通过遗传和生化研究的结合来确定Gbp 2和Hrb 1在这个过程中的单独功能。通过高通量方法（例如质谱）我们将鉴定与Hrb 1相比主要发现Gbp 2的蛋白复合物。此外，我们将使用mRNA降解因子的突变体，例如标记用于降解的假mRNA或核外泌体突变体和mRNA输出突变体的TRAMP复合物的组分，以富集SR-这些复合物中的蛋白质不仅可以研究它们的相互作用，还可以研究它们的潜在修饰，这可能为降解或输出提供开关。在降解突变体中，还将通过RIP实验和随后的RNA-Seq分析来分析Gbp 2和Hrb 1与哪些mRNA结合。通过这种方式，我们将表征它们与缺陷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Mlp 1和Mlp 2介导的NPC的最后控制步骤。这些研究将确定Gbp 2和Hrb 1在核质量控制中的各自功能，并阐明其潜在机制。