U1 snRNP and polyadenylation in plants – a novel liaison

U1 snRNP 和植物中的聚腺苷酸化——一种新的联系

• 批准号: 426553355
• 负责人: Professorin Dr. Dorothee Staiger
• 金额: --
• 依托单位: Department of Gene Expression
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/426553355?language=en
• 关键词: U1; snRNP; polyadenylation; plants; novel

The various steps of posttranscriptional regulation including splicing, 3´end cleavage and poly-adenylation, and decay are tightly coordinated to shape the plant transcriptome. We have shown that active 5´splice sites affect polyadenylation of miRNA precursors in Arabidopsis thaliana. Moreover, we observed interactions between U1 snRNP, a ribonucleoprotein particle rec-ognizing 5´splice sites, and the CFI complex as part of the 3´end cleavage and polyadenylation machinery. Here, we investigate coupling between splicing and 3´end processing globally. We propose that the crosstalk between U1 snRNP and the CFI complex plays an important role in de-fining the use of polyadenylation sites. We will determine in vivo targets of the CFI subunits ge-nome-wide using individual nucleotide resolution crosslinking and immunoprecipitation recently established for plants and determine binding motifs. In parallel, we will perform 3´end sequencing on mutants defective in the CFI subunits. This will reveal for the first time which of the CFI subunits control polyadenylation. Cross-referencing the in vivo binding data and transcriptomic data will enable us to determine CFI binding sites involved in the choice of poly(A) sites and CFI sites located in the vicinity of 5´splice sites. From these data, we will select candidate genes for a functional analysis. To identify proteins mediating the crosstalk between U1 snRNP and CFI we will immunoprecipitate tagged CFI subunits and identify co-purified proteins by mass spec-trometry. A function of the proteins that are part of U1 snRNP or interact with both, U1 snRNP and CFI in the control of polyadenylation will be analyzed. For this, corresponding mutants will be obtained assayed for alterations in polyadenylation. Our results will greatly improve our knowledge of polyadenylation control in Arabidopsis and unravel novel functions of U1 snRNP besides its role in splicing. This will significantly advance our understanding of gene regulation in general, a process fundamental in plant adaptation to changing environments. Our project is based on a complementary expertise in RNA processing and interest in polyadenylation in both groups that will lead to a fruitful collaboration. The project will also benefit from a mutual ex-change of state-of-the art techniques developed in each group.

转录后调控的各个步骤包括剪接、3 ′端切割和多聚腺苷酸化以及衰变，这些步骤紧密协调以形成植物转录组。我们已经证明，活跃的5 ′剪接位点影响拟南芥中miRNA前体的聚腺苷酸化。此外，我们观察到U1 snRNP（一种识别5 ′剪接位点的核糖核蛋白颗粒）与CFI复合物之间的相互作用，作为3 ′端切割和多聚腺苷酸化机制的一部分。在这里，我们研究剪接和3 '端加工之间的耦合。我们建议U1 snRNP和CFI复合物之间的串扰起着重要的作用，在定义使用的聚腺苷酸化位点。我们将使用最近建立的用于植物的单个核苷酸分辨率、交联和免疫沉淀来确定基因组范围内的CFI亚基的体内靶点，并确定结合基序。同时，我们将对CFI亚基缺陷的突变体进行3 ′端测序。这将首次揭示哪些CFI亚基控制多聚腺苷酸化。交叉参考体内结合数据和转录组学数据将使我们能够确定参与选择poly（A）位点和位于5 ′剪接位点附近的CFI位点的CFI结合位点。从这些数据中，我们将选择候选基因进行功能分析。为了鉴定介导U1 snRNP和CFI之间串扰的蛋白质，我们将免疫沉淀标记的CFI亚基，并通过质谱法鉴定共纯化的蛋白质。 将分析作为U1 snRNP的一部分或与U1 snRNP和CFI两者相互作用的蛋白质在多聚腺苷酸化控制中的功能。为此，将获得相应的突变体，测定多聚腺苷酸化的改变。我们的研究结果将大大提高我们的知识，在拟南芥中的多聚腺苷酸化控制和解开新的功能，U1 snRNP除了其在剪接中的作用。这将大大促进我们对基因调控的理解，这是植物适应不断变化的环境的一个基本过程。我们的项目是基于互补的专业知识，在RNA加工和兴趣的聚腺苷酸化在这两个群体，将导致一个富有成效的合作。该项目还将受益于每个小组开发的最先进技术的相互交流。