U1 snRNP and polyadenylation in plants – a novel liaison

转录后调控的各个步骤，包括剪接、3′端切割、多聚腺苷化和衰变，紧密协调形成植物转录组。我们已经证明，活性5´剪接位点影响拟南芥miRNA前体的聚腺苷化。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 ’端切割和聚腺苷酸化机制的一部分。在这里，我们研究了拼接和3 '端加工之间的耦合。我们提出U1 snRNP和CFI复合物之间的串扰在定义聚腺苷化位点的使用中起重要作用。我们将利用最近在植物中建立的单个核苷酸分辨率交联和免疫沉淀来确定CFI亚基全基因组的体内靶标，并确定结合基序。同时，我们将对CFI亚基缺陷突变体进行3′端测序。这将首次揭示哪一个CFI亚基控制聚腺苷酸化。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poly(A)位点选择的CFI结合位点和位于5´剪接位点附近的CFI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。我们将分析作为U1 snRNP的一部分或与U1 snRNP和CFI相互作用的蛋白在控制聚腺苷化中的作用。为此，将获得相应的突变体，检测聚腺苷酸化的改变。我们的研究结果将大大提高我们对拟南芥多聚腺苷化控制的认识，并揭示U1 snRNP除了在剪接中的作用之外的新功能。这将大大提高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。我们的项目是基于两个小组在RNA加工和聚腺苷化方面的互补专业知识，这将导致富有成效的合作。该项目还将受益于双方相互交流各自开发的最先进技术。

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.