Molecular mechanism of U1 snRNP-mediated suppression of 3 prime end processing

U1 snRNP介导的3素末端加工抑制的分子机制

• 批准号: 244876233
• 负责人: Privatdozent Dr. Jens Bohne
• 金额: --
• 依托单位: Institut für Virologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/244876233?language=en
• 关键词: Molecular; mechanism; U1; snRNP; mediated

The regulation of gene expression is the key to understand the wealth of sequence information available today. RNA is the central molecule in this process. The past years illustrated that both alternative mRNA processing and non-coding RNAs contribute substantially to the genomic complexity. Recently, it has been recognized that alternative usage of polyadenylation signals (PAS) is even more frequent than alternative splicing. Thus, the heterogeneity of mRNA 37nds is important for gene regulation. First, selection of different terminal exons leads to variations in the C-termini of proteins and second, differential length of 3/ untranslated regions (UTRs) determines seed regions for miRNAs or other RNA motifs regulating RNA stability or localization. So, alternative polyadenylation (APA) must be tightly controlled. Interestingly, U1 snRNP was shown to protect mRNAs from premature cleavage and polyadenylation (PCPA) and to contribute to variations in 3/UTR length besides its role in splicing.In parallel to the discovery of the new surveillance mechanism associated with U1 snRNP, we revealed the molecular pathomechanism of a previously unclassified 3/UTR mutation leading to a new complex immunodeficiency syndrome. A point mutation in the 3/UTR of the gene encoding p14/robld3 creates a 5/ splice site (SS), which is recognized by U1 snRNP and leads to a failure of 3/ end processing in the absence of splicing. Thus, suppression of intronic polyadenylation by U1 snRNP is important for the cell, but deleterious for p14 expression. It was previously assumed that the U1-70k subunit of U1 snRNP directly inhibits poly A polymerase. We could show that this is an unlikely scenario of PCPA suppression. We have preliminary data that U1 snRNP already inhibits cleavage of pre-mRNAs by targeting a factor of the 3/ end processing complex or by modulating the properties of RNA polymerase II. To address the molecular mechanism of PCPA suppression, we constructed two elegant reporter systems. One is a so-called tandem reporter characterized by a duplication of the p14 3/UTR. In addition, we have experience with a bi-directional Tet construct. A symmetric doxycycline inducible promoter drives expression of gene used for normalization and a p14 expression cassette simultaneously. This construct can be targeted to a pre-selected genomic locus by recombination. Many experimental questions like the identification of the RNA decay pathway or the component of U1 executing the suppression can be answered. Based on our data from histone reporter genes, we propose that U1 snRNP inhibits recognition of the PAS consensus sequence or targets a factor involved in cleavage of the pre-mRNA.The molecular analysis of this additional U1 snRNP function will have important implications for our understanding of a surveillance mechanism, which protects mRNAs genome-wide from premature transcriptional termination, but also allows for the accumulation of different mRNA isoforms.

基因表达的调控是理解当今丰富的序列信息的关键。RNA是这个过程中的核心分子。近年来的研究表明，mRNA的替代加工和非编码RNA都对基因组的复杂性有很大的贡献。最近，人们已经认识到，多聚腺苷酸化信号（PAS）的选择性使用甚至比选择性剪接更频繁。因此，mRNA 37 nds的异质性对于基因调控是重要的。首先，不同末端外显子的选择导致蛋白质C末端的变化，其次，3/非翻译区（UTR）的不同长度决定了调节RNA稳定性或定位的miRNA或其他RNA基序的种子区域。因此，必须严格控制选择性多聚腺苷酸化（阿帕）。有趣的是，U1 snRNP被证明可以保护mRNA免受过早切割和多聚腺苷酸化（PCPA），并有助于3/UTR长度的变化，除了其在splicing.In平行的新的监视机制与U1 snRNP的发现，我们揭示了一个以前未分类的3/UTR突变导致一种新的复杂免疫缺陷综合征的分子病理机制。编码p14/robld 3的基因的3/UTR中的点突变产生5/剪接位点（SS），其被U1 snRNP识别并在不存在剪接的情况下导致3/末端加工失败。因此，U1 snRNP对内含子多聚腺苷酸化的抑制对细胞是重要的，但对p14表达是有害的。先前假设U1 snRNP的U1- 70 k亚基直接抑制聚腺苷酸聚合酶。我们可以证明，这是一个不太可能的PCPA抑制的情况。我们有初步的数据表明，U1 snRNP已经通过靶向3/末端加工复合物的因子或通过调节RNA聚合酶II的性质来抑制前mRNA的切割。为了解决PCPA抑制的分子机制，我们构建了两个优雅的报告系统。一种是所谓的串联报告基因，其特征在于p14 3/UTR的重复。此外，我们有双向泰特结构的经验。对称多西环素诱导型启动子同时驱动用于标准化的基因和p14表达盒的表达。该构建体可以通过重组靶向预选的基因组基因座。许多实验问题，如RNA衰变途径的鉴定或U1执行抑制的组分可以回答。基于我们从组蛋白报告基因获得的数据，我们提出U1 snRNP抑制PAS共有序列的识别或靶向参与前体mRNA切割的因子。对U1 snRNP额外功能的分子分析将对我们理解一种监视机制具有重要意义，该机制保护全基因组的mRNA免于过早转录终止，而且允许不同mRNA同种型的积累。