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

基因表达的调控是理解当今丰富的序列信息的关键。RNA是这一过程中的中心分子。过去的几年表明，选择性的mRNA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，多聚腺苷化信号(PAS)的替代使用甚至比替代剪接更频繁。因此，mRNA37nds的异质性对基因调控具有重要意义。首先，不同末端外显子的选择会导致蛋白质的C-末端发生变化，其次，3/非翻译区(UTRs)的不同长度决定了miRNAs或其他调节RNA稳定性或定位的RNA基序的种子区域。因此，选择性多聚腺苷酸化(APA)必须严格控制。有趣的是，U1 SnRNP除了在剪接中的作用外，还被证明可以保护mRNAs免受过早切割和多聚腺苷酸化(PCPA)的影响，并导致3/UTR长度的变化。在发现与U1 SnRNP相关的新的监视机制的同时，我们揭示了以前未分类的3/UTR突变导致新的复杂免疫缺陷综合征的分子病理机制。编码p14/robld3的基因的3/UTR点突变产生了一个5/剪接点(SS)，该点突变可被U1 SnRNP识别，并导致在没有剪接的情况下3/端处理失败。因此，U1SnRNP抑制内含子多腺化对细胞是重要的，但对p14的表达是有害的。以前的假设是，U1SnRNP的U1-70k亚基直接抑制Poly A聚合酶。我们可以证明，这是一种不太可能抑制PCPA的情况。我们有初步数据表明，U1 SnRNP已经通过靶向3/末端加工复合体的一个因子或通过调节RNA聚合酶II的性质来抑制前mRNAs的切割。为了研究PCPA抑制的分子机制，我们构建了两个优雅的报告系统。一种是以复制p143/UTR为特征的所谓串联报告基因。此外，我们还拥有双向TET构建的经验。一个对称的多西环素可诱导启动子同时驱动用于归一化的基因的表达和一个p14表达盒。该构建物可通过重组作用于预先选定的基因组位点。许多实验问题都可以得到解答，比如识别RNA衰变途径或执行抑制的U1组分。基于我们来自组蛋白报告基因的数据，我们认为U1 SnRNP抑制PAS共识序列的识别或针对参与切割前mRNAs的因子。对这一额外的U1 SnRNP功能的分子分析将对我们理解监督机制具有重要意义，该机制保护全基因组的mRNAs免于过早转录终止，但也允许不同的mRNA亚型的积累。

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.