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是这个过程的中心分子。过去几年的研究表明，替代mRNA加工和非编码rna对基因组复杂性都有重要贡献。最近，人们认识到聚腺苷酸化信号（PAS）的选择性使用甚至比选择性剪接更频繁。因此，mRNA 37nds的异质性对基因调控很重要。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（UTRs）长度的差异决定了mirna或其他RNA基序的种子区域，这些基序调节RNA的稳定性或定位。因此，必须严格控制选择性聚腺苷化（APA）。有趣的是，U1 snRNP被证明可以保护mrna免于过早切割和聚腺苷化（PCPA），除了在剪接中起作用外，还有助于3/UTR长度的变化。在发现与U1 snRNP相关的新监测机制的同时，我们揭示了先前未分类的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基因的3/UTR点突变产生5/ splice site (SS)，该位点被U1 snRNP识别，并导致在没有剪接的情况下3/ end加工失败。因此，U1 snRNP抑制内含子聚腺苷化对细胞是重要的，但对p14表达是有害的。以前认为U1 snRNP的U1-70k亚基直接抑制聚A聚合酶。我们可以证明这是一种不太可能抑制PCPA的情况。我们有初步的数据表明，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的特性来抑制前mrna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。一种是所谓的串联报告基因，其特征是p143 /UTR的复制。此外，我们有双向Tet结构的经验。对称多西环素诱导启动子同时驱动用于规范化的基因和p14表达盒的表达。这种结构可以通过重组靶向一个预先选择的基因组位点。许多实验问题，如RNA衰变途径的识别或U1执行抑制的成分可以得到回答。基于组蛋白报告基因的数据，我们提出U1 snRNP抑制PAS共识序列的识别或靶向一个参与前mrna切割的因子。这种额外的U1 snRNP功能的分子分析将对我们理解监视机制具有重要意义，该机制保护mRNA全基因组免于过早转录终止，但也允许不同mRNA同种异构体的积累。

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.