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

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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.