Alternative splicing of pre-mRNAs is the major contributor in the human system to generate complex proteomes from a comparatively low number of genes. Several modes of alternative splicing are known today which are tightly regulated to ensure accurate protein expression. We have identified intronic CA repeat and CA-rich sequences as a new class of regulatory elements acting as enhancers or silencers on alternative splicing. Their function is mediated by the heterogenous ribonucleoprotein (hnRNP) L which has been characterised as the main CA binding protein. Considering that CA repetitive sequences are very common in the human genome, the identification and analysis of hnRNP L target genes should give further insights into the mechanism of alternative splicing regulation. In this work, I studied alternative splicing regulation of three recently identified hnRNP L target genes. First, in the SLC2A2 gene CA repeats were identified as an intronic splicing silencer element. Their function was shown to depend on hnRNP L as the trans-acting factor. Further studies on the mechanism of splicing regulation demonstrated that hnRNP L interfered with 5 splice site recognition by the U1 snRNP. Second, I characterised a short intronic CA-rich cluster in the TJP1 gene as an hnRNP L-dependent splicing silencer. In contrast to SLC2A2, hnRNP L was shown to compete with U2AF65 for binding to the polypyrimidine tract thus impairing 3 splice site recognition. Third, an intronic CA repeat in the ITGA2 gene either activated splicing of the corresponding exon or repressed recognition of a cryptic exon in a sequence-dependent manner. Furthermore, a combined microarray and RNAi analysis revealed new modes of hnRNP L-mediated splicing regulation and, moreover, a novel role for hnRNP L in alternative polyadenylation. In the ASAH1 gene, hnRNP L repressed usage of an internal poly(A) site. Taken together, I have studied different mechanism of splicing regulation on the basis of three genes, SLC2A2, TJP1, and ITGA2. The results revealed new functions of CA repeat and CA-rich sequences and hnRNP L in alternative splicing regulation. In sum, hnRNP L was shown to be a global and versatile regulator protein in the human system with roles in alterative splicing and polyadenylation.
