Molecular Mechanisms and Conservation of Pre-mRNA Splicing

The eukaryotic genome is a large and complex network of molecules that work together to create diversity across many different species. Pre-mRNA splicing is a vital step in the processing of RNA into functionally diverse proteins. Splicing has a vast history and understanding the combination of many different factors and elements is fundamental to studying gene expression. Gene evolution and the evolutionary pressures that encode a set of exonic sequences maintain efficient pre-mRNA splicing and ultimately dictate the selection of amino acids that define a protein. Exonic sequences have been regulated in a way to demonstrate the co-existence of coding and splicing pressures. Through the design of an exon conservation database, evolutionary conservation patterns were identified that influenced the final sequence of an exon. This information led to important predictions about splicing patterns in human disease. The database allowed for the identification of essential architectural parameters of the human genome. In addition, analysis of nucleotide variations at the wobble position identified splice altering SNPs and how these SNPs influenced exon inclusion. Regulation of alternatively spliced exons requires a coordinated effort by many cis and trans-acting factors. Understanding how these factors work together is important for the mechanism of alternative splicing regulation. SR proteins and hnRNPs have previously demonstrated a position-dependent method of regulation. It was shown that U1 snRNP, at activating or repressive conditions, displayed dynamic changes in its compositional integrity. This demonstrated that U1 snRNP integrity is therefore modulated by the presence of position-dependent interactions with splicing regulatory factors, further suggestive of U1 snRNP as a molecular gatekeeper for splicing initiation. Polyadenylation is a fundamental step in the 3’ end processing of mRNA. Alternative polyadenylation is another contributor to genomic diversity. The coordinated efforts between splicing and polyadenylation have been demonstrated for terminal exons, however, understanding the influence these two processes have on upstream exons was unknown. Genome-wide analyses allowed for the identification of an important role that a polyadenylation factor (CstF64) has on alternative splicing. In addition, the coupling that was seen between alternative polyadenylation and alternative splicing was in fact limited to terminal exons