Most human genes are interrupted by non-coding sequences known as introns. The nascent transcript must be processed to remove the introns and to join the coding sequence into a contiguous mature mRNA molecule. The human genome project provided an estimate that only 1 to 1.5% of the approximately 3x10/9 base pair (bp) genome is spanned by coding sequences or exons, whereas introns occupy approximately 24% of the genome. It is apparent that the cellular machinery is presented with a formidable task to precisely locate the splice sites within a gene's transcript. Another dimension of complexity is added when certain exon sequences are skipped or included, a phenomenon known as alternative splicing. Alternative splicing occurs in a large number of human genes and it plays important roles in regulating gene expression during development and differentiation. Alternative splicing is also responsible for generating molecular diversity in certain cells, such as neurons. Splicing of pre-mRNA involves an ordered assembly of components, including small nuclear ribonucleoprotein particles (snRNP) and additional protein splicing factors, resulting in the formation of a large 50-60S complex, termed the spliceosome. We are interested in understanding the molecular mechanism of splice site recognition from a structural standpoint. In this proposal, we focus our study on several well characterized human splicing factors that are important for splice site selection, namely, SR proteins, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and the U2 auxiliary factor (U2AF).
