Quaternary structure of 3´ splice site recognition

In the early stages of eukaryotic splicing, two splicing factors (SF1, U2AF) cooperatively bind to pre-mRNA intron sequences close to the 3` splice site. Several fundamental questions arise from the fact that the modular complex invokes weak binary protein-protein/RNA interactions to achieve 3`-splice site recognition: What is the structural basis for recognition of the 3` splice site, how do the associated splicing factors induce a unique conformation to the intron RNA? Does the dynamic assembly of the spliceosome allow the subsequent replacement of SF1 by the U2 snRNP? And, how might other splicing regulators compete for interactions with the intron and thus establish alternative splice sites? These questions are expected to be answered through quaternary structure determination of the 3` splice site recognition complex SF1-U2AF/RNA. Recent methodological developments carried out by the applicant and in the Sattler group will be combined and applied to study the large macromolecular assembly. New methodology will be established and will open up the field for a wide range of large molecular complexes that were not routinely accessible. The recognition of the 3` splice site and the competitions of splicing regulators for the intron are of tremendous biological and medicinal importance. Splice site recognition is a complex and tightly controlled process and is linked to ~15% of human genetic disorders, in which functional splice sites are altered. Furthermore, the regulation of alternative splicing, which plays important roles, for example, in the regulation of gene expression during sex determination, nervous system differentiation, and apoptotic cell death, frequently involves interference with spliceosome assembly. The proposed research will give rise to novel details on 3` splice site recognition, and approach the long-term goal of understanding regulation of pre-mRNA (alternative) splicing.