Structure And Dynamics Of Protein-RNA Recognition In The Regulation Of Gene Expression

Abstract

We are studying mechanisms of biomolecular recognition in the regulation of pre-mRNA splicing and RNA-based gene silencing. Splicing of nuclear pre-mRNA, i.e. the removal of non-coding, intervening intron sequences, is a key step in the regulation of eukaryotic gene expression. It contributes to gene regulation and protein diversity by joining of alternative exons. Early spliceosome assembly is tightly regulated and involves the recognition of characteristic intron RNA sequences. The biogenesis of these pre-spliceosomal intermediates is dynamic and involves cooperative protein-protein and protein-RNA interactions.For the structural analysis of such multi-domain proteins and protein complexes, we have developed an approach for determining the quaternary arrangement based on solution NMR and Small Angle Scattering methods. We combine orientational information derived from NMR residual dipolar couplings (RDCs) and (long-range) distance restraints derived from paramagnetic relaxation enhancement (PRE) using spin-labeled proteins and/or RNA. These data can be combined with a novel target function in CNS for direct refinement against small angle X-ray and/or neutron scattering (SAXS/SANS) data. The RDC, PRE and SAS data can be jointly used for structure calculation in ARIA/CNS and be supplement with additional information from chemical shift perturbation or biochemical data.Results will be presented on polypyrimidine-tract recognition by U2 auxiliary factor, 65 kDa (U2AF65), which reveals a novel mechanism of RNA recognition that couples RNA binding affinity to a population shift of alternate conformations.