Structure and molecular recognition of proteins linked to pre-mRNA splicing and transcriptional regulation

Gene expression is a highly regulated process in our eukaryotic cells. To accomplish tight and dynamic control, regulatory functions affect protein production at various stages. The structural and biochemical work presented in this doctoral thesis, focuses on proteins involved in pre-mRNA splicing, one of the key steps in mRNA maturation, as well as on proteins engaged in chromatin remodeling. Notably, post-translational modifications, such methylation of arginine or lysine residues, have been shown to play critical roles for these processes. Chapter 1 and 2 serves as an introduction to regulation of gene expression and to structural biology, respectively. The aim is to give an overview of the current knowledge of the fundamental regulatory processes on the way from genes to proteins. The intention is to stress molecular aspects, and to point out how different pathways are intricately interconnected. Structural biology consists of rather different and complementary techniques. Here, mainly basic aspects of nuclear magnetic resonance (NMR), and its use to study the structure, dynamics and interactions of biomolecules, are covered. Chapter 3 describes the three-dimensional structure of the so-called TSN domain of Tudor-SN, comprising an extended Tudor domain fold. The structure was determined by X-ray crystallography. NMR 15N relaxation data and residual dipolar coupling measurements show that TSN adopts a compact fold, and that the two subdomains tumble together in solution, consistent with the crystal structure. Using NMR titrations, the TSN domain was found to bind peptides containing symmetrically dimethylated arginines (sDMA). The interaction involves an aromatic cage of the Tudor domain. Dimethylarginine-modified proteins have important functions in various cellular pathways, including the spliceosome. My results suggest how Tudor-SN might interact with the spliceosome, where it has been reported to enhance assembly and splicing efficiency. Chapter 4 reports the NMR-derived solution structure of the Tudor domain of Drosophila Polycomblike (Pcl), which is involved in transcriptional regulation at the level of chromatin remodeling. It was hypothesized that Pcl may act as a targeting factor of a repressive complex by recognition of methylated histone tails through its Tudor domain. Our data, however, show that the Pcl Tudor domain has an atypical aromatic cage, which does not bind to any of the predicted putative Tudor ligands, rendering a role in targeting rather unlikely. A structural comparison to Tudor-SN highlights a hydrophobic surface patch as a potential interaction site, where binding of other domains or proteins in the repressive complex could occur. In Chapter 5, data on the recently discovered trimeric RES (retention and splicing) complex are presented. RES is involved in splicing and nuclear export of messenger mRNAs. I present a preliminary biophysical characterization, and provide evidence that the interaction of two of the components involves a novel, extended variation of a so-called UHM-ULM (U2AF Homology Motif- UHM Ligand Motif) protein-protein interaction. 15N relaxation experiments indicate that approximately 25 amino acids in the ULM peptide tightly interact with the UHM domain. Chemical shift analysis suggests that a helix is formed in the ULM peptide upon binding. NMR data has been acquired for a structural elucidation of this protein-peptide complex. Finally, Chapter 6 briefly covers additional short projects I was involved in during my PhD. Many of them included validation of small-molecule ligands that had been found to interact with their targets in different kinds of primary screens.