Structural investigation of protein splicing factors that promote the formation of the spliceosomes catalytic centre

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Pre-mRNA splicing occurs in two chemical steps that are catalysed by a large, dynamic RNA-protein complex called the spliceosome. Initially assembled in a catalytically inactive form, the spliceosome undergoes massive compositional and conformational remodelling, through which remote RNA elements are re-configured into a functional catalytic centre. This intricate process- the formation of the catalytic centre - requires the recruitment of numerous proteins. Among the most important of these is the protein Cwc2 as well as eight proteins that are pre-assembled into the so-called nineteen complex (NTC). Current data suggest that the NTC, one of the major building blocks of the spliceosome, performs several functional tasks at various stages of the splicing cycle. Despite the many biochemical and genetic studies conducted so far, an integrated picture of the function of the NTC remains elusive, mainly owing to the absence of a 3D model at atomic resolution. One of our major goals is therefore the reconstitution of the NTC and the determination of its intramolecular architecture by X-ray crystallography. In view of the difficulty of crystallising this 550-kDa complex, we would first set our sights on the crystallisation of the NTC subcomplexes NTCPrp19 and NTCSyf1.Although the NTC is crucial for the formation of the RNA-based catalytic centre, the only physical connection between the NTC and RNA is mediated by Cwc2, a protein that makes direct contact with the RNA-based catalytic centre. As the Cwc2-NTC interaction might explain how the NTC indirectly induces a functional conformation of the catalytic centre, our aim is to investigate the NTC-Cwc2 relation in atomic detail. The crystal structures of these proteins and protein complexes will be complemented by various biochemical and biophysical studies, in order to obtain deeper mechanistic insight into the function of the entire spliceosome.