Higher order structure and remodelling of the spliceosomal RNA network

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Nuclear pre-mRNA splicing is catalysed by the spliceosome, which consists of the snRNPs U1, U2, U4/U6 and U5, and non-snRNP proteins. During spliceosome assembly, a complex and highly dynamic RNA network is formed between the pre-mRNA and snRNAs. One major goal of our project is to identify higher order interactions of the snRNAs in the active centre of the spliceosome using site-directed UV crosslinkung and hydroxyl radical probing approaches. As a general experimental strategy, we will incorporate into spliceosomes snRNA or pre-mRNA molecules that contain either diverse photo-sensitive crosslinkers or the hydroxyl radical generating reagent Fe(II)-BABE (1-(pbromoacetamidobenzyl) EDTA) at defined positions. In addition we will perform structure probing of the RNAs using chemical probes and purified, native spliceosomes. We will also investigate the role of the modified nucleotides of the U2RNA in splicing. As a second major goal, we plan to elucidate by site-directed UV crosslinking approaches snRNA-protein interactions at the catalytic core of the spliceosome. This will allow us not only to identify proteins which are potentially involved in splicing catalysis, but also to gather important information about the remodelling of the spliceosomal RNP structure.