Mechanisms of spliceosome remodeling and splice site selection during the catalytic stage of mammalian pre-mRNA splicing

The spliceosome produces mRNAs in two sequential transesterifications – branching and exon ligation. The post-branching C complex is remodeled by the ATPase Prp16 into the C* complex.

Prp16 allows binding of the exon ligation factors Cactin and FAM32A, which stabilise docking of the 3’-splice site (3’SS) at the active site.

While in yeast Prp18 is constitutively bound to C* spliceosomes, in humans Prp18 and other exon ligation factors of unknown function appear to engage the C* spliceosome in a transcript-specific manner.

Here, I will reconstitute the C to C* transition in vitro in mammals and use biochemistry and cryo-electron microscopy to isolate and visualize novel intermediates and dissect the structural mechanisms by which Cactin, FAM32A, Prp18, and additional exon ligation factors cooperate to promote Prp16-mediated remodeling and to ensure correct 3’SS choice.

In parallel, iCLIP studies will reveal where these exon ligation factors bind across all human pre-mRNAs and how RNA sequences around the splice sites determine the association of subsets of factors on specific pre-mRNAs.

This work will elucidate how Prp16 drives remodeling of the human catalytic spliceosome and will investigate whether exon ligation factors regulate alternative splicing in a sequence-specific manner while proofreading 3'SS recognition during exon ligation.