Abstract
Brr2p is one of eight RNA helicases involved in pre-mRNA splicing. Detailed understanding of the functions of Brr2p and other spliceosomal helicases has been limited by lack of knowledge of their in vivo substrates. To address this, sites of direct Brr2p-RNA interaction were identified by in vivo UV cross-linking in budding yeast. Cross-links identified in the U4 and U6 small nuclear RNAs (snRNAs) suggest U4/U6 stem I as a Brr2p substrate during spliceosome activation. Further Brr2p cross-links were identified in loop 1 of the U5 snRNA and near splice sites and 3' ends of introns, suggesting the possibility of a previously uncharacterized function for Brr2p in the catalytic center of the spliceosome. Consistent with this, mutant brr2-G858R reduced second-step splicing efficiency and enhanced cross-linking to 3' ends of introns. Furthermore, RNA sequencing indicated preferential inhibition of splicing of introns with structured 3' ends. The Brr2-G858Rp cross-linking pattern in U6 was consistent with an open conformation for the catalytic center of the spliceosome during first-to-second-step transition. We propose a previously unsuspected function for Brr2p in driving conformational rearrangements that lead to competence for the second step of splicing.