U2 snRNA Conformation is Regulated by Cus2 to Facilitate Dead-Box Protein Loading

Abstract

The spliceosome undergoes dynamic changes in composition and conformation during assembly and splicing. We are interested in studying how the spliceosomal U2 snRNP loads onto pre-mRNA branchsites during assembly. This involves multiple conformations of the U2 snRNA (stem IIa vs. stem IIc), the Prp5 DEAD-box protein, and the Cus2 RRM protein. Our results show that Cus2 binds to WT or stem IIa stabilized model RNAs but not to stem IIc. In contrast, Prp5 binds indiscriminately to all three types of U2 RNA and each equally stimulates Prp5's ATPase. Prp5 appears to either displace or out compete Cus2 for U2 RNA binding in an ATP-independent process. Using single molecule FRET, we show the U2 core RNA is dynamic with interconverting populations of high- (likely stem IIc) and mid-FRET states (likely stem IIa). Addition of Cus2 depletes the high-FRET state, consistent with conformational selection of U2 stem IIa by Cus2. Together, our data support spliceosomal proteins acting in concert to facilitate loading of the DEAD-box onto a particular RNA structure. This represents a new paradigm for protein:RNA interactions that is distinct from ordered binding of structure specific RNA-binding proteins (i.e., ribosome assembly) or from positional control of DEAD-box protein loading during exon junction complex formation.