Talk abstracts

Talk on Friday 02:00-02:12pm submitted by Sandy Tretbar

Conformational control of the U2 snRNA by Cus2p

Sandy Tretbar (a University of Wisconsin-Madison, Biochemistry Department, 433 Babcock Dr, Madison, WI, 53706), Aaron A. Hoskins (a University of Wisconsin-Madison, Biochemistry Department, 433 Babcock Dr, Madison, WI, 53706)

Tremendous RNA rearrangements occur during yeast spliceosome assembly and catalysis. These include both intermolecular rearrangements between different snRNAs and/or the pre-mRNA substrate or intramolecular conformational dynamics within an individual snRNA. One example of intermolecular dynamics involves structural transitions within the U2 snRNA. Two mutually exclusive conformations of the U2 snRNA promote either binding to the pre-mRNA branchsite (stem IIa) or the catalytic steps of splicing (stem IIc). Two key players in this process are yeast Cus2p and Prp5p. Prp5p hydrolyzes ATP to promote duplex formation between the U2 snRNA and the pre-mRNA; however, this function is only required in the presence of Cus2p. The detailed biochemical relationship between Cus2p, Prp5p, and U2 snRNA conformation remains unexplored.
Using an in vitro model system, we show that Cus2p binds to the stem II region of the U2 snRNA (U2 core) with a clear preference for binding to the stem IIa conformation. Prp5p appears to remodel the Cus2p/U2 core interaction resulting in formation of a Prp5/U2 core complex. Preliminary single molecule FRET analysis reveals that U2 core RNA is dynamic and that Cus2p stabilizes a particular FRET conformation of the RNA consistent with stem IIa formation. We hypothesize that Cus2p may function as a chaperone to conformationally select the stem IIa form of the U2 snRNA. Cus2p then holds the snRNA in this conformation until arrival of Prp5p in preparation for branchsite duplex formation. Significantly, this demonstrates how two different classes of RNA binding proteins (the RRMs of Cus2p and the DEAD-box of Prp5p) can work collaboratively to regulate and control RNA conformation.

Keywords: U2 snRNA, Cus2p, Prp5p