THE DEAH-BOX SPLICING FACTOR PRP16 UNWINDS RNA DUPLEXES IN-VITRO

Background: During pre-mRNA splicing, dynamic rearrangement of RNA sec ondary structure within the spliceosome is crucial for intron recognit ion and formation of the catalytic core. Splicing factors belonging to the DExD/DExH-box family of RNA-dependent ATPases are thought to have a central role in directing these rearrangements by unwinding RNA hel ices. Proof of this hypothesis has, however, been conspicuously lackin g. Results: Prp16 is a DEAH-box protein that functions in the second s tep of splicing in vitro. Using various RNA duplexes as substrate, we have shown that Prp16 has an ATP-dependent RNA unwinding activity. Thi s activity is independent of sequence in either the single-stranded or duplexed regions of the RNA substrate. A mutation (prp16-1) near the ATP-binding motif of Prp16 inhibits both the RNA-dependent ATPase acti vity and the ATP-dependent RNA unwinding activity. Conclusions: Our fi ndings provide strong biochemical evidence that Prp16 can disrupt a du plexed RNA structure on the spliceosome. Because the purified protein lacks sequence specificity in unwinding RNA duplexes, targeting of the unwinding activity of Prp16 in the spliceosome is likely to be determ ined by other interacting protein factors. The demonstration of unwind ing activity will also help our understanding of how the fidelity of b ranchpoint recognition is controlled by Prp16. (C) Current Biology Ltd ISSN 0960-9822.