The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro.
BACKGROUND
During pre-mRNA splicing, dynamic rearrangement of RNA secondary structure within the spliceosome is crucial for intron recognition 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 helices. Proof of this hypothesis has, however, been conspicuously lacking.


RESULTS
Prp16 is a DEAH-box protein that functions in the second step of splicing in vitro. Using various RNA duplexes as substrate, we have shown that Prp16 has an ATP-dependent RNA unwinding activity. This 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 activity and the ATP-dependent RNA unwinding activity.


CONCLUSIONS
Our findings provide strong biochemical evidence that Prp16 can disrupt a duplexed 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 determined by other interacting protein factors. The demonstration of unwinding activity will also help our understanding of how the fidelity of branchpoint recognition is controlled by Prp16.
Wang
Y
Y
Department of Biochemistry, University of California, San Francisco, California 94143-0448, USA.
Wagner
J D
JD
Guthrie
C
C
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics; D020365:RNA Helicases; D012326:RNA Splicing / Q000502:physiology*; D000072260:RNA Splicing Factors; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D012265:Ribonucleotides / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D005656:Fungal Proteins; D000072260:RNA Splicing Factors; D012330:RNA, Double-Stranded; D012342:RNA, Small Nuclear; D011993:Recombinant Fusion Proteins; D012265:Ribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; D020365:RNA Helicases