RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2.
BACKGROUND
The dynamic rearrangements of RNA structure which occur during pre-mRNA splicing are thought to be mediated by members of the DExD/H-box family of RNA-dependent ATPases. Although three DExD/H-box splicing factors have recently been shown to unwind synthetic RNA duplexes in purified systems, in no case has the natural biological substrate been identified. A duplex RNA target of particular interest is the extensive base-pairing interaction between U4 and U6 small nuclear RNAs. Because these helices must be disrupted to activate the spliceosome for catalysis, this rearrangement is believed to be tightly regulated in vivo.


RESULTS
We have immunopurified Brr2, a DEIH-box ATPase, in a native complex containing U1, U2, U5 and duplex U4/U6 small nuclear ribonucleoprotein particles (snRNPs). Addition of hydrolyzable ATP to this complex results in the disruption of U4/U6 base-pairing, and the release of free U4 and U6 snRNPs. A mutation in the helicase-like domain of Brr2 (brr2-1) prevents these RNA rearrangements. Notably, U4/U6 dissociation and release occur in the absence of exogenously added pre-mRNA.


CONCLUSIONS
Disruption of U4/U6 base-pairing in native snRNPs requires ATP hydrolysis and Brr2. This is the first assignment of a DExD/H-box splicing factor to a specific biological unwinding event. The unwinding function of Brr2 can be antagonized by the annealing activity of Prp24. We propose the existence of a dynamic cycle, uncoupled from splicing, that interconverts free and base-paired U4/U6 snRNPs.
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
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Raghunathan
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Guthrie
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D002457:Cell Extracts; D003080:Cold Temperature; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D012313:RNA / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism
D002457:Cell Extracts; D005656:Fungal Proteins; D016601:RNA-Binding Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D012313:RNA; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D020365:RNA Helicases