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The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA.

MPG-Autoren
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Mozaffari-Jovin,  S.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

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Hsiao,  H. H.
Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society;

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Will,  C. L.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

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Urlaub,  H.
Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society;

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Lührmann,  R.
Department of Cellular Biochemistry, MPI for biophysical chemistry, Max Planck Society;

Volltexte (frei zugänglich)

1570032.pdf
(Verlagsversion), 947KB

Ergänzendes Material (frei zugänglich)

1570032-Suppl.doc
(Ergänzendes Material), 9MB

Zitation

Mozaffari-Jovin, S., Santos, K. F., Hsiao, H. H., Will, C. L., Urlaub, H., Wahl, M. C., et al. (2012). The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA. Genes and Development, 26(23), 2422-2434. doi:10.1101/gad.200949.112.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0010-25FA-F
Zusammenfassung
The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2's interaction with the U4 snRNA. Our data reveal a novel mechanism whereby Prp8 negatively regulates Brr2 and potentially prevents premature U4/U6 unwinding during splicing. They also support the idea that the RH domain acts as a platform for the exchange of U6 snRNA for U1 at the 5′ splice site. Our results provide insights into the mechanism whereby Brr2 unwinds U4/U6 and show how this activity is potentially regulated prior to spliceosome activation.