Abstract
To identify splicing factors in proximity of the 5' splice site (5'SS), we followed a crosslinking profile of site-specifically modified, photoreactive RNA substrates. Upon U4/U5/U6 snRNP addition, the 5'SS RNA crosslinks in an ATP-dependent manner to U6 snRNA, an unidentified protein p27, and the 100-kDa U5 snRNP protein, a human ortholog of an ATPase/RNA helicase yPrp28p. The 5'SS:hPrp28p crosslink maps to the highly conserved TAT motif in proximity of the ATP-binding site in hPrp28p. We propose that hPrp28p acts as a helicase to unwind the 5'SS:U1 snRNA duplex, and at the same time as a 5'SS translocase, which, upon NTP-dependent conformational change, positions the 5'SS for pairing with U6 snRNA within the spliceosome. This repositioning of the 5'SS takes place regardless of whether the 5'SS is originally duplexed with U1 snRNA.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphatases / chemistry*
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Adenosine Triphosphate / chemistry
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Adenosine Triphosphate / metabolism
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Benzophenones / chemistry
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Cross-Linking Reagents
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Electrophoresis, Polyacrylamide Gel
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HeLa Cells / metabolism
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Humans
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Introns
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Oligoribonucleotides / chemistry
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Precipitin Tests
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Protein Binding
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RNA / analysis
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RNA / chemistry
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RNA Helicases / metabolism*
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RNA Precursors / metabolism
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RNA Splice Sites / genetics*
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RNA Splice Sites / physiology
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Ribonucleoprotein, U5 Small Nuclear / genetics
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Ribonucleoprotein, U5 Small Nuclear / metabolism*
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Spliceosomes / metabolism
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Trans-Splicing / genetics
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Ultraviolet Rays
Substances
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Benzophenones
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Cross-Linking Reagents
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Oligoribonucleotides
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RNA Precursors
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RNA Splice Sites
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Ribonucleoprotein, U5 Small Nuclear
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RNA
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benzophenone
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Adenosine Triphosphate
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Adenosine Triphosphatases
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RNA Helicases