Abstract
Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-type spliceosomes. This protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded protein precipitated the 14 kDa protein cross-linked to the branch adenosine, confirming the identity of the p14 cDNA. A combination of immunoblotting, protein microsequencing and immunoprecipitation revealed that p14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. p14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that p14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.
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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Amino Acid Sequence
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Animals
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Binding Sites
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Cloning, Molecular
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Conserved Sequence
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Cross-Linking Reagents
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Evolution, Molecular
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HeLa Cells
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Humans
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Molecular Sequence Data
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Peptides / classification
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Peptides / genetics
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Peptides / metabolism
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Phosphoproteins / metabolism*
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RNA Precursors / metabolism*
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RNA Splicing Factors
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RNA Splicing*
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RNA-Binding Proteins / classification
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism*
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Ribonucleoprotein, U2 Small Nuclear / classification
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Ribonucleoprotein, U2 Small Nuclear / genetics
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Ribonucleoprotein, U2 Small Nuclear / metabolism*
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Ribonucleoproteins, Small Nuclear / classification
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Ribonucleoproteins, Small Nuclear / genetics
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Ribonucleoproteins, Small Nuclear / metabolism*
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Sequence Homology, Amino Acid
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Spliceosomes / metabolism
Substances
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Cross-Linking Reagents
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Peptides
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Phosphoproteins
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RNA Precursors
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RNA Splicing Factors
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RNA-Binding Proteins
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Ribonucleoprotein, U2 Small Nuclear
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Ribonucleoproteins, Small Nuclear
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SF3B1 protein, human
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U11 small nuclear ribonucleoprotein, human
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U11-U12 small nuclear ribonucleoprotein
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U2 small nuclear ribonucleoprotein p14