Identification of a human protein that recognizes the 3' splice site during the second step of pre-mRNA splicing.
Accurate splicing of precursor mRNAs (pre-mRNAs) requires recognition of the 5' and 3' splice sites at the intron boundaries. Interactions between several splicing factors and the 5' splice site, which occur prior to the first step of splicing, have been well described. In contrast, recognition of the 3' splice site, which is cleaved during the second catalytic step, is poorly understood, particularly in higher eukaryotes. Here, using site-specific photo-crosslinking, we find that the conserved AG dinucleotide at the 3' splice site is contacted specifically by a 70 kDa polypeptide (p70). The p70-3' splice site crosslink has kinetics and biochemical requirements similar to those of splicing, was detected only in the mature spliceosome and occurs subsequent to the first step. Thus, p70 has all the properties expected of a factor that functionally interacts with the 3' splice site during the second step of splicing. Using antisera to various known splicing factors, we find that p70 corresponds to a previously reported 69 kDa protein of unknown function associated with the Sm core domain of spliceosomal small nuclear ribonucleoproteins.
Wu
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Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605, USA.
Green
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D003432:Cross-Linking Reagents / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D015151:Immunoblotting; D007438:Introns; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D017381:Spliceosomes / Q000378:metabolism*; D013388:Succinimides / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D015226:Dinucleoside Phosphates; D009687:Nuclear Proteins; D010455:Peptides; D012322:RNA Precursors; D013388:Succinimides; C015752:N-chlorosuccinimide