Abstract
Step 2 of pre-mRNA splicing has characteristics that are suggestive of a 5' to 3' scanning process from the branch point to locate the 3' splice site. Specifically, the 3' splice site is almost always at the first AG downstream of the branch point even when the two elements are separated by hundreds of nucleotides. Insertion of new AGs between the branch and 3' splice site, or mutation of the wild-type 3' splice site, usually results in use of the new first AG as the 3' splice site. Finally, insertion of stable secondary structure between the branch point and 3' splice site, but distant from both elements, results in a block to step 2. We have sought to complement this circumstantial evidence by detecting physical contacts between the spliceosome and the RNA substrate in regions that are not themselves important for splicing, other than that they lie between the branch point/polypyrimidine tract and the 3' splice site. We have blocked step 2 of splicing by insertion of hairpin structures between the branch point and 3' splice site and applied methylene blue-mediated crosslinking, which is specific for protein-dsRNA interactions. Using this approach, we have detected a 116-kDa crosslinked protein that appears after step 1 of splicing with all transcripts containing a hairpin downstream of the branch point. The protein was identified as the 116-kDa U5 snRNP protein, which is a GTP-binding protein involved in step 2 of splicing. The crosslinking characteristics of U5 p116 are consistent with it having a role in locating the 3' splice site AG prior to step 2 of splicing.
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