CROSS-LINKING OF THE U5 SNRNP-SPECIFIC 116-KDA PROTEIN TO RNA HAIRPINS THAT BLOCK STEP 2 OF SPLICING

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' sp lice site. Specifically, the 3' splice site is almost always at the fi rst AG downstream of the branch point even when the two elements are s eparated 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. Fi nally, insertion of stable secondary structure between the branch poin t and 3' splice site, but distant from both elements, results in a blo ck to step 2. We have sought to complement this circumstantial evidenc e by detecting physical contacts between the spliceosome and the RNA s ubstrate in regions that are not themselves important for splicing, ot her than that they lie between the branch point/polypyrimidine tract a nd 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 p rotein-dsRNA interactions. Using this approach, we have detected a 116 -kDa crosslinked protein that appears after step 1 of splicing with al l transcripts containing a hairpin downstream of the branch point. The protein was identified as the 116-kDa U5 snRNP protein, which is a GT P-binding protein involved in step 2 of splicing. The crosslinking cha racteristics of U5 pile are consistent with it having a role in locati ng the 3' splice site AG prior to step 2 of splicing.