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Sequential recognition of the pre-mRNA branch point by U2AF65 and a novel spliceosome-associated 28-kDa protein
R K Gaur1, J Valcárcel, M R Green
1Howard Hughes Medical Institute Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605, USA.
Abstract:
Splicing of pre-mRNAs occurs via a lariat intermediate in which an intronic adenosine, embedded within a branch point sequence, forms a 2',5'-phosphodiester bond (RNA branch) with the 5' end of the intron. How the branch point is recognized and activated remains largely unknown. Using site-specific photochemical cross-linking, we have identified two proteins that specifically interact with the branch point during the splicing reaction. U2AF65, an essential splicing factor that binds to the adjacent polypyrimidine tract, crosslinks to the branch point at the earliest stage of spliceosome formation in an ATP-independent manner. A novel 28-kDa protein, which is a constituent of the mature spliceosome, contacts the branch point after the first catalytic step. Our results indicate that the branch point is sequentially recognized by distinct splicing factors in the course of the splicing reaction.
Insights
Researchers identified two proteins that bind to the RNA branch point during pre-mRNA splicing. This reveals sequential recognition of the branch point by splicing factors, U2AF65 and a novel 28-kDa protein, during spliceosome assembly.
Area of Science:
- Molecular Biology
- RNA Splicing Mechanisms
Background:
- Pre-mRNA splicing involves a lariat intermediate with a 2',5'-phosphodiester bond at the branch point.
- Recognition and activation of the branch point sequence are poorly understood.
Purpose of the Study:
- To identify proteins interacting with the pre-mRNA branch point during splicing.
- To elucidate the mechanism of branch point recognition and activation.
Main Methods:
- Site-specific photochemical cross-linking was employed to capture protein-RNA interactions.
- Analysis of protein binding at different stages of spliceosome formation.
Main Results:
- U2AF65, a known splicing factor, crosslinks to the branch point early in spliceosome formation, independently of ATP.
- A novel 28-kDa protein interacts with the branch point after the first catalytic step of splicing.
- These findings demonstrate sequential protein recognition of the branch point.
Conclusions:
- The branch point is recognized by distinct splicing factors in a stepwise manner.
- U2AF65 and the novel 28-kDa protein play crucial roles in branch point recognition during splicing.