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ABSTRACT
Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3′ splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155’s downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.
ACKNOWLEDGMENTS
We are grateful to Z. Zhou and K. Chua for useful discussions and critical reading of the manuscript and to M. Rosbash and N. Abovich for useful discussions and for the mBBP clone. We are grateful to Ron McKinney for excellent technical assistance with the two-hybrid assays of the S. pombe constructs.
This work was supported by an NIH grant to R.R. and an NIH grant (R01GM47487) and ACS grant (JFRA-545) to J.P.