Kinetic role for mammalian SF1/BBP in spliceosome assembly and function after polypyrimidine tract recognition by U2AF

Two sequences important for pre-mRNA splicing precede the 3' end of introns in higher eukaryotes, the branch point (BP) and the polypyrimidine (Py) tr act. Initial recognition of these signals involves cooperative binding of t he splicing factor SF1/mammalian branch point binding protein (mBBP) to the BP and of U2AF(65) to the By tract. Both factors are required for recruitm ent of the U2 small nuclear ribonucleoprotein particle (U2 snRNP) to the BP in reactions reconstituted from purified components. In contrast, extensiv e depletion of ST1/BBP in Saccharomyces cerevisiae does not compromise spli ceosome assembly or splicing significantly. As BP sequences are less conser ved in mammals, these discrepancies could reflect more stringent requiremen ts for SF1/BBP in this system. We report here that extensive depletion of S F1/mBBP from nuclear extracts of HeLa cells results in only modest reductio n of their activity in spliceosome assembly and splicing. Some of these eff ects reflect differences in the kinetics of U2 snRNP binding. Although U2AF (65) binding was reduced in the depleted extracts, the defects caused by SF 1/mBBP depletion could not be fully restored by an increase in occupancy of the Py tract by exogenously added U2AF65, arguing for a role of SF1/mBBP i n U2 snRNP recruitment distinct from promoting U2AF65 binding.