title: SC35-mediated reconstitution of splicing in U2AF-depleted nuclear extract.
abstract: Assembly of the mammalian spliceosome is known to proceed in an ordered fashion through several discrete complexes, but the mechanism of this assembly process may not be universal. In an early step, pre-mRNAs are committed to the splicing pathway through association with U1 small nuclear ribonucleoprotein (snRNP) and non-snRNP splicing factors, including U2AF and members of the SR protein family. As a means of studying the steps of spliceosome assembly, we have prepared HeLa nuclear extracts specifically depleted of the splicing factor U2AF. Surprisingly, the SR protein SC35 can functionally substitute for U2AF65 in the reconstitution of pre-mRNA splicing in U2AF-depleted extracts. This reconstitution is substrate-specific and is reminiscent of the SC35-mediated reconstitution of splicing in extracts depleted of U1 snRNP. However, SC35 reconstitution of splicing in U2AF-depleted extracts is dependent on the presence of functional U1 snRNP. These observations suggest that there are at least three distinguishable mechanisms for the binding of U2 snRNP to the pre-mRNA, including U2AF-dependent and -independent pathways.
authors.affiliation: Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge 02139-4307, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: MacMillan
authors.forename: P S
authors.initials: PS
authors.lastname: McCaw
authors.forename: J D
authors.initials: JD
authors.lastname: Crispino
authors.forename: P A
authors.initials: PA
authors.lastname: Sharp
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000378:metabolism*; D000072668:Splicing Factor U2AF
chemical_list: D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors