title: U1 snRNP-ASF/SF2 interaction and 5' splice site recognition: characterization of required elements.
abstract: Members of the SR family of proteins, can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. We have previously shown that purified U1 snRNP and ASF/SF2 form a ternary complex with pre-mRNA, which is dependent on a functional 5' splice site. In this manuscript we dissect the requirements for the formation of this complex. Sequences in the pre-mRNA, domains in ASF/SF2 and components of the U1 snRNP particle are shown to be required for complex formation. We had shown that sequences at the 5' splice site of PIP7. A are necessary and now we show these are sufficient for complex formation. Furthermore, we show that one functional RNA binding domain and the RS domain are both required for ASF/SF2 to participate in complex formation. The RNA binding domains were redundant in this assay, suggesting that either domain can interact with the pre-messenger RNA. Finally, our experiments show no function for the U1-specific A protein in complex formation, whereas a function for U1-specific C protein was strongly suggested. The study of the earliest interactions between pre-mRNA and splicing factors suggests a model for 5' splice site recognition.
authors.affiliation: Department of Molecular Cancer Biology, Levine Science Research Center, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: S F
authors.initials: SF
authors.lastname: Jamison
authors.forename: Z
authors.initials: Z
authors.lastname: Pasman
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: C
authors.initials: C
authors.lastname: Will
authors.forename: R
authors.initials: R
authors.lastname: Lührmann
authors.forename: J L
authors.initials: JL
authors.lastname: Manley
authors.forename: M A
authors.initials: MA
authors.lastname: Garcia-Blanco
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics* / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D000068103:Serine-Arginine Splicing Factors