U1 SNRNP-ASF SF2 INTERACTION AND 5'-SPLICE-SITE RECOGNITION - CHARACTERIZATION OF REQUIRED ELEMENTS/

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 pre viously shown that purified U1 snRNP and ASF/SF2 form a ternary comple x 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 formati on, We had shown that sequences at the 5' splice site of PIP7.A are ne cessary and now we show these are sufficient for complex formation. Fu rthermore, we show that one functional RNA binding domain and the RS d omain are both required for ASF/SF2 to participate in complex formatio n, The RNA binding domains were redundant in this assay, suggesting th at 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.