SR proteins can compensate for the loss of U1 snRNP functions in vitro.
SR proteins are essential splicing factors that also influence 5' splice site choice. We show that addition of excess mixed SR proteins to a HeLa in vitro splicing system stimulates utilization of a novel 5' splice site (site 125) within the intron of the standard adenovirus pre-mRNA substrate. When U1 snRNPs are debilitated by sequestering the 5' end of U1 snRNA with a 2'-O-methyl oligoribonucleotide, excess SR proteins not only rescue splicing at the normal site and site 125 but also activate yet another 5' splice site (site 47) in the adenovirus intron. One SR protein, SC35, is sufficient to exhibit the above activities. The possibility that excess SR proteins recruit residual unblocked U1 snRNPs to participate in 5' splice site recognition has been ruled out by psoralen cross-linking studies, which demonstrate that the 2'-O-methyl oligoribonucleotide effectively blocks 5' splice site/U1 interaction. Native gel analysis reveals a nearly normal splicing complex profile in the 2'-O-methyl oligoribonucleotide pretreated, SR protein-supplemented extract. These results indicate that SR proteins can replace some functions of the U1 snRNP but underscore the contribution of U1 to the fidelity of 5' splice site selection.
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut.
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D000256:Adenoviridae / Q000235:genetics; D017398:Alternative Splicing; D001483:Base Sequence; D005363:Ficusin / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology*; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000502:physiology*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000502:physiology*; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000502:physiology*
D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D000068103:Serine-Arginine Splicing Factors; D005363:Ficusin