Overexpression of the SR proteins ASF/SF2 and SC35 influences alternative splicing in vivo in diverse ways.
The SR proteins are a family of essential splicing factors highly conserved throughout metazoa. Here we examine the effects of two prototypical SR proteins, ASF/SF2 and SC35, when overexpressed by transfection in cultured cells together with plasmids encoding alternatively spliced model transcripts. As expected from past work, both proteins were found to affect alternative splicing, but differences as well as similarities in their behavior were observed. With adenovirus E1a pre-mRNA, ASF/SF2 caused shifts in alternative splicing similar to those observed previously, and the effects of mutations, in the protein and the pre-mRNA, were largely consistent with in vitro results. For example, the C-terminal RS domain was not required to alter splice site selection. SC35 overexpression also altered E1a splicing, but the pattern was distinct from that detected with ASF/SF2, indicating that the two proteins can function differently in vivo. Unexpectedly, with SV40 early pre-mRNA, overexpression of either protein resulted in a marked inhibition of splicing, with the downstream small t 5' splice site more sensitive than the upstream large T 5' splice site. This is essentially the opposite of what has been observed when the concentration of these proteins is increased in vitro. The RS domain was necessary but not sufficient for this effect. Finally, overexpression of SC35, but not ASF/SF2, resulted in substantial accumulation of the unspliced SV40 pre-mRNA, which was efficiently transported to the cytoplasm. This finding suggests that SC35 may play an unanticipated role in mRNA stability and/or transport.
D017500:Adenovirus E1A Proteins / Q000235:genetics; D017398:Alternative Splicing; D001483:Base Sequence; D015153:Blotting, Western; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins; D000068103:Serine-Arginine Splicing Factors; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
D017500:Adenovirus E1A Proteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C073288:SRSF2 protein, human; D000068103:Serine-Arginine Splicing Factors