Arginine/serine repeats are sufficient to constitute a splicing activation domain.
SR proteins are essential pre-mRNA splicing factors that have been shown to bind a number of exonic splicing enhancers where they function to stimulate the splicing of adjacent introns. Members of the SR protein family contain one or two N-terminal RNA binding domains, as well as a C-terminal arginine-serine (RS) rich domain. The RS domains mediate protein-protein interactions with other RS domain containing proteins and are essential for many, but not all, SR protein functions. Hybrid proteins containing an RS domain fused to the bacteriophage MS2 coat protein are sufficient to activate enhancer-dependent splicing in HeLa cell nuclear extract when bound to the pre-mRNA. Here we report progress towards determining the protein sequence requirements for RS domain function. We show that the RS domains from non-SR proteins can also function as splicing activation domains when tethered to the pre-mRNA. Truncation experiments with the RS domain of the human SR protein 9G8 identified a 29 amino acid segment, containing 26 arginine or serine residues, that is sufficient to activate splicing when fused to MS2. We also show that synthetic domains composed solely of RS dipeptides are capable of activating splicing, although their potency is proportional to their size.
Philipps
Dana
D
Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3301, USA.
Celotto
Alicia M
AM
Wang
Qiao-Qiao
QQ
Tarng
Rosa S
RS
Graveley
Brenton R
BR
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012694:Serine / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism; D000072668:Splicing Factor U2AF; D018411:Spodoptera
D036022:Capsid Proteins; D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; C490688:SNRNP70 protein, human; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human; C000607494:U2AF2 protein, human; D012694:Serine; D001120:Arginine