PSF and p54nrb bind a conserved stem in U5 snRNA.
PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prpl8. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 3' side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.
Peng
Rui
R
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA.
Dye
Billy T
BT
Pérez
Ismael
I
Barnard
Daron C
DC
Thompson
Amanda B
AB
Patton
James G
JG
D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003429:Cross Reactions; D004268:DNA-Binding Proteins; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D050810:Octamer Transcription Factors; D000072670:PTB-Associated Splicing Factor; D011687:Purines / Q000378:metabolism; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins / Q000276:immunology; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D050810:Octamer Transcription Factors; C080779:PRP18 protein, S cerevisiae; D000072670:PTB-Associated Splicing Factor; D011687:Purines; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405485:U5 small nuclear RNA; C030985:purine