Identification of nuclear and cytoplasmic mRNA targets for the shuttling protein SF2/ASF.
The serine and arginine-rich protein family (SR proteins) are highly conserved regulators of pre-mRNA splicing. SF2/ASF, a prototype member of the SR protein family, is a multifunctional RNA binding protein with roles in pre-mRNA splicing, mRNA export and mRNA translation. These observations suggest the intriguing hypothesis that SF2/ASF may couple splicing and translation of specific mRNA targets in vivo. Unfortunately the paucity of endogenous mRNA targets for SF2/ASF has hindered testing of this hypothesis. Here, we identify endogenous mRNAs directly cross-linked to SF2/ASF in different sub-cellular compartments. Cross-Linking Immunoprecipitation (CLIP) captures the in situ specificity of protein-RNA interaction and allows for the simultaneous identification of endogenous RNA targets as well as the locations of binding sites within the RNA transcript. Using the CLIP method we identified 326 binding sites for SF2/ASF in RNA transcripts from 180 protein coding genes. A purine-rich consensus motif was identified in binding sites located within exon sequences but not introns. Furthermore, 72 binding sites were occupied by SF2/ASF in different sub-cellular fractions suggesting that these binding sites may influence the splicing or translational control of endogenous mRNA targets. We demonstrate that ectopic expression of SF2/ASF regulates the splicing and polysome association of transcripts derived from the SFRS1, PABC1, NETO2 and ENSA genes. Taken together the data presented here indicate that SF2/ASF has the capacity to co-regulate the nuclear and cytoplasmic processing of specific mRNAs and provide further evidence that the nuclear history of an mRNA may influence its cytoplasmic fate.
Sanford
Jeremy R
JR
MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK. sanford@biology.ucsc.edu
Coutinho
Pedro
P
Hackett
Jamie A
JA
Wang
Xin
X
Ranahan
William
W
Caceres
Javier F
JF
D017398:Alternative Splicing; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D009687:Nuclear Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000068103:Serine-Arginine Splicing Factors
D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors