Global Promotion of Alternative Internal Exon Usage by mRNA 3' End Formation Factors.
The mechanisms that regulate alternative precursor mRNA (pre-mRNA) splicing are largely unknown. Here, we perform an RNAi screen to identify factors required for alternative splicing regulation by RBFOX2, an RNA-binding protein that promotes either exon inclusion or exclusion. Unexpectedly, we find that two mRNA 3' end formation factors, cleavage and polyadenylation specificity factor (CPSF) and SYMPK, are RBFOX2 cofactors for both inclusion and exclusion of internal exons. RBFOX2 interacts with CPSF/SYMPK and recruits it to the pre-mRNA. RBFOX2 and CPSF/SYMPK then function together to regulate binding of the early intron recognition factors U2AF and U1 small nuclear ribonucleoprotein particle (snRNP). Genome-wide analysis reveals that CPSF also mediates alternative splicing of many internal exons in the absence of RBFOX2. Accordingly, we show that CPSF/SYMPK is also a cofactor of NOVA2 and heterologous nuclear ribonucleoprotein A1 (HNRNPA1), RNA-binding proteins that also regulate alternative splicing. Collectively, our results reveal an unanticipated role for mRNA 3' end formation factors in global promotion of alternative splicing.
Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Ashish
A
Misra
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Jianhong
J
Ou
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA; Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Lihua J
LJ
Zhu
Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address: michael.green@umassmed.edu.
Michael R
MR
Green
D017398:Alternative Splicing; D001483:Base Sequence; D039223:Cleavage And Polyadenylation Specificity Factor / Q000502:physiology*; D005091:Exons; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D017423:Sequence Analysis, RNA; D000072668:Splicing Factor U2AF
D039223:Cleavage And Polyadenylation Specificity Factor; D009687:Nuclear Proteins; C454924:RBFOX2 protein, human; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; C101043:SYMPK protein, human; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D049452:Green Fluorescent Proteins