FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35.
We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3' end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.
Chusainow
Janet
J
Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
Ajuh
Paul M
PM
Trinkle-Mulcahy
Laura
L
Sleeman
Judith E
JE
Ellenberg
Jan
J
Lamond
Angus I
AI
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D000072668:Splicing Factor U2AF; D014162:Transfection
D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human; C000607494:U2AF2 protein, human; C054562:yellow fluorescent protein, Bacteria