Spatial mapping of splicing factor complexes involved in exon and intron definition.
We have analyzed the interaction between serine/arginine-rich (SR) proteins and splicing components that recognize either the 5' or 3' splice site. Previously, these interactions have been extensively characterized biochemically and are critical for both intron and exon definition. We use fluorescence resonance energy transfer (FRET) microscopy to identify interactions of individual SR proteins with the U1 small nuclear ribonucleoprotein (snRNP)-associated 70-kD protein (U1 70K) and with the small subunit of the U2 snRNP auxiliary factor (U2AF35) in live-cell nuclei. We find that these interactions occur in the presence of RNA polymerase II inhibitors, demonstrating that they are not exclusively cotranscriptional. Using FRET imaging by means of fluorescence lifetime imaging microscopy (FLIM), we map these interactions to specific sites in the nucleus. The FLIM data also reveal a previously unknown interaction between HCC1, a factor related to U2AF65, with both subunits of U2AF. Spatial mapping using FLIM-FRET reveals differences in splicing factors interactions within complexes located in separate subnuclear domains.
Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, UK.
Jonathan D
JD
Ellis
David
D
Llères
Marco
M
Denegri
Angus I
AI
Lamond
Javier F
JF
Cáceres
D002460:Cell Line; D002470:Cell Survival; D019281:Dimerization; D005091:Exons / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007438:Introns / Q000235:genetics*; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D000072668:Splicing Factor U2AF; D014158:Transcription, Genetic
C084105:HCC1 autoantigen; D009687:Nuclear Proteins; D021122:Protein Subunits; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; C490688:SNRNP70 protein, human; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human; C000607494:U2AF2 protein, human