Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65).
U2 snRNP auxiliary factor (U2AF) promotes U2 snRNP binding to pre-mRNAs and consists of two subunits of 65 and 35 kDa, U2AF(65) and U2AF(35). U2AF(65) binds to the polypyrimidine (Py) tract upstream from the 3' splice site and plays a key role in assisting U2 snRNP recruitment. It has been proposed that U2AF(35) facilitates U2AF(65) binding through a network of protein-protein interactions with other splicing factors, but the requirement and function of U2AF(35) remain controversial. Here we show that recombinant U2AF(65) is sufficient to activate the splicing of two constitutively spliced pre-mRNAs in extracts that were chromatographically depleted of U2AF. In contrast, U2AF(65), U2AF(35), and the interaction between them are required for splicing of an immunoglobulin micro; pre-RNA containing an intron with a weak Py tract and a purine-rich exonic splicing enhancer. Remarkably, splicing activation by U2AF(35) occurs without changes in U2AF(65) cross-linking to the Py tract. These results reveal substrate-specific requirements for U2AF(35) and a novel function for this factor in pre-mRNA splicing.
Guth
S
S
Gene Expression Programme, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.
Martínez
C
C
Gaur
R K
RK
Valcárcel
J
J
D000818:Animals; D001609:Beta-Globulins / Q000235:genetics; D002457:Cell Extracts; D004268:DNA-Binding Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007075:Immunoglobulin M / Q000235:genetics; D051379:Mice; D009687:Nuclear Proteins; D011743:Pyrimidines / Q000378:metabolism*; D012322:RNA Precursors; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D000072668:Splicing Factor U2AF; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics; D014764:Viral Proteins
D001609:Beta-Globulins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D007075:Immunoglobulin M; C051096:MLTF protein, adenovirus; D009687:Nuclear Proteins; D011743:Pyrimidines; D012322:RNA Precursors; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; D014157:Transcription Factors; C076753:U2AF1 protein, human; C000607494:U2AF2 protein, human; D014764:Viral Proteins; C000607495:Zrsr1 protein, mouse