Analysis of mutant phenotypes and splicing defects demonstrates functional collaboration between the large and small subunits of the essential splicing factor U2AF in vivo.
The heterodimeric splicing factor U2AF plays an important role in 3' splice site selection, but the division of labor between the two subunits in vivo remains unclear. In vitro assays led to the proposal that the human large subunit recognizes 3' splice sites with extensive polypyrimidine tracts independently of the small subunit. We report in vivo analysis demonstrating that all five domains of spU2AFLG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in Schizosaccharomyces pombe. As this is not predicted by the model for metazoan 3' splice site recognition, we sought introns for which the spU2AFLG and spU2AFSM make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3' pyrimidine tract. These and other studies performed in fission yeast support a model for 3' splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.
Webb
Christopher J
CJ
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4960, USA.
Lakhe-Reddy
Sujata
S
Romfo
Charles M
CM
Wise
Jo Ann
JA
D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002470:Cell Survival; D017124:Conserved Sequence; D019281:Dimerization; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D010649:Phenylalanine / Q000378:metabolism; D010957:Plasmids; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000072668:Splicing Factor U2AF; D013696:Temperature; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques
D009687:Nuclear Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D010649:Phenylalanine