The splicing factor U2AF small subunit is functionally conserved between fission yeast and humans.
The small subunit of U2AF, which functions in 3' splice site recognition, is more highly conserved than its heterodimeric partner yet is less thoroughly investigated. Remarkably, we find that the small subunit of Schizosaccharomyces pombe U2AF (U2AF(SM)) can be replaced in vivo by its human counterpart, demonstrating that the conservation extends to function. Precursor mRNAs accumulate in S. pombe following U2AF(SM) depletion in a time frame consistent with a role in splicing. A comprehensive mutational analysis reveals that all three conserved domains are required for viability. Notably, however, a tryptophan in the pseudo-RNA recognition motif implicated in a key contact with the large subunit by crystallographic data is dispensable whereas amino acids implicated in RNA recognition are critical. Mutagenesis of the two zinc-binding domains demonstrates that they are neither equivalent nor redundant. Finally, two- and three-hybrid analyses indicate that mutations with effects on large-subunit interactions are rare whereas virtually all alleles tested diminished RNA binding by the heterodimer. In addition to demonstrating extraordinary conservation of U2AF small-subunit function, these results provide new insights into the roles of individual domains and residues.
Webb
Christopher J
CJ
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4960, USA.
Wise
Jo Ann
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012326:RNA Splicing; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D000072668:Splicing Factor U2AF; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D009687:Nuclear Proteins; D021122:Protein Subunits; D012261:Ribonucleoproteins; D029702:Schizosaccharomyces pombe Proteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human