Alternative modes of binding by U2AF65 at the polypyrimidine tract.
During initial recognition of an intron in pre-mRNA, the 3' end of the intron is bound by essential splicing factors. Notably, the consensus RNA sequences bound by these proteins are highly degenerate in humans. This raises the question of 3' splicing factor function in introns lacking canonical binding sites. Investigating the introns of the model organism Neurospora crassa revealed a different organization at the 3' end of the intron compared to most eukaryotic organisms. The predicted branch point sequences of Neurospora introns are much closer to the 3' splice site compared to those in human introns. In addition, Neurospora introns lack the canonical polypyrimidine tract found at the end of introns in most eukaryotic organisms. The large subunit of the U2 snRNP associated factor (U2AF65), which is essential for splicing of human introns and specifically recognizes the polypyrimidine tract, is also present in Neurospora. We show that Neurospora U2AF65 binds RNA with low affinity and specificity, apparently evolving with its disappearing binding site. The arginine/serine rich domain at the N-terminus of Neurospora U2AF65 regulates its RNA binding. We find that this regulated binding can be recapitulated in human U2AF65 which has been mutated to decrease both affinity and overall charge. Finally, we show that the addition of the small U2AF subunit (U2AF35) to U2AF65 with weakened RNA binding affinity significantly enhances the affinity of the resulting U2AF heterodimer.
Henscheid
Kristy L
KL
Institute of Molecular Biology, 1229 University of Oregon, Eugene, Oregon 97403, USA.
Voelker
Rodger B
RB
Berglund
J Andrew
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D000072668:Splicing Factor U2AF
D005656:Fungal Proteins; D009687:Nuclear Proteins; D012331:RNA, Fungal; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D038941:Polypyrimidine Tract-Binding Protein