Biochemical properties of a novel U2AF65 protein isoform generated by alternative RNA splicing.
A variety of RNA binding proteins with one or more RNA recognition (RNP-CS) motifs play essential roles in the pre-mRNA splicing process. One such factor, the U2 snRNP auxiliary factor large subunit (U2AF65), contains three RNP-CS motifs each of which is required for high affinity binding to polypyrimidine tracts. Here we report the isolation of a natural cDNA variant of human U2AF65, U2AF65 (S), which is shortened by a 12 nucleotide in frame deletion between RNP-CS2 and -CS3 motifs. A portion of the U2AF65 (S) cDNA was reported previously but was not characterized further. We observe that the U2AF65 (S) variant predominates in a variety of tissues and cell lines, and is generated together with the U2AF65 (L) form (2) by alternative 5' splice site selection from a single gene. The corresponding histidine-tagged recombinant proteins bind with similar affinities to model RNA substrates containing strong or weak polypyrimidine tracts. Both U2AF65 (S) and (L) protein isoforms reconstitute splicing activity with similar kinetic profiles in U2AF-depleted (splicing-deficient) HeLa nuclear extracts. Finally, the thermal stabilities of the protein isoforms are essentially equivalent. Thus, the presence or absence of the peptide segment, VSPP (residues 345-348), in the linker region between RNP-CS2 and -CS3 does not detract from the intrinsic RNA binding and splicing properties of the U2AF65 protein. The biological implications of alternative splicing for the function and evolution of RNA binding proteins are discussed.
Ding
F
F
Howard Hughes Medical Institute, University of Pittsburgh, Pennsylvania 15260, USA.
Hagan
J P
JP
Wang
Z
Z
Grabowski
P J
PJ
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D000072668:Splicing Factor U2AF
D018076:DNA, Complementary; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human