Substrate-dependent differences in U2AF requirement for splicing in adenovirus-infected cell extracts.
U2AF has been characterized as an essential splicing factor required for efficient recruitment of U2 small nuclear ribonucleoprotein to the 3'-splice site in a pre-mRNA. The U2AF65 subunit binds to the pyrimidine tract of the pre-mRNA, whereas the U2AF(35) subunit contacts the 3'-splice site AG. Here we show that U2AF35 appears to be completely dispensable for splicing in nuclear extracts prepared from adenovirus late-infected cells (Ad-NE). As a consequence, the viral IIIa and cellular IgM introns, which both have suboptimal 3'-splice sites and require U2AF35 for splicing in nuclear extracts from uninfected cells, are transformed to U2AF35-independent introns in Ad-NE. Furthermore, we present evidence that two parallel pathways of 3'-splice site recognition exist in Ad-NE. We show that the viral 52,55K intron, which has an extended pyrimidine tract, requires U2AF for activity in Ad-NE. In contrast, the IgM intron, which has a weak 3'-splice site sequence context, undergoes the first catalytic step of splicing in U2AF-depleted Ad-NE, suggesting that spliceosome assembly occurs through a novel U2AF-independent pathway in Ad-NE.
Lützelberger
Martin
M
Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden.
Backström
Ellenor
E
Akusjärvi
Göran
G
D000256:Adenoviridae / Q000235:genetics*; D000257:Adenoviridae Infections / Q000235:genetics* / Q000378:metabolism*; D002457:Cell Extracts; D006367:HeLa Cells; D006801:Humans; D007075:Immunoglobulin M / Q000235:genetics; D007438:Introns / Q000502:physiology; D009687:Nuclear Proteins / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D022821:RNA Splice Sites / Q000502:physiology; D012326:RNA Splicing / Q000502:physiology*; D012342:RNA, Small Nuclear / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D000072668:Splicing Factor U2AF
D002457:Cell Extracts; D007075:Immunoglobulin M; D009687:Nuclear Proteins; D012322:RNA Precursors; D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C405484:U2 small nuclear RNA; C076753:U2AF1 protein, human; C000607494:U2AF2 protein, human