title: Targeting of U2AF65 to sites of active splicing in the nucleus.
abstract: U2AF65 is an essential splicing factor that promotes binding of U2 small nuclear (sn)RNP at the pre-mRNA branchpoint. Here we describe a novel monoclonal antibody that reacts specifically with U2AF65. Using this antibody, we show that U2AF65 is diffusely distributed in the nucleoplasm with additional concentration in nuclear speckles, which represent subnuclear compartments enriched in splicing snRNPs and other splicing factors. Furthermore, transient expression assays using epitope-tagged deletion mutants of U2AF65 indicate that targeting of the protein to nuclear speckles is not affected by removing either the RNA binding domain, the RS domain, or the region required for interaction with U2AF35. The association of U2AF65 with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF65 is localized to nuclear speckles in early G1 cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF65 in speckles is independent of the presence of pre-mRNA in the nucleus, which is consistent with the idea that speckles represent storage sites for inactive splicing factors. After adenovirus infection, U2AF65 redistributes from the speckles and is prefferentially detected at sites of viral transcription. By combining adenoviral infection with transient expression of deletion mutants, we show a specific requirement of the RS domain for recruitment of U2AF65 to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.
authors.affiliation: Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal.
authors.forename: M
authors.initials: M
authors.lastname: Gama-Carvalho
authors.forename: R D
authors.initials: RD
authors.lastname: Krauss
authors.forename: L
authors.initials: L
authors.lastname: Chiang
authors.forename: J
authors.initials: J
authors.lastname: Valcárcel
authors.forename: M R
authors.initials: MR
authors.lastname: Green
authors.forename: M
authors.initials: M
authors.lastname: Carmo-Fonseca
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D005260:Female; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008938:Mitosis / Q000502:physiology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D012326:RNA Splicing / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D000072668:Splicing Factor U2AF
chemical_list: D000911:Antibodies, Monoclonal; D002843:Chromatin; D009687:Nuclear Proteins; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; C000607495:Zrsr1 protein, mouse