Targeting of U2AF65 to Sites of Active Splicing in the Nucleus

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 additiona...

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Main Authors:	Gama-Carvalho, Margarida, Krauss, Randy D., Chiang, Lijian, Valcárcel, Juan, Green, Michael R., Carmo-Fonseca, Maria
Format:	Online
Language:	English
Published:	The Rockefeller University Press 1997
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2136214/

id	pubmed-2136214
recordtype	oai_dc
spelling	pubmed-21362142008-05-01 Targeting of U2AF65 to Sites of Active Splicing in the Nucleus Gama-Carvalho, Margarida Krauss, Randy D. Chiang, Lijian Valcárcel, Juan Green, Michael R. Carmo-Fonseca, Maria Article 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. The Rockefeller University Press 1997-06-02 /pmc/articles/PMC2136214/ /pubmed/9166400 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Gama-Carvalho, Margarida Krauss, Randy D. Chiang, Lijian Valcárcel, Juan Green, Michael R. Carmo-Fonseca, Maria
spellingShingle	Gama-Carvalho, Margarida Krauss, Randy D. Chiang, Lijian Valcárcel, Juan Green, Michael R. Carmo-Fonseca, Maria Targeting of U2AF65 to Sites of Active Splicing in the Nucleus
author_facet	Gama-Carvalho, Margarida Krauss, Randy D. Chiang, Lijian Valcárcel, Juan Green, Michael R. Carmo-Fonseca, Maria
author_sort	Gama-Carvalho, Margarida
title	Targeting of U2AF65 to Sites of Active Splicing in the Nucleus
title_short	Targeting of U2AF65 to Sites of Active Splicing in the Nucleus
title_full	Targeting of U2AF65 to Sites of Active Splicing in the Nucleus
title_fullStr	Targeting of U2AF65 to Sites of Active Splicing in the Nucleus
title_full_unstemmed	Targeting of U2AF65 to Sites of Active Splicing in the Nucleus
title_sort	targeting of u2af65 to sites of active splicing in the nucleus
description	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.
publisher	The Rockefeller University Press
publishDate	1997
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2136214/
_version_	1611419920355033088

Targeting of U2AF65 to Sites of Active Splicing in the Nucleus

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