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© The Rockefeller University Press, 0021-9525/1997//975 $5.00
The Journal of Cell Biology, Volume 137, Number 5, , 1997 975-987

Targeting of U2AF⁶⁵ to Sites of Active Splicing in the Nucleus

Margarida Gama-Carvalho^*, Randy D. Krauss, Lijian Chiang, Juan Valcárcel, Michael R. Green, and Maria Carmo-Fonseca^*

^* Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal; and Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, Massachusetts 01605

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.

Randy D. Krauss's present address is Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118-2394.

Juan Valcárcel's present address is Gene Expression Program, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.

Please address all correspondence to Dr. Maria Carmo-Fonseca, Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal. Tel.: (351) 1-7934340; Fax: (351) 1-7951780.

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