Targeting of U2AF(65) to Sites of Active Splicing in the Nucleus

U2AF(65) 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 U2AF(65). Using this antibody, we show that U2AF(65) 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 U2AF(65) 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 U2AF(35). The association of U2AF(65) with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF(65) is localized to nuclear speckles in early G(1) cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF(65) 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, U2AF(65) 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 U2AF(65) to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF(65) may play an important role in targeting this protein to spliceosomes in vivo.