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Targeting of U2AF65 to sites of active splicing in the nucleus.

Gama-Carvalho M, Krauss RD, Chiang L, Valcárcel J, Green MR, Carmo-Fonseca M - J. Cell Biol. (1997)

Bottom Line: 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.This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal.

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.

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Epitope mapping of U2AF65. (A) Diagram showing the  reactivity of mAb MC3 with different GST–U2AF65 fusion proteins as determined by immunoblot analysis. The grey bars represent the U2AF65 fragment fused to the GST protein. Hatched regions represent internal deletions of U2AF65. (B) Diagram  showing the mapped epitope (amino acids 138–161, bar) in relation to the functional organization of U2AF65. The mapped region overlaps with the beginning of the RNA binding domain  (amino acids 151–462).
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Figure 2: Epitope mapping of U2AF65. (A) Diagram showing the reactivity of mAb MC3 with different GST–U2AF65 fusion proteins as determined by immunoblot analysis. The grey bars represent the U2AF65 fragment fused to the GST protein. Hatched regions represent internal deletions of U2AF65. (B) Diagram showing the mapped epitope (amino acids 138–161, bar) in relation to the functional organization of U2AF65. The mapped region overlaps with the beginning of the RNA binding domain (amino acids 151–462).

Mentions: Using a series of GST-U2AF65 deletion mutants (Valcárcel et al., 1996), we conclude that the epitope recognized by mAb MC3 maps between amino acids 138 and 161 (Fig. 2).


Targeting of U2AF65 to sites of active splicing in the nucleus.

Gama-Carvalho M, Krauss RD, Chiang L, Valcárcel J, Green MR, Carmo-Fonseca M - J. Cell Biol. (1997)

Epitope mapping of U2AF65. (A) Diagram showing the  reactivity of mAb MC3 with different GST–U2AF65 fusion proteins as determined by immunoblot analysis. The grey bars represent the U2AF65 fragment fused to the GST protein. Hatched regions represent internal deletions of U2AF65. (B) Diagram  showing the mapped epitope (amino acids 138–161, bar) in relation to the functional organization of U2AF65. The mapped region overlaps with the beginning of the RNA binding domain  (amino acids 151–462).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2136214&req=5

Figure 2: Epitope mapping of U2AF65. (A) Diagram showing the reactivity of mAb MC3 with different GST–U2AF65 fusion proteins as determined by immunoblot analysis. The grey bars represent the U2AF65 fragment fused to the GST protein. Hatched regions represent internal deletions of U2AF65. (B) Diagram showing the mapped epitope (amino acids 138–161, bar) in relation to the functional organization of U2AF65. The mapped region overlaps with the beginning of the RNA binding domain (amino acids 151–462).
Mentions: Using a series of GST-U2AF65 deletion mutants (Valcárcel et al., 1996), we conclude that the epitope recognized by mAb MC3 maps between amino acids 138 and 161 (Fig. 2).

Bottom Line: 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.This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Histology and Embryology, Faculty of Medicine, University of Lisbon, 1699 Lisboa Codex, Portugal.

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.

Show MeSH
Related in: MedlinePlus