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ZNF265--a novel spliceosomal protein able to induce alternative splicing.

Adams DJ, van der Weyden L, Mayeda A, Stamm S, Morris BJ, Rasko JE - J. Cell Biol. (2001)

Bottom Line: Transfection of HT-1080 cells with ZNF265-EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain.Alignment with other RS domain-containing proteins revealed a high degree of SR dipeptide conservation.These data show that ZNF265 functions as a novel component of the mRNA processing machinery.

View Article: PubMed Central - PubMed

Affiliation: The University of Sydney, Basic & Clinical Genomics Laboratory, Department of Physiology and Institute for Biomedical Research, Sydney, NSW 2006, Australia.

ABSTRACT
The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-beta1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF(35). Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265-EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain-containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery.

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Interaction of ZNF265 with the essential spliceosomal factors U1-70K and U2AF35. (A) Activation-domain plasmids (pACT) p80 coilin (1), 9G8 (2), SC35 (3), SRp20 (4), SRp30c (5), SRp40 (6), U1-70K (7), U2AF65 (8), U2AF35 (9), WT1 (10), ASF/SF2 (11), and negative control SNF4 (12), were transformed into AH109 yeast containing the pGBK-ZNF265 binding-domain plasmid, cultured on SD-L-W-A-H plates, and transferred to filters. The ability of the yeast containing pGBK-ZNF265 and either pACT-U1-70K or pACT-U2AF35 to grow on autotrophic media (I: brown) and produce β-gal (II: blue) was observed. The inability of yeast containing pACT plasmids alone to produce β-gal (III) was shown as a control. (B) Results of coimmunoprecipitation performed using anti-ZNF265 to pulldown U1-70K and U2AF35 in association with ZNF265 from HeLa cell nuclear extracts. Immunoprecipitates were analyzed by Western blotting using antibodies against U1-70K or U2AF35 (arrow points to band of predicted size). (C) Relative strength of interaction of ZNF265 with U2AF35 or U1-70K, shown as β-gal activity relative to that for interaction of T-antigen with p53 (mean ± SE, n = 4, *P < 0.0001). (Control) β-gal activity of AH109 yeast containing pGBK-ZNF265 alone.
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fig3: Interaction of ZNF265 with the essential spliceosomal factors U1-70K and U2AF35. (A) Activation-domain plasmids (pACT) p80 coilin (1), 9G8 (2), SC35 (3), SRp20 (4), SRp30c (5), SRp40 (6), U1-70K (7), U2AF65 (8), U2AF35 (9), WT1 (10), ASF/SF2 (11), and negative control SNF4 (12), were transformed into AH109 yeast containing the pGBK-ZNF265 binding-domain plasmid, cultured on SD-L-W-A-H plates, and transferred to filters. The ability of the yeast containing pGBK-ZNF265 and either pACT-U1-70K or pACT-U2AF35 to grow on autotrophic media (I: brown) and produce β-gal (II: blue) was observed. The inability of yeast containing pACT plasmids alone to produce β-gal (III) was shown as a control. (B) Results of coimmunoprecipitation performed using anti-ZNF265 to pulldown U1-70K and U2AF35 in association with ZNF265 from HeLa cell nuclear extracts. Immunoprecipitates were analyzed by Western blotting using antibodies against U1-70K or U2AF35 (arrow points to band of predicted size). (C) Relative strength of interaction of ZNF265 with U2AF35 or U1-70K, shown as β-gal activity relative to that for interaction of T-antigen with p53 (mean ± SE, n = 4, *P < 0.0001). (Control) β-gal activity of AH109 yeast containing pGBK-ZNF265 alone.

Mentions: To test whether ZNF265 could interact with other RS domain–containing proteins we conducted a yeast two-hybrid screen against representative spliceosomal proteins that included many with RS domains, namely U1-70K, U2AF35, U2AF65, SC35, p80 Coilin, WT1, 9G8, SF2/ASF, SRp20, SRp30c, and SRp40. Interaction was seen with U1-70K and U2AF35, as determined by growth on SD-L-W-A-H plates, and the production of a blue precipitate on a β-gal filter assay (Fig. 3 A). Interaction of ZNF265 with U1-70K and U2AF35 was confirmed by coimmunoprecipitation (Fig. 3 B). Liquid β-gal assay, which provides a semiquantitative estimation of interaction strength, showed that ZNF265 interacted more strongly with U1-70K than with U2AF35 (Fig. 3 C). A U1-70K cDNA clone was also isolated in a yeast two-hybrid screen against a human fetal brain cDNA library using ZNF265 as “bait.” Analysis of this clone revealed that residues 180–437 of U1-70K were responsible for mediating the interaction of U1-70K with the RS domain of ZNF265 (unpublished data). It is notable that this region contains the residues necessary for the binding of SF2/ASF to U1-70K (Cao and Garcia-Blanco, 1998). Several cDNA clones for the SR protein kinase Clk1 were also isolated from this screen. Because ZNF265 contains the Clk1 consensus phosphorylation site R/KXR/KXR/KXSXXR (Colwill et al., 1996; Moeslein et al., 1999), there may be a role for phosphorylation in the regulation of ZNF265.


ZNF265--a novel spliceosomal protein able to induce alternative splicing.

Adams DJ, van der Weyden L, Mayeda A, Stamm S, Morris BJ, Rasko JE - J. Cell Biol. (2001)

Interaction of ZNF265 with the essential spliceosomal factors U1-70K and U2AF35. (A) Activation-domain plasmids (pACT) p80 coilin (1), 9G8 (2), SC35 (3), SRp20 (4), SRp30c (5), SRp40 (6), U1-70K (7), U2AF65 (8), U2AF35 (9), WT1 (10), ASF/SF2 (11), and negative control SNF4 (12), were transformed into AH109 yeast containing the pGBK-ZNF265 binding-domain plasmid, cultured on SD-L-W-A-H plates, and transferred to filters. The ability of the yeast containing pGBK-ZNF265 and either pACT-U1-70K or pACT-U2AF35 to grow on autotrophic media (I: brown) and produce β-gal (II: blue) was observed. The inability of yeast containing pACT plasmids alone to produce β-gal (III) was shown as a control. (B) Results of coimmunoprecipitation performed using anti-ZNF265 to pulldown U1-70K and U2AF35 in association with ZNF265 from HeLa cell nuclear extracts. Immunoprecipitates were analyzed by Western blotting using antibodies against U1-70K or U2AF35 (arrow points to band of predicted size). (C) Relative strength of interaction of ZNF265 with U2AF35 or U1-70K, shown as β-gal activity relative to that for interaction of T-antigen with p53 (mean ± SE, n = 4, *P < 0.0001). (Control) β-gal activity of AH109 yeast containing pGBK-ZNF265 alone.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196870&req=5

fig3: Interaction of ZNF265 with the essential spliceosomal factors U1-70K and U2AF35. (A) Activation-domain plasmids (pACT) p80 coilin (1), 9G8 (2), SC35 (3), SRp20 (4), SRp30c (5), SRp40 (6), U1-70K (7), U2AF65 (8), U2AF35 (9), WT1 (10), ASF/SF2 (11), and negative control SNF4 (12), were transformed into AH109 yeast containing the pGBK-ZNF265 binding-domain plasmid, cultured on SD-L-W-A-H plates, and transferred to filters. The ability of the yeast containing pGBK-ZNF265 and either pACT-U1-70K or pACT-U2AF35 to grow on autotrophic media (I: brown) and produce β-gal (II: blue) was observed. The inability of yeast containing pACT plasmids alone to produce β-gal (III) was shown as a control. (B) Results of coimmunoprecipitation performed using anti-ZNF265 to pulldown U1-70K and U2AF35 in association with ZNF265 from HeLa cell nuclear extracts. Immunoprecipitates were analyzed by Western blotting using antibodies against U1-70K or U2AF35 (arrow points to band of predicted size). (C) Relative strength of interaction of ZNF265 with U2AF35 or U1-70K, shown as β-gal activity relative to that for interaction of T-antigen with p53 (mean ± SE, n = 4, *P < 0.0001). (Control) β-gal activity of AH109 yeast containing pGBK-ZNF265 alone.
Mentions: To test whether ZNF265 could interact with other RS domain–containing proteins we conducted a yeast two-hybrid screen against representative spliceosomal proteins that included many with RS domains, namely U1-70K, U2AF35, U2AF65, SC35, p80 Coilin, WT1, 9G8, SF2/ASF, SRp20, SRp30c, and SRp40. Interaction was seen with U1-70K and U2AF35, as determined by growth on SD-L-W-A-H plates, and the production of a blue precipitate on a β-gal filter assay (Fig. 3 A). Interaction of ZNF265 with U1-70K and U2AF35 was confirmed by coimmunoprecipitation (Fig. 3 B). Liquid β-gal assay, which provides a semiquantitative estimation of interaction strength, showed that ZNF265 interacted more strongly with U1-70K than with U2AF35 (Fig. 3 C). A U1-70K cDNA clone was also isolated in a yeast two-hybrid screen against a human fetal brain cDNA library using ZNF265 as “bait.” Analysis of this clone revealed that residues 180–437 of U1-70K were responsible for mediating the interaction of U1-70K with the RS domain of ZNF265 (unpublished data). It is notable that this region contains the residues necessary for the binding of SF2/ASF to U1-70K (Cao and Garcia-Blanco, 1998). Several cDNA clones for the SR protein kinase Clk1 were also isolated from this screen. Because ZNF265 contains the Clk1 consensus phosphorylation site R/KXR/KXR/KXSXXR (Colwill et al., 1996; Moeslein et al., 1999), there may be a role for phosphorylation in the regulation of ZNF265.

Bottom Line: Transfection of HT-1080 cells with ZNF265-EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain.Alignment with other RS domain-containing proteins revealed a high degree of SR dipeptide conservation.These data show that ZNF265 functions as a novel component of the mRNA processing machinery.

View Article: PubMed Central - PubMed

Affiliation: The University of Sydney, Basic & Clinical Genomics Laboratory, Department of Physiology and Institute for Biomedical Research, Sydney, NSW 2006, Australia.

ABSTRACT
The formation of the active spliceosome, its recruitment to active areas of transcription, and its role in pre-mRNA splicing depends on the association of a number of multifunctional serine/arginine-rich (SR) proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zinc finger protein with conserved pre-mRNA splicing protein motifs. Here we show that ZNF265 immunoprecipitates from splicing extracts in association with mRNA, and that it is able to alter splicing patterns of Tra2-beta1 transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybrid analysis and immunoprecipitation indicated interaction of ZNF265 with the essential splicing factor proteins U1-70K and U2AF(35). Confocal microscopy demonstrated colocalization of ZNF265 with the motor neuron gene product SMN, the snRNP protein U1-70K, the SR protein SC35, and with the transcriptosomal components p300 and YY1. Transfection of HT-1080 cells with ZNF265-EGFP fusion constructs showed that nuclear localization of ZNF265 required the RS domain. Alignment with other RS domain-containing proteins revealed a high degree of SR dipeptide conservation. These data show that ZNF265 functions as a novel component of the mRNA processing machinery.

Show MeSH
Related in: MedlinePlus