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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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Function of ZNF265 in the spliceosome. (A) In vitro splicing reactions were performed using labeled β-globin pre-mRNA and immunoprecipitated with the indicated antibodies, pre-immune serum, anti-SF2/ASF/anti-hnRNP A1, and increasing amounts of anti-ZNF265 (shown by triangle), immobilized on protein G-Sepharose. The immunoprecipitated complexes were washed extensively and RNA was extracted and analyzed by denaturing PAGE followed by autoradiography. 1/20 of total RNA recovered from the supernatant of an immunoprecipitation with control preimmune serum reflects the initial relative abundance of predicted pre-mRNA, intermediates and products, which are schematically depicted on left hand side. (B) Ability of ZNF265 to stimulate exon exclusion of alternatively spliced Tra-β1 pre-mRNA. At top is schematic diagram of the Tra2-β1mini- gene construct and splice products (introns: A, B, C; exons: 1, 2, 3, 4). HEK 293 cells were transfected with 3 μg of total plasmid DNA and, as indicated below abscissa, an increasing proportion of the expression plasmid C2-ZNF265. Representative ethidium bromide stained gel is shown, with schematic diagram of β1, β3, and β4 isoforms detected by this assay depicted on the right. (M) 100-bp marker. Relative abundance of the β4, β1, and β3 isoforms from each lane (mean ± SD) from three experiments is shown in the panels.
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fig5: Function of ZNF265 in the spliceosome. (A) In vitro splicing reactions were performed using labeled β-globin pre-mRNA and immunoprecipitated with the indicated antibodies, pre-immune serum, anti-SF2/ASF/anti-hnRNP A1, and increasing amounts of anti-ZNF265 (shown by triangle), immobilized on protein G-Sepharose. The immunoprecipitated complexes were washed extensively and RNA was extracted and analyzed by denaturing PAGE followed by autoradiography. 1/20 of total RNA recovered from the supernatant of an immunoprecipitation with control preimmune serum reflects the initial relative abundance of predicted pre-mRNA, intermediates and products, which are schematically depicted on left hand side. (B) Ability of ZNF265 to stimulate exon exclusion of alternatively spliced Tra-β1 pre-mRNA. At top is schematic diagram of the Tra2-β1mini- gene construct and splice products (introns: A, B, C; exons: 1, 2, 3, 4). HEK 293 cells were transfected with 3 μg of total plasmid DNA and, as indicated below abscissa, an increasing proportion of the expression plasmid C2-ZNF265. Representative ethidium bromide stained gel is shown, with schematic diagram of β1, β3, and β4 isoforms detected by this assay depicted on the right. (M) 100-bp marker. Relative abundance of the β4, β1, and β3 isoforms from each lane (mean ± SD) from three experiments is shown in the panels.

Mentions: In vitro splicing reactions showed that ZNF265 is immunoprecipitated in a complex that includes spliced mRNA (Fig. 5 A). This result indicates that ZNF265 binds directly or indirectly to mRNA, but much less to pre-mRNA. This property is shared with other splicing factors, such as SF2/ASF and RNPS1 (Hanamura et al., 1998; Mayeda et al., 1999), both of which synergistically stimulate general splicing. Here we show in splicing assays in cultured cells that ZNF265 can regulate alternative splicing in a concentration-dependent manner (Fig. 5 B). Namely, overexpression of ZNF265 resulted in exclusion of exons 2 and 3 from the Tra2-β1 pre-mRNA, which led to an increase in the production of the β3 alternatively spliced isoform. Our in vivo splicing result suggests that ZNF265 may have the ability to antagonize the alternative splicing activity of SR proteins on Tra2-β1 pre-mRNA. Splicing factor SR protein-mediated antagonism of alternative 5′ splice site selection has been reported for human hnRNP A1 protein in that hnRNP A1 causes activation of distal alternative 5′ splice site and exon exclusion in vitro and in vivo (Mayeda and Krainer, 1992; Mayeda et al., 1993; Cáceres et al., 1994; Yang et al., 1994). In contrast to hnRNP A1 that does not cause inhibition of general constitutive splicing, we have shown that addition of recombinant ZNF265 to SR protein-deficient HeLa cell S100 extracts supplemented with recombinant SF2/ASF may antagonize constitutive splicing of a β-globin pre-mRNA substrate and repress its splicing (our unpublished data). In Drosophila, RSF1 protein antagonizes and represses splicing by binding to SF2/ASF and preventing it from interacting with U1-70K (Labourier et al., 1999). It is possible that ZNF265 may also interfere with SF2/ASF-mediated constitutive splicing by binding directly to U1-70K.


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)

Function of ZNF265 in the spliceosome. (A) In vitro splicing reactions were performed using labeled β-globin pre-mRNA and immunoprecipitated with the indicated antibodies, pre-immune serum, anti-SF2/ASF/anti-hnRNP A1, and increasing amounts of anti-ZNF265 (shown by triangle), immobilized on protein G-Sepharose. The immunoprecipitated complexes were washed extensively and RNA was extracted and analyzed by denaturing PAGE followed by autoradiography. 1/20 of total RNA recovered from the supernatant of an immunoprecipitation with control preimmune serum reflects the initial relative abundance of predicted pre-mRNA, intermediates and products, which are schematically depicted on left hand side. (B) Ability of ZNF265 to stimulate exon exclusion of alternatively spliced Tra-β1 pre-mRNA. At top is schematic diagram of the Tra2-β1mini- gene construct and splice products (introns: A, B, C; exons: 1, 2, 3, 4). HEK 293 cells were transfected with 3 μg of total plasmid DNA and, as indicated below abscissa, an increasing proportion of the expression plasmid C2-ZNF265. Representative ethidium bromide stained gel is shown, with schematic diagram of β1, β3, and β4 isoforms detected by this assay depicted on the right. (M) 100-bp marker. Relative abundance of the β4, β1, and β3 isoforms from each lane (mean ± SD) from three experiments is shown in the panels.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196870&req=5

fig5: Function of ZNF265 in the spliceosome. (A) In vitro splicing reactions were performed using labeled β-globin pre-mRNA and immunoprecipitated with the indicated antibodies, pre-immune serum, anti-SF2/ASF/anti-hnRNP A1, and increasing amounts of anti-ZNF265 (shown by triangle), immobilized on protein G-Sepharose. The immunoprecipitated complexes were washed extensively and RNA was extracted and analyzed by denaturing PAGE followed by autoradiography. 1/20 of total RNA recovered from the supernatant of an immunoprecipitation with control preimmune serum reflects the initial relative abundance of predicted pre-mRNA, intermediates and products, which are schematically depicted on left hand side. (B) Ability of ZNF265 to stimulate exon exclusion of alternatively spliced Tra-β1 pre-mRNA. At top is schematic diagram of the Tra2-β1mini- gene construct and splice products (introns: A, B, C; exons: 1, 2, 3, 4). HEK 293 cells were transfected with 3 μg of total plasmid DNA and, as indicated below abscissa, an increasing proportion of the expression plasmid C2-ZNF265. Representative ethidium bromide stained gel is shown, with schematic diagram of β1, β3, and β4 isoforms detected by this assay depicted on the right. (M) 100-bp marker. Relative abundance of the β4, β1, and β3 isoforms from each lane (mean ± SD) from three experiments is shown in the panels.
Mentions: In vitro splicing reactions showed that ZNF265 is immunoprecipitated in a complex that includes spliced mRNA (Fig. 5 A). This result indicates that ZNF265 binds directly or indirectly to mRNA, but much less to pre-mRNA. This property is shared with other splicing factors, such as SF2/ASF and RNPS1 (Hanamura et al., 1998; Mayeda et al., 1999), both of which synergistically stimulate general splicing. Here we show in splicing assays in cultured cells that ZNF265 can regulate alternative splicing in a concentration-dependent manner (Fig. 5 B). Namely, overexpression of ZNF265 resulted in exclusion of exons 2 and 3 from the Tra2-β1 pre-mRNA, which led to an increase in the production of the β3 alternatively spliced isoform. Our in vivo splicing result suggests that ZNF265 may have the ability to antagonize the alternative splicing activity of SR proteins on Tra2-β1 pre-mRNA. Splicing factor SR protein-mediated antagonism of alternative 5′ splice site selection has been reported for human hnRNP A1 protein in that hnRNP A1 causes activation of distal alternative 5′ splice site and exon exclusion in vitro and in vivo (Mayeda and Krainer, 1992; Mayeda et al., 1993; Cáceres et al., 1994; Yang et al., 1994). In contrast to hnRNP A1 that does not cause inhibition of general constitutive splicing, we have shown that addition of recombinant ZNF265 to SR protein-deficient HeLa cell S100 extracts supplemented with recombinant SF2/ASF may antagonize constitutive splicing of a β-globin pre-mRNA substrate and repress its splicing (our unpublished data). In Drosophila, RSF1 protein antagonizes and represses splicing by binding to SF2/ASF and preventing it from interacting with U1-70K (Labourier et al., 1999). It is possible that ZNF265 may also interfere with SF2/ASF-mediated constitutive splicing by binding directly to U1-70K.

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