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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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Role of the RS domain of ZNF265 in nuclear localization. (Left) EGFP fusion protein constructs used for the expression of ZNF265. Wild-type ZNF265 sequence (1st row) and 5 mutant sequences (2nd–6th row) were used. (Right) EGFP fluorescence (green) and DAPI (blue) detection in HT-1080 cells at 48 h posttransfection. Bar, 10 μm.
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fig2: Role of the RS domain of ZNF265 in nuclear localization. (Left) EGFP fusion protein constructs used for the expression of ZNF265. Wild-type ZNF265 sequence (1st row) and 5 mutant sequences (2nd–6th row) were used. (Right) EGFP fluorescence (green) and DAPI (blue) detection in HT-1080 cells at 48 h posttransfection. Bar, 10 μm.

Mentions: To determine the region of ZNF265 necessary for its nuclear localization, cDNA expression plasmids were generated from which specific domains were deleted. Compared with the nuclear localization of the wild-type ZNF265 fusion protein (C2-ZNF265), fusions containing the zinc finger with (C2-Mut3) or without (C2-Mut2) the putative nuclear localization signal (NLS) showed a predominantly cytoplasmic distribution (Fig. 2) . In contrast, nuclear localization was preserved when the RS domain was retained, either with (C2-Mut4) or without (C2-Mut5) the NLS. Consistent with this observation, nuclear localization was not affected by mutation of the NLS (C2-Mut6). Thus, nuclear localization is dictated by the RS domain of ZNF265, consistent with the behavior of other RS proteins such as SC35 (Hedley et al., 1995), SF2/ASF, SRp20, and 9G8 (Cáceres et al., 1997, 1998).


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)

Role of the RS domain of ZNF265 in nuclear localization. (Left) EGFP fusion protein constructs used for the expression of ZNF265. Wild-type ZNF265 sequence (1st row) and 5 mutant sequences (2nd–6th row) were used. (Right) EGFP fluorescence (green) and DAPI (blue) detection in HT-1080 cells at 48 h posttransfection. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Role of the RS domain of ZNF265 in nuclear localization. (Left) EGFP fusion protein constructs used for the expression of ZNF265. Wild-type ZNF265 sequence (1st row) and 5 mutant sequences (2nd–6th row) were used. (Right) EGFP fluorescence (green) and DAPI (blue) detection in HT-1080 cells at 48 h posttransfection. Bar, 10 μm.
Mentions: To determine the region of ZNF265 necessary for its nuclear localization, cDNA expression plasmids were generated from which specific domains were deleted. Compared with the nuclear localization of the wild-type ZNF265 fusion protein (C2-ZNF265), fusions containing the zinc finger with (C2-Mut3) or without (C2-Mut2) the putative nuclear localization signal (NLS) showed a predominantly cytoplasmic distribution (Fig. 2) . In contrast, nuclear localization was preserved when the RS domain was retained, either with (C2-Mut4) or without (C2-Mut5) the NLS. Consistent with this observation, nuclear localization was not affected by mutation of the NLS (C2-Mut6). Thus, nuclear localization is dictated by the RS domain of ZNF265, consistent with the behavior of other RS proteins such as SC35 (Hedley et al., 1995), SF2/ASF, SRp20, and 9G8 (Cáceres et al., 1997, 1998).

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