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Tissue type-specific expression of the dsRNA-binding protein 76 and genome-wide elucidation of its target mRNAs.

Neplioueva V, Dobrikova EY, Mukherjee N, Keene JD, Gromeier M - PLoS ONE (2010)

Bottom Line: The association of RNA-binding proteins with their targets is regulated through changing subcellular distribution, post-translational modification or association with other proteins.Our data suggest that physiologic DRBP76 expression, isoform distribution and subcellular localization are profoundly altered upon malignant transformation.Thus, the functional role of DRBP76 in co- or post-transcriptional gene regulation may contribute to the neoplastic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurosurgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT

Background: RNA-binding proteins accompany all steps in the life of mRNAs and provide dynamic gene regulatory functions for rapid adjustment to changing extra- or intracellular conditions. The association of RNA-binding proteins with their targets is regulated through changing subcellular distribution, post-translational modification or association with other proteins.

Methodology: We demonstrate that the dsRNA binding protein 76 (DRBP76), synonymous with nuclear factor 90, displays inherently distinct tissue type-specific subcellular distribution in the normal human central nervous system and in malignant brain tumors of glial origin. Altered subcellular localization and isoform distribution in malignant glioma indicate that tumor-specific changes in DRBP76-related gene products and their regulatory functions may contribute to the formation and/or maintenance of these tumors. To identify endogenous mRNA targets of DRBP76, we performed RNA-immunoprecipitation and genome-wide microarray analyses in HEK293 cells, and identified specific classes of transcripts encoding critical functions in cellular metabolism.

Significance: Our data suggest that physiologic DRBP76 expression, isoform distribution and subcellular localization are profoundly altered upon malignant transformation. Thus, the functional role of DRBP76 in co- or post-transcriptional gene regulation may contribute to the neoplastic phenotype.

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Immunoprecipitation studies of DRBP76.A. Left panel. Control mouse IgG and anti-DRBP76 IP from HEK293 cells, normal primate CNS and GBM tissues. Co-IP of NF45 was analyzed as well. Middle/Right panel. Silver stain of anti-DRBP76 immunoprecipitated material from normal human/simian brain and from HEK293 and HTB-14 cell lines, respectively, analyzed by immunoblot. B. IP with anti-DRBP76, but not isotype-matched control IgG, effectively removes DRBP76 from HEK293 cell lysates. C. RT-PCR of GCase and GAPDH transcript from RNA isolated from immunoprecipitated DRBP76 RNP.
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pone-0011710-g003: Immunoprecipitation studies of DRBP76.A. Left panel. Control mouse IgG and anti-DRBP76 IP from HEK293 cells, normal primate CNS and GBM tissues. Co-IP of NF45 was analyzed as well. Middle/Right panel. Silver stain of anti-DRBP76 immunoprecipitated material from normal human/simian brain and from HEK293 and HTB-14 cell lines, respectively, analyzed by immunoblot. B. IP with anti-DRBP76, but not isotype-matched control IgG, effectively removes DRBP76 from HEK293 cell lysates. C. RT-PCR of GCase and GAPDH transcript from RNA isolated from immunoprecipitated DRBP76 RNP.

Mentions: To confirm the identity of DRBP76 immuno-reactive material in cytoplasmic extracts from CNS tissues and to prepare for RIP-Chip analyses of DRBP76, we performed DRBP76 immunoprecipitation (IP) and co-IP analyses of NF45 (Fig. 3A). DRBP76 antibody, but not mouse isotype-matched control IgG, efficiently precipitated the previously observed distinct immunoblot signals from HEK293 cell- and primate brain cytosolic extracts, and yielded co-IP of NF45 in both instances (Fig. 3A, left panel). Silver stain analyses of the DRBP76 IPs from human/macaque normal brain revealed the presence of the characteristic DRBP76 double-band detected in IP immunoblots (Fig. 3A, middle panel). Corroborating the direct immunoblot results (Fig. 1E) and the immunofluorescence data (Fig. 2U–W), DRBP76 IP from cytoplasmic extracts of GBM tissues did not reveal proteins in the ∼75–90 kDa size range (Fig. 3A). We speculate that a faint band at ∼110 kDa in both IPs from normal brain and GBM represents ILF3, since our cytoplasmic extracts typically contain nuclear contaminants (Fig. 1E). Similar IPs from cell lysates produced the expected bands, i.e. the ∼90 kDa and ∼110 kDa NFAR proteins typically isolated from transformed cell lines (Fig. 3A, right panel).


Tissue type-specific expression of the dsRNA-binding protein 76 and genome-wide elucidation of its target mRNAs.

Neplioueva V, Dobrikova EY, Mukherjee N, Keene JD, Gromeier M - PLoS ONE (2010)

Immunoprecipitation studies of DRBP76.A. Left panel. Control mouse IgG and anti-DRBP76 IP from HEK293 cells, normal primate CNS and GBM tissues. Co-IP of NF45 was analyzed as well. Middle/Right panel. Silver stain of anti-DRBP76 immunoprecipitated material from normal human/simian brain and from HEK293 and HTB-14 cell lines, respectively, analyzed by immunoblot. B. IP with anti-DRBP76, but not isotype-matched control IgG, effectively removes DRBP76 from HEK293 cell lysates. C. RT-PCR of GCase and GAPDH transcript from RNA isolated from immunoprecipitated DRBP76 RNP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2909144&req=5

pone-0011710-g003: Immunoprecipitation studies of DRBP76.A. Left panel. Control mouse IgG and anti-DRBP76 IP from HEK293 cells, normal primate CNS and GBM tissues. Co-IP of NF45 was analyzed as well. Middle/Right panel. Silver stain of anti-DRBP76 immunoprecipitated material from normal human/simian brain and from HEK293 and HTB-14 cell lines, respectively, analyzed by immunoblot. B. IP with anti-DRBP76, but not isotype-matched control IgG, effectively removes DRBP76 from HEK293 cell lysates. C. RT-PCR of GCase and GAPDH transcript from RNA isolated from immunoprecipitated DRBP76 RNP.
Mentions: To confirm the identity of DRBP76 immuno-reactive material in cytoplasmic extracts from CNS tissues and to prepare for RIP-Chip analyses of DRBP76, we performed DRBP76 immunoprecipitation (IP) and co-IP analyses of NF45 (Fig. 3A). DRBP76 antibody, but not mouse isotype-matched control IgG, efficiently precipitated the previously observed distinct immunoblot signals from HEK293 cell- and primate brain cytosolic extracts, and yielded co-IP of NF45 in both instances (Fig. 3A, left panel). Silver stain analyses of the DRBP76 IPs from human/macaque normal brain revealed the presence of the characteristic DRBP76 double-band detected in IP immunoblots (Fig. 3A, middle panel). Corroborating the direct immunoblot results (Fig. 1E) and the immunofluorescence data (Fig. 2U–W), DRBP76 IP from cytoplasmic extracts of GBM tissues did not reveal proteins in the ∼75–90 kDa size range (Fig. 3A). We speculate that a faint band at ∼110 kDa in both IPs from normal brain and GBM represents ILF3, since our cytoplasmic extracts typically contain nuclear contaminants (Fig. 1E). Similar IPs from cell lysates produced the expected bands, i.e. the ∼90 kDa and ∼110 kDa NFAR proteins typically isolated from transformed cell lines (Fig. 3A, right panel).

Bottom Line: The association of RNA-binding proteins with their targets is regulated through changing subcellular distribution, post-translational modification or association with other proteins.Our data suggest that physiologic DRBP76 expression, isoform distribution and subcellular localization are profoundly altered upon malignant transformation.Thus, the functional role of DRBP76 in co- or post-transcriptional gene regulation may contribute to the neoplastic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurosurgery, Department of Surgery, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT

Background: RNA-binding proteins accompany all steps in the life of mRNAs and provide dynamic gene regulatory functions for rapid adjustment to changing extra- or intracellular conditions. The association of RNA-binding proteins with their targets is regulated through changing subcellular distribution, post-translational modification or association with other proteins.

Methodology: We demonstrate that the dsRNA binding protein 76 (DRBP76), synonymous with nuclear factor 90, displays inherently distinct tissue type-specific subcellular distribution in the normal human central nervous system and in malignant brain tumors of glial origin. Altered subcellular localization and isoform distribution in malignant glioma indicate that tumor-specific changes in DRBP76-related gene products and their regulatory functions may contribute to the formation and/or maintenance of these tumors. To identify endogenous mRNA targets of DRBP76, we performed RNA-immunoprecipitation and genome-wide microarray analyses in HEK293 cells, and identified specific classes of transcripts encoding critical functions in cellular metabolism.

Significance: Our data suggest that physiologic DRBP76 expression, isoform distribution and subcellular localization are profoundly altered upon malignant transformation. Thus, the functional role of DRBP76 in co- or post-transcriptional gene regulation may contribute to the neoplastic phenotype.

Show MeSH
Related in: MedlinePlus