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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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Significant enrichment of DRBP76-associated mRNAs encoding histone proteins in HEK293 cells.A. Transcripts rank-ordered by t-scores representing differential enrichment in DRBP76 IP vs. mock IP from HEK 293 cells. Black lines indicate the rank of each individual histone mRNA detected. B. Validation of histone mRNA targets by RT-PCR.
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pone-0011710-g006: Significant enrichment of DRBP76-associated mRNAs encoding histone proteins in HEK293 cells.A. Transcripts rank-ordered by t-scores representing differential enrichment in DRBP76 IP vs. mock IP from HEK 293 cells. Black lines indicate the rank of each individual histone mRNA detected. B. Validation of histone mRNA targets by RT-PCR.

Mentions: We examined the putative DRBP76 target mRNAs, using gene set enrichment analysis (GSEA) to explore known relationships among them and to determine if proteins encoded by these mRNAs are functionally related. GSEA analysis revealed significantly enriched functional categories vital to cellular metabolism (Fig. 5B). These include mRNAs encoding histone proteins, ribosomal proteins, and proteins involved in oxidative phosphorylation. Particularly striking was the enrichment of mRNAs encoding histone proteins. In a list ranked by t-scores representing differential enrichment between of transcripts DRBP76 vs. mock IP, the distribution of histone transcripts was significantly biased for being enriched in the DRBP76 IP (Fig. 6A). Furthermore, gene sets representing mRNAs containing KRCTCNNNNMANAGC or TTTNNANAGCYR motifs were both significantly enriched in the DRBP76 IPs. These motifs are present in histone mRNAs, and overlap with the terminal conserved stem loop (SL) in their 3′UTR [31]. To validate DRBP76 histone mRNA targets, we performed RT-PCR analysis of total RNA isolated from HEK293 cytoplasmic extracts and corresponding RIPs obtained with α-DRBP76 or isotype matched control mIgG antibodies (Fig. 6B). RT-PCR amplified Hist1H4A (∼48.5-fold enriched on microarray) and Hist2H2ac (∼4.9 fold enriched on microarray) from DRBP76 IP, but not from control mouse IgG IP (Fig. 6B).


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)

Significant enrichment of DRBP76-associated mRNAs encoding histone proteins in HEK293 cells.A. Transcripts rank-ordered by t-scores representing differential enrichment in DRBP76 IP vs. mock IP from HEK 293 cells. Black lines indicate the rank of each individual histone mRNA detected. B. Validation of histone mRNA targets by RT-PCR.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2909144&req=5

pone-0011710-g006: Significant enrichment of DRBP76-associated mRNAs encoding histone proteins in HEK293 cells.A. Transcripts rank-ordered by t-scores representing differential enrichment in DRBP76 IP vs. mock IP from HEK 293 cells. Black lines indicate the rank of each individual histone mRNA detected. B. Validation of histone mRNA targets by RT-PCR.
Mentions: We examined the putative DRBP76 target mRNAs, using gene set enrichment analysis (GSEA) to explore known relationships among them and to determine if proteins encoded by these mRNAs are functionally related. GSEA analysis revealed significantly enriched functional categories vital to cellular metabolism (Fig. 5B). These include mRNAs encoding histone proteins, ribosomal proteins, and proteins involved in oxidative phosphorylation. Particularly striking was the enrichment of mRNAs encoding histone proteins. In a list ranked by t-scores representing differential enrichment between of transcripts DRBP76 vs. mock IP, the distribution of histone transcripts was significantly biased for being enriched in the DRBP76 IP (Fig. 6A). Furthermore, gene sets representing mRNAs containing KRCTCNNNNMANAGC or TTTNNANAGCYR motifs were both significantly enriched in the DRBP76 IPs. These motifs are present in histone mRNAs, and overlap with the terminal conserved stem loop (SL) in their 3′UTR [31]. To validate DRBP76 histone mRNA targets, we performed RT-PCR analysis of total RNA isolated from HEK293 cytoplasmic extracts and corresponding RIPs obtained with α-DRBP76 or isotype matched control mIgG antibodies (Fig. 6B). RT-PCR amplified Hist1H4A (∼48.5-fold enriched on microarray) and Hist2H2ac (∼4.9 fold enriched on microarray) from DRBP76 IP, but not from control mouse IgG IP (Fig. 6B).

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