Limits...
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.

Show MeSH

Related in: MedlinePlus

NFAR protein isoform- and subcellular distribution in normal and neoplastic tissues.A. Intron-exon organization of the ILF3 gene [25], [33]. Exons are indicated by black boxes (top); alternative splicing yields diverse transcripts encoding DRBP76/ILF3 proteins with distinct C-termini (bottom; coding sequences are indicated by solid boxes). Hatched lines indicate proposed alternative splicing of exon 3 [25]. B. DRBP76 immunoblot of HeLa or HEK293 cell lysates (lanes 1–2) or non-fractionated murine tissue extracts of the indicated origin (lanes 3–8). (Lanes 9–10) DRBP76 immunoblot of normal Cynomologus brain (cyno) and NHA cell lysate. C. DRBP76 immunoblots of HEK293 and HTB-14 glioma cell lysates (lanes 1, 2) and total extracts prepared from normal cortex (3, 6 = frontal; 4, 7 = temporal; 5 = parietal) of human (h.s.) or simian (cyno) brain or human GBM tissue samples (8–13). Asterisks indicate histopathologically confirmed contamination with normal CNS. D. NFAR protein immunoblot analyzed with 7% tris-acetate gel electrophoresis. Simian (cyno) and fractionated cytoplasmic and nuclear murine (m.) brain extracts were studied in parallel with HEK293 lysates. Protein bands are labeled in (E). E. Evaluation of NFAR proteins in fractionated, cytoplasmic extracts from human normal brain and GBM tissues. Immunoblot filters were deliberately over-exposed to reveal all DRBP76-immunoreactive material. sc35 is a nuclear spliceosome component. F. DRBP76 immunoblot from fractionated extracts of the indicated cell lines. SH-SY5Y (SY5Y) cells were untreated or treated to induce a neuronal phenotype with ATRA.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2909144&req=5

pone-0011710-g001: NFAR protein isoform- and subcellular distribution in normal and neoplastic tissues.A. Intron-exon organization of the ILF3 gene [25], [33]. Exons are indicated by black boxes (top); alternative splicing yields diverse transcripts encoding DRBP76/ILF3 proteins with distinct C-termini (bottom; coding sequences are indicated by solid boxes). Hatched lines indicate proposed alternative splicing of exon 3 [25]. B. DRBP76 immunoblot of HeLa or HEK293 cell lysates (lanes 1–2) or non-fractionated murine tissue extracts of the indicated origin (lanes 3–8). (Lanes 9–10) DRBP76 immunoblot of normal Cynomologus brain (cyno) and NHA cell lysate. C. DRBP76 immunoblots of HEK293 and HTB-14 glioma cell lysates (lanes 1, 2) and total extracts prepared from normal cortex (3, 6 = frontal; 4, 7 = temporal; 5 = parietal) of human (h.s.) or simian (cyno) brain or human GBM tissue samples (8–13). Asterisks indicate histopathologically confirmed contamination with normal CNS. D. NFAR protein immunoblot analyzed with 7% tris-acetate gel electrophoresis. Simian (cyno) and fractionated cytoplasmic and nuclear murine (m.) brain extracts were studied in parallel with HEK293 lysates. Protein bands are labeled in (E). E. Evaluation of NFAR proteins in fractionated, cytoplasmic extracts from human normal brain and GBM tissues. Immunoblot filters were deliberately over-exposed to reveal all DRBP76-immunoreactive material. sc35 is a nuclear spliceosome component. F. DRBP76 immunoblot from fractionated extracts of the indicated cell lines. SH-SY5Y (SY5Y) cells were untreated or treated to induce a neuronal phenotype with ATRA.

Mentions: The interleukin enhancer binding factor 3 (ILF3) gene encodes a variety of related proteins in cultured cells with apparent sizes of 110 kDa (termed ILF3 [4] or nuclear factor associated with dsRNA-2 (NFAR-2) [5]) and 90 kDa (termed nuclear factor (NF) 90 [6], [7], DRBP76 [8] or NFAR-1 [5]). Diverse NFAR proteins originate from alternative splicing in the 3′ portion of ILF3 transcripts (Fig. 1A). For clarity, we will refer to the 110 kDa and 90 kDa proteins as ILF3 and DRBP76, respectively. Both proteins are known to associate with nuclear factor 45 (NF45), forming the nuclear factor of activated T-cells (NFAT; [9]). NFAR proteins contain nucleic acid binding motifs [8] and were originally identified because of their association with the interleukin-2 promoter [7]. They have been implicated in transcriptional regulation, although their precise role in this process remains poorly defined [10]. In addition, DRBP76 contains two double-stranded (ds) RNA binding motifs [11]. Complementing its involvement in transcriptional regulation, many lines of evidence point to a role for DRBP76 in post-transcriptional gene regulation due to its RNA-binding capacity.


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)

NFAR protein isoform- and subcellular distribution in normal and neoplastic tissues.A. Intron-exon organization of the ILF3 gene [25], [33]. Exons are indicated by black boxes (top); alternative splicing yields diverse transcripts encoding DRBP76/ILF3 proteins with distinct C-termini (bottom; coding sequences are indicated by solid boxes). Hatched lines indicate proposed alternative splicing of exon 3 [25]. B. DRBP76 immunoblot of HeLa or HEK293 cell lysates (lanes 1–2) or non-fractionated murine tissue extracts of the indicated origin (lanes 3–8). (Lanes 9–10) DRBP76 immunoblot of normal Cynomologus brain (cyno) and NHA cell lysate. C. DRBP76 immunoblots of HEK293 and HTB-14 glioma cell lysates (lanes 1, 2) and total extracts prepared from normal cortex (3, 6 = frontal; 4, 7 = temporal; 5 = parietal) of human (h.s.) or simian (cyno) brain or human GBM tissue samples (8–13). Asterisks indicate histopathologically confirmed contamination with normal CNS. D. NFAR protein immunoblot analyzed with 7% tris-acetate gel electrophoresis. Simian (cyno) and fractionated cytoplasmic and nuclear murine (m.) brain extracts were studied in parallel with HEK293 lysates. Protein bands are labeled in (E). E. Evaluation of NFAR proteins in fractionated, cytoplasmic extracts from human normal brain and GBM tissues. Immunoblot filters were deliberately over-exposed to reveal all DRBP76-immunoreactive material. sc35 is a nuclear spliceosome component. F. DRBP76 immunoblot from fractionated extracts of the indicated cell lines. SH-SY5Y (SY5Y) cells were untreated or treated to induce a neuronal phenotype with ATRA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2909144&req=5

pone-0011710-g001: NFAR protein isoform- and subcellular distribution in normal and neoplastic tissues.A. Intron-exon organization of the ILF3 gene [25], [33]. Exons are indicated by black boxes (top); alternative splicing yields diverse transcripts encoding DRBP76/ILF3 proteins with distinct C-termini (bottom; coding sequences are indicated by solid boxes). Hatched lines indicate proposed alternative splicing of exon 3 [25]. B. DRBP76 immunoblot of HeLa or HEK293 cell lysates (lanes 1–2) or non-fractionated murine tissue extracts of the indicated origin (lanes 3–8). (Lanes 9–10) DRBP76 immunoblot of normal Cynomologus brain (cyno) and NHA cell lysate. C. DRBP76 immunoblots of HEK293 and HTB-14 glioma cell lysates (lanes 1, 2) and total extracts prepared from normal cortex (3, 6 = frontal; 4, 7 = temporal; 5 = parietal) of human (h.s.) or simian (cyno) brain or human GBM tissue samples (8–13). Asterisks indicate histopathologically confirmed contamination with normal CNS. D. NFAR protein immunoblot analyzed with 7% tris-acetate gel electrophoresis. Simian (cyno) and fractionated cytoplasmic and nuclear murine (m.) brain extracts were studied in parallel with HEK293 lysates. Protein bands are labeled in (E). E. Evaluation of NFAR proteins in fractionated, cytoplasmic extracts from human normal brain and GBM tissues. Immunoblot filters were deliberately over-exposed to reveal all DRBP76-immunoreactive material. sc35 is a nuclear spliceosome component. F. DRBP76 immunoblot from fractionated extracts of the indicated cell lines. SH-SY5Y (SY5Y) cells were untreated or treated to induce a neuronal phenotype with ATRA.
Mentions: The interleukin enhancer binding factor 3 (ILF3) gene encodes a variety of related proteins in cultured cells with apparent sizes of 110 kDa (termed ILF3 [4] or nuclear factor associated with dsRNA-2 (NFAR-2) [5]) and 90 kDa (termed nuclear factor (NF) 90 [6], [7], DRBP76 [8] or NFAR-1 [5]). Diverse NFAR proteins originate from alternative splicing in the 3′ portion of ILF3 transcripts (Fig. 1A). For clarity, we will refer to the 110 kDa and 90 kDa proteins as ILF3 and DRBP76, respectively. Both proteins are known to associate with nuclear factor 45 (NF45), forming the nuclear factor of activated T-cells (NFAT; [9]). NFAR proteins contain nucleic acid binding motifs [8] and were originally identified because of their association with the interleukin-2 promoter [7]. They have been implicated in transcriptional regulation, although their precise role in this process remains poorly defined [10]. In addition, DRBP76 contains two double-stranded (ds) RNA binding motifs [11]. Complementing its involvement in transcriptional regulation, many lines of evidence point to a role for DRBP76 in post-transcriptional gene regulation due to its RNA-binding capacity.

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