The RNA Binding Protein Nuclear Factor 90 Functions as Both a Positive and Negative Regulator of Gene Expression in Mammalian Cells

Reichman, Trevor; Muñiz, Luis C.; Mathews, Michael B.

doi:10.1128/mcb.22.1.343-356.2002

Cited by 101 publications

(111 citation statements)

References 41 publications

(55 reference statements)

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“…The resulting peptides were analyzed by mass spectrometry (16), which identified the 80 -90-kDa protein as the short form of ILF3 (also called NF90, NFAR-1, MPP4, and DRBP76). ILF3 is a nuclear protein that contains two dsRBD and an RGG domain and that influences gene expression (11,(17)(18)(19)(20)(21)(22). Importantly, the RNA/protein complex was dissociated upon addition of anti-ILF3 antibodies whereas mock antibodies had no effect (Fig.…”

Section: Ilf3mentioning

confidence: 99%

Minihelix-containing RNAs Mediate Exportin-5-dependent Nuclear Export of the Double-stranded RNA-binding Protein ILF3

Gwizdek

Ossareh-Nazari

Brownawell

et al. 2004

Journal of Biological Chemistry

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The karyopherin-related nuclear transport factor exportin-5 preferentially recognizes and transports RNAs containing minihelix motif, a structural cis-acting export element that comprises a double-stranded stem (>14 nucleotides) with a base-paired 5 end and a 3-8-nucleotide protruding 3 end. This structural motif is present in various small cellular and viral polymerase III transcripts such as the adenovirus VA1 RNA (VA1). Here we show that the double-stranded RNA-binding protein, ILF3 (interleukin enhancer binding factor 3) preferentially binds minihelix motif. Gel retardation assays and glutathione S-transferase pull-down experiments revealed that ILF3, exportin-5, RanGTP, and VA1 RNA assembled in a quaternary complex in which the RNA moiety bridges the interaction between ILF3 and exportin-5. Formation of this complex is facilitated by the ability of both exportin-5 and ILF3 to mutually increase their apparent affinity for VA1 RNA. Using microinjection in the nucleus of HeLa cells and transfection experiments, we show here that formation of the cooperative RanGTP-dependent RNA/ILF3/exportin-5 complex promotes the co-transport of VA1 and ILF3 from the nucleus to the cytoplasm. Exportin-5 thus appears as the first example of a nuclear export receptor that mediates RNA export but also promotes transport of proteinaceous cargo through appropriate and specific RNA adaptors.Transfer of macromolecules through nuclear pore complexes depends on both structural components of nuclear pore complexes and soluble factors that ensure nuclear transport of a wide diversity of cellular proteins and RNAs. Selectivity of transport is mediated by the recognition of specific signals within the cargoes by specific transport receptors through a direct or adaptor-mediated interaction. Transport receptors interact with nucleoporins thereby mediating docking to and translocation through the nuclear pore complexes. Most of the transport receptors belong to the karyopherin ␤ family (also known as importin ␤ family) and use the GTPase Ran to control cargo association. The asymmetric distribution of the Ran regulatory proteins provides a steep gradient of RanGDP (cytoplasmic)/RanGTP (nuclear) across the nuclear envelope that ensures the directionality of nuclear transport (1, 2). Indeed, nuclear import receptors (importins) bind their cargoes in the cytoplasm in the absence of RanGTP and unload them upon binding to RanGTP in the nucleus. In contrast, export receptors (exportins) require RanGTP to interact with their cargoes in the nucleus, and those export complexes are destabilized by dissociation of RanGTP and GTP hydrolysis in the cytoplasm (3, 4).Study of nuclear export pathways of diverse viral RNAs has greatly contributed to the identification of cellular factors implicated in nuclear transport processes. In particular, we recently reported nuclear export mechanisms of the adenovirus VA1 RNA (5, 6). This RNA is abundantly transcribed by RNA polymerase III and efficiently exported to the cytoplasm in the late phase of the viral infect...

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Section: Ilf3mentioning

confidence: 99%

Minihelix-containing RNAs Mediate Exportin-5-dependent Nuclear Export of the Double-stranded RNA-binding Protein ILF3

Gwizdek

Ossareh-Nazari

Brownawell

et al. 2004

Journal of Biological Chemistry

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“…NF90 and its homologs selectively regulate gene expression in mammalian cells, both positively and negatively (Saunders et al 2001b;Krasnoselskaya-Riz et al 2002;Reichman et al 2002). The dsRNA-binding region is essential for these regulatory actions but the role of RNA has not been directly established.…”

Section: Introductionmentioning

confidence: 99%

RNA binding and intramolecular interactions modulate the regulation of gene expression by nuclear factor 110

Reichman¹,

Mathews²

2003

RNA

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Nuclear factor 110 (NF110) belongs to the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA) binding proteins that regulate gene expression at the transcriptional level in vertebrates. The proteins are identical at their N terminus, which functions as a negative regulatory region, but have distinct C termini as a result of alternate splicing. Maximal transcriptional activity of NF110 requires its C-terminal domain and a central domain that contains a nuclear localization signal and two dsRNA-binding motifs (dsRBMs). We find that dsRNA binding is reduced by RGG and GQSY motifs present in the C-terminal region. To directly evaluate the role of RNA binding in transactivation, we conducted site-directed mutagenesis to substitute conserved residues in one or both of the dsRBMs. The mutations reduced the ability of NF110 to stimulate gene expression to an extent that paralleled the mutants' reduced ability to bind dsRNA. Full activity was restored when the dsRBM-containing region of NF110 was replaced with the RNA-binding region of the protein kinase PKR. Finally, NF110-mediated transactivation was inhibited by cotransfection of a plasmid encoding an artificial highly structured RNA. These data suggest that NF110 and its homologs are regulated by cis-acting domains present in some of the protein isoforms, and via interactions with RNAs that bind to their dsRBMs. We propose a model in which structured RNAs regulate gene expression by modulating transcription through interactions with members of the NF90 protein family.

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“…1. Additionally, a common complex partner of NF45 and a known dsRNA binding protein [26][27][28], NF90, was predicted to be part of the mRNP complexes and was tested for its ability to bind PGHS-1 mRNA sequence. (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1) after affinity purification, were used for validation experiments. An additional protein, NF90, was included in the investigation since it is a common binding partner of NF45 and a known dsRNA binding protein [26][27][28]. The affinity purification results were further validated by immunoprecipitation assays using antibodies specific to nucleolin, NF45, NF90 and serpin B1 followed by RT-PCR experiments to detect PGHS-1 mRNA.…”

Section: Discussionmentioning

confidence: 99%

“…NF45 and NF90 were initially purified as components of an NFAT complex that interacts with the antigen receptor response element-2 (ARRE-2) sequence within the promoter region of the interleukin-2 (IL-2) gene and act as transcriptional enhancer [37,38]. It is suggested that NF45 acts as a regulator of NF90 activity [28]. Originally identified as DNA-binding proteins [26,36,37], NF90 and NF45 do not contain a recognized sequence-specific DNA-binding domain and the complex does not appear to interact with DNA directly [26,39].…”

Section: Discussionmentioning

confidence: 99%

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Characterization of proteins associating with 5’ terminus of PGHS-1 mRNA

Bunimov

Laneuville

2010

Cellular and Molecular Biology Letters

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Abbreviations used: ARRE-2 -antigen receptor response element-2; ATCC -American Type Culture Collection; DRBP76 -double-stranded RNA-binding protein-76; dsRNAdouble stranded RNA; FBS -fetal bovine serum; GAPDH -glyceraldehyde 3-phosphate dehydrogenase; IL-2 -interleukin-2; IP -immunoprecipitation; IRES -internal ribosome entry site; LC-MS -liquid chromatography mass-spectrometry; Luc -luciferase; MKP-1 -mitogen-activated protein kinase phosphatase 1; MNEI -monocyte/neutrophil elastase inhibitor; mRNP -messenger ribonucleoprotein; NCL -nucleolin; NF45 -nuclear factor 45; NF90 -nuclear factor 90; NFAT -nuclear factor of activated T-cells; ORF -open reading frame; PG -prostaglandins; PGHS -prostaglandin endoperoxide H synthase; PMA -phorbol 12-myristate 13-acetate; SB1 or serpin B1 -serine protease inhibitor; TPA -12-O-tetradecanoylphorbol -13-acetate; Tx -thromboxanes; UTR -untranslated region Immunoprecipitation experiments using MEG-01 protein extracts validated mass spectrometry data and confirmed binding of nucleolin, serpin B1, NF45 and NF90. The RNA fraction was extracted from immunoprecipitated mRNP complexes and association of RNA binding proteins, serpin B1, NF45 and NF90, to PGHS-1 mRNA target sequence was confirmed by RT-PCR. Together these data suggest that serpin B1, NF45 and NF90 associate with PGHS-1 mRNA and can potentially participate in the formation a single or a number of PGHS-1 ribonucleoprotein complexes, through nucleolin that possibly serves as a docking base for other protein complex members.

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The RNA Binding Protein Nuclear Factor 90 Functions as Both a Positive and Negative Regulator of Gene Expression in Mammalian Cells

Cited by 101 publications

References 41 publications

Minihelix-containing RNAs Mediate Exportin-5-dependent Nuclear Export of the Double-stranded RNA-binding Protein ILF3

Minihelix-containing RNAs Mediate Exportin-5-dependent Nuclear Export of the Double-stranded RNA-binding Protein ILF3

RNA binding and intramolecular interactions modulate the regulation of gene expression by nuclear factor 110

Characterization of proteins associating with 5’ terminus of PGHS-1 mRNA

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