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

ABSTRACT Nuclear factor 90 (NF90) was originally isolated in a complex that binds to the antigen recognition response element (ARRE-2) present in the interleukin-2 promoter. To characterize the transcriptional properties of NF90 in mammalian cells, we examined its ability to modulate promoter function in cellular transfection assays. NF90-Gal4 fusion proteins inhibited transcription from the adenovirus major late promoter in a fashion that was dependent on Gal4 targeting. Conversely, NF90 activated the cytomegalovirus immediate-early promoter, to which it was not targeted. These effects required distinct but overlapping domains in the C terminus of NF90, which contains a functional nuclear localization signal and two double-stranded-RNA binding motifs. NF90 is present in cellular complexes together with the NF45 protein. Transfection assays showed that NF45 binds NF90 strongly and stimulates its ability to activate but not to inhibit gene expression. This report characterizes NF90 as both a positive and negative regulator of gene expression, depending on the promoter context, and suggests a role for NF45 as a regulator of NF90.

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Protein localization to the nucleolus: a search for targeting domains in nucleolin

Journal of Cell Science ◽

10.1242/jcs.105.3.799 ◽

1993 ◽

Vol 105 (3) ◽

pp. 799-806 ◽

Cited By ~ 8

Author(s):

M.S. Schmidt-Zachmann ◽

E.A. Nigg

Keyword(s):

Mammalian Cells ◽

Rna Binding ◽

Protein Localization ◽

Site Directed Mutagenesis ◽

Rdna Transcription ◽

Binding Motifs ◽

Mutant Proteins ◽

C Terminus ◽

Localization Signal ◽

Functional Nuclear Localization Signal

Nucleolin, a major nucleolar phosphoprotein, is presumed to function in rDNA transcription, rRNA packaging and ribosome assembly. Its primary sequence was highly conserved during evolution and suggests a multi-domain structure. To identify structural elements required for nuclear uptake and nucleolar accumulation of nucleolin, we used site-directed mutagenesis to introduce point- and deletion-mutations into a chicken nucleolin cDNA. Following transient expression in mammalian cells, the intracellular distribution of the corresponding wild-type and mutant proteins was determined by indirect immunofluorescence microscopy. We found that nucleolin contains a functional nuclear localization signal (KRKKEMANKSAPEAKKKK) that conforms exactly to the consensus proposed recently for a bipartite signal (Robbins, J., Dilworth, S.M., Laskey, R.A. and Dingwall, C. (1991) Cell 64, 615–623). Concerning nucleolar localization, we found that the N-terminal 250 amino acids of nucleolin are dispensible, but deletion of either the centrally located RNA-binding motifs (the RNP domain) or the glycine/arginine-rich C terminus (the GR domain) resulted in an exclusively nucleoplasmic distribution. Although both of these latter domains were required for correct subcellular localization of nucleolin, they were not sufficient to target non-nucleolar proteins to the nucleolus. From these results we conclude that nucleolin does not contain a single, linear nucleolar targeting signal. Instead, we propose that the protein uses a bipartite NLS to enter the nucleus and then accumulates within the nucleolus by virtue of binding to other nucleolar components (probably rRNA) via its RNP and GR domains.

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Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants

Biochemical Journal ◽

10.1042/bj20050694 ◽

2005 ◽

Vol 393 (1) ◽

pp. 245-254 ◽

Cited By ~ 32

Author(s):

Catherine Martel ◽

Paolo Macchi ◽

Luc Furic ◽

Michael A. Kiebler ◽

Luc Desgroseillers

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Mammalian Cells ◽

Rna Binding ◽

Binding Activity ◽

Nuclear Trafficking ◽

Binding Domains ◽

Bipartite Nuclear Localization Signal ◽

Localization Signal

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

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Sla1, a Schizosaccharomyces pombe Homolog of the Human La Protein, Induces Ectopic Meiosis when Its C Terminus Is Truncated

Eukaryotic Cell ◽

10.1128/ec.2.6.1274-1287.2003 ◽

2003 ◽

Vol 2 (6) ◽

pp. 1274-1287 ◽

Cited By ~ 9

Author(s):

Kaori Tanabe ◽

Noriko Ito ◽

Tomomi Wakuri ◽

Fumiyo Ozoe ◽

Makoto Umeda ◽

...

Keyword(s):

Schizosaccharomyces Pombe ◽

Rna Binding ◽

Rna Binding Proteins ◽

Small Region ◽

Full Length ◽

Rna Recognition Motifs ◽

La Protein ◽

C Terminus ◽

Localization Signal ◽

Recognition Motifs

ABSTRACT Sla1 is a Schizosaccharomyces pombe homolog of the human La protein. La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved. In this study, we show that the S. pombe La homolog (Sla1) is involved in regulating sexual development. Sla1 truncated in the C terminus (Sla1ΔC) induced ectopic sporulation in the ras1Δ strain and several other sporulation-deficient mutants. The C terminus contains a nuclear localization signal. While full-length Sla1 localizes in the nucleus, Sla1ΔC is found throughout the cell, suggesting the cytoplasmic localization of Sla1ΔC is involved in its sporulation-inducing activity. Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation. The La motif (RRM1) is not involved in this activity. Strikingly, Sla1ΔC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation. Sla1ΔC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1ΔC requires Mei2 to induce haploid meiosis. Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant. Two-hybrid analysis indicated that Pat1 interacts with Sla1ΔC but not full-length Sla1. Thus, Sla1ΔC may block Pat1 activity. This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis. Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced. The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins.

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Posttranscriptional gene regulation by RNA-binding proteins during oxidative stress: implications for cellular senescence

Biological Chemistry ◽

10.1515/bc.2008.022 ◽

2008 ◽

Vol 389 (3) ◽

pp. 243-255 ◽

Cited By ~ 156

Author(s):

Kotb Abdelmohsen ◽

Yuki Kuwano ◽

Hyeon Ho Kim ◽

Myriam Gorospe

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Cellular Senescence ◽

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Profiles ◽

Expression Patterns ◽

Oxidant Damage ◽

Regulated Gene Expression

AbstractTo respond adequately to oxidative stress, mammalian cells elicit rapid and tightly controlled changes in gene expression patterns. Besides alterations in the subsets of transcribed genes, two posttranscriptional processes prominently influence the oxidant-triggered gene expression programs: mRNA turnover and translation. Here, we review recent progress in our knowledge of theturnover andtranslationregulatory (TTR) mRNA-bindingproteins (RBPs) that influence gene expression in response to oxidative damage. Specifically, we identify oxidant damage-regulated mRNAs that are targets of TTR-RBPs, we review the oxidant-triggered signaling pathways that govern TTR-RBP function, and we examine emerging evidence that TTR-RBP activity is altered with senescence and aging. Given the potent influence of TTR-RBPs upon oxidant-regulated gene expression profiles, we propose that the senescence-associated changes in TTR-RBPs directly contribute to the impaired responses to oxidant damage that characterize cellular senescence and advancing age.

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A Trypanosoma cruzi Zinc Finger protein that controls expression of epimastigote specific genes and affects metacyclogenesis

10.1101/2020.07.07.189688 ◽

2020 ◽

Author(s):

Thais Silva Tavares ◽

Fernanda Lins Brandão Mügge ◽

Viviane Grazielle-Silva ◽

Bruna Mattioly Valente ◽

Wanessa Moreira Goes ◽

...

Keyword(s):

Gene Expression ◽

Trypanosoma Cruzi ◽

Zinc Finger ◽

Rna Binding ◽

Environmental Changes ◽

Zinc Finger Protein ◽

Negative Regulator ◽

Transcriptional Level ◽

Control Of Gene Expression ◽

Genes Encoding

SummaryTrypanosoma cruzi has three biochemically and morphologically distinct developmental stages that are programed to rapidly respond to environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, Trypanosomatid genomes contain protein coding genes that are transcribed into polycistronic pre-mRNAs and control of gene expression relies on mechanisms acting at the post-transcriptional level. Transcriptome analyses comparing epimastigote, trypomastigote and intracellular amastigote stages revealed changes in gene expression that reflect the parasite adaptation to distinct environments. Several genes encoding RNA binding proteins (RBP), known to act as key post-transcriptional regulatory factors, were also differentially expressed. We characterized one T. cruzi RBP (TcZH3H12) that contains a zinc finger domain, and whose transcripts are upregulated in epimastigotes compared to trypomastigotes and amastigotes. TcZC3H12 knockout epimastigotes showed decreased growth rates and increased capacity to differentiate into metacyclic trypomastigotes. Comparative transcriptome analysis revealed a TcZC3H12-dependent expression of epimastigote specific genes encoding amino acid transporters and proteins associated with differentiation (PAD), among others. RNA immunoprecipitation assays showed that transcripts from the PAD family interact with TcZC3H12. Taken together, these findings suggest that TcZC3H12 positively regulates the expression of genes involved in epimastigote proliferation and also acts as a negative regulator of metacyclogenesis.

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New Insights into the Interplay between Non-Coding RNAs and RNA-Binding Protein HnRNPK in Regulating Cellular Functions

Cells ◽

10.3390/cells8010062 ◽

2019 ◽

Vol 8 (1) ◽

pp. 62 ◽

Cited By ~ 12

Author(s):

Yongjie Xu ◽

Wei Wu ◽

Qiu Han ◽

Yaling Wang ◽

Cencen Li ◽

...

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Rna Binding ◽

Binding Protein ◽

Genomic Structure ◽

Rna Binding Protein ◽

Stem Cell Differentiation ◽

Cellular Functions ◽

Non Coding Rnas ◽

Conserved Gene

The emerging data indicates that non-coding RNAs (ncRNAs) epresent more than the “junk sequences” of the genome. Both miRNAs and long non-coding RNAs (lncRNAs) are involved in fundamental biological processes, and their deregulation may lead to oncogenesis and other diseases. As an important RNA-binding protein (RBP), heterogeneous nuclear ribonucleoprotein K (hnRNPK) is known to regulate gene expression through the RNA-binding domain involved in various pathways, such as transcription, splicing, and translation. HnRNPK is a highly conserved gene that is abundantly expressed in mammalian cells. The interaction of hnRNPK and ncRNAs defines the novel way through which ncRNAs affect the expression of protein-coding genes and form autoregulatory feedback loops. This review summarizes the interactions of hnRNPK and ncRNAs in regulating gene expression at transcriptional and post-transcriptional levels or by changing the genomic structure, highlighting their involvement in carcinogenesis, glucose metabolism, stem cell differentiation, virus infection and other cellular functions. Drawing connections between such discoveries might provide novel targets to control the biological outputs of cells in response to different stimuli.

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Post-transcriptional regulation of gene expression by Unr

Biochemical Society Transactions ◽

10.1042/bst20140271 ◽

2015 ◽

Vol 43 (3) ◽

pp. 323-327 ◽

Cited By ~ 12

Author(s):

Swagat Ray ◽

Pól Ó Catnaigh ◽

Emma C. Anderson

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Protein Interactions ◽

Rna Binding ◽

Regulation Of Gene Expression ◽

Negative Regulator ◽

Cellular Translation ◽

Cellular Mrnas ◽

Internal Initiation ◽

Post Transcriptional Regulation

Unr (upstream of N-ras) is a eukaryotic RNA-binding protein that has a number of roles in the post-transcriptional regulation of gene expression. Originally identified as an activator of internal initiation of picornavirus translation, it has since been shown to act as an activator and inhibitor of cellular translation and as a positive and negative regulator of mRNA stability, regulating cellular processes such as mitosis and apoptosis. The different post-transcriptional functions of Unr depend on the identity of its mRNA and protein partners and can vary with cell type and changing cellular conditions. Recent high-throughput analyses of RNA–protein interactions indicate that Unr binds to a large subset of cellular mRNAs, suggesting that Unr may play a wider role in translational responses to cellular signals than previously thought.

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Identification and Functional Characterization of a Novel Nuclear Localization Signal Present in the Yeast Nab2 Poly(A)+ RNA Binding Protein

Molecular and Cellular Biology ◽

10.1128/mcb.18.3.1449 ◽

1998 ◽

Vol 18 (3) ◽

pp. 1449-1458 ◽

Cited By ~ 46

Author(s):

Ray Truant ◽

Robert A. Fridell ◽

R. Edward Benson ◽

Hal Bogerd ◽

Bryan R. Cullen

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Mammalian Cells ◽

Rna Binding ◽

Yeast Cells ◽

Vitro Assay ◽

Localization Signal

ABSTRACT The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin α/importin α, which acts as the NLS receptor, and karyopherin β1/importin β, which binds karyopherin α and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin β1, termed karyopherin β2 or transportin, and does not require a karyopherin α-like adapter protein. A yeast homolog of karyopherin β2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin β1, but not the Kap104p homolog karyopherin β2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin α. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

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