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Gary Brewer

Publications and source records attributed to Gary Brewer.

16 recordsLinked to original sources
PubMed2006

Upf1/Upf2 regulation of 3' untranslated region splice variants of AUF1 links nonsense-mediated and A+U-rich element-mediated mRNA decay.

AUF1 is an RNA-binding protein that targets mRNAs containing A+U-rich elements (AREs) for rapid cytoplasmic turnover. Alternative pre-mRNA splicing produces five variants of AUF1 mRNA that differ in the composition of their 3'-untranslated regions (3'-UTRs). Previous work suggested that this heterogeneity in 3'-UTR sequence could regulate AUF1 expression by two potential mechanisms. First, AUF1 may regulate its own expression by binding to AREs in 3'-UTR splice variants that retain intron 9. The second potential mechanism, and the focus of this report, is regulation of a subset of 3'-UTR splice variants by the nonsense-mediated mRNA decay (NMD) pathway. Two of the five AUF1 mRNA 3'-UTR variants position the translational termination codon more than 50 nucleotides upstream of an exon-exon junction, creating a potential triggering signal for NMD in mammalian cells. Disruption of cellular NMD pathways by RNA interference-mediated knockdown of Upf1/Rent1 or Upf2/Rent2 or transfection of a dominant-negative Upf1 mutant specifically enhanced expression of these two candidate NMD substrate mRNAs in cells, involving stabilization of each transcript. Ribonucleoprotein immunoprecipitation experiments revealed that both Upf1 and Upf2 can associate with an NMD-sensitive AUF1 mRNA 3'-UTR variant in cells. Finally, quantitation of AUF1 mRNA 3'-UTR splice variants during murine embryonic development showed that the expression of NMD-sensitive AUF1 mRNAs is specifically enhanced as development proceeds, contributing to dynamic changes in AUF1 3'-UTR structures during embryogenesis. Together, these studies provide the first evidence of linkage between the nonsense- and ARE-mediated mRNA decay pathways, which may constitute a new mechanism regulating the expression of ARE-containing mRNAs.

3' Untranslated Regions↗
PubMed2006

Posttranscriptional derepression of GADD45alpha by genotoxic stress.

The growth arrest- and DNA damage-inducible gene GADD45alpha is potently upregulated in response to stress stimuli. Here, two RNA binding proteins, the mRNA decay-promoting AUF1 and the translational suppressor TIAR, were found to interact specifically with the 3' untranslated region (UTR) of the GADD45alpha mRNA in HeLa cells. These associations were prominent in unstimulated cells, decreasing dramatically after treatment with the genotoxin methyl methanesulfonate (MMS). Analysis of both endogenous and chimeric GADD45alpha mRNA revealed that in untreated cells AUF1 strongly reduced GADD45alpha mRNA stability, whereas TIAR potently inhibited GADD45alpha translation. After genotoxic stress, AUF1 and TIAR dissociated from the GADD45alpha mRNA, thereby allowing coordinated elevations in both GADD45alpha mRNA half-life and translation rate, respectively. We propose that the posttranscriptional derepression of GADD45alpha critically contributes to its potent upregulation after DNA damage.

3' Untranslated Regions↗
PubMed2005

The RNA-binding protein IMP-3 is a translational activator of insulin-like growth factor II leader-3 mRNA during proliferation of human K562 leukemia cells.

IMP-3, a member of the insulin-like growth factor-II (IGF-II) mRNA-binding protein (IMP) family, is expressed mainly during embryonic development and in some tumors. Thus, IMP-3 is considered to be an oncofetal protein. The functional significance of IMP-3 is not clear. To identify the functions of IMP-3 in target gene expression and cell proliferation, RNA interference was employed to knock down IMP-3 expression. Using human K562 leukemia cells as a model, we show that IMP-3 protein associates with IGF-II leader-3 and leader-4 mRNAs and H19 RNA but not c-myc and beta-actin mRNAs in vivo by messenger ribonucleoprotein immunoprecipitation analyses. IMP-3 knock down significantly decreased levels of intracellular and secreted IGF-II without affecting IGF-II leader-3, leader-4, c-myc, or beta-actin mRNA levels and H19 RNA levels compared with the negative control siRNA treatment. Moreover, IMP-3 knock down specifically suppressed translation of chimeric IGF-II leader-3/luciferase mRNA without altering reporter mRNA levels. Together, these results suggest that IMP-3 knock down reduced IGF-II expression by inhibiting translation of IGF-II mRNA. IMP-3 knock down also markedly inhibited cell proliferation. The addition of recombinant human IGF-II peptide to these cells restored cell proliferation rates to normal. IMP-3 and IMP-1, two members of the IMP family with significant structural similarity, appear to have some distinct RNA targets and functions in K562 cells. Thus, we have identified IMP-3 as a translational activator of IGF-II leader-3 mRNA. IMP-3 plays a critical role in regulation of cell proliferation via an IGF-II-dependent pathway in K562 leukemia cells.

5' Untranslated Regions↗
PubMed2005

The c-Yes 3'-UTR contains adenine/uridine-rich elements that bind AUF1 and HuR involved in mRNA decay in breast cancer cells.

c-Yes is a member of the c-Src family of tyrosine kinases and has been implicated in intracellular signaling, cell morphology, and adhesion. Changes in its expression have also been associated with the aggressiveness of human breast and colon cancer cells. In MDA-MB-231 human breast cancer cells, overexpression of the small heat shock protein 27 (hsp27) results in a downregulation of c-Yes levels, concomitant with increased in vitro invasiveness and in vivo metastatic behavior. Very little is known, however, about the mechanisms regulating c-Yes expression. Here, we demonstrate that hsp27-induced c-Yes downregulation is not due to a reduction in transcriptional activity. However, the 3'-untranslated region (3'-UTR) of the c-Yes gene may be involved in its own regulation, since this region affects heterologous reporter gene activity in transactivation assays. This down-regulatory effect maps to three adenine/uridine-rich elements (AREs) that bind to cellular HuR and AUF1 (hnRNP D), two ARE-binding proteins (ARE-BPs) implicated in accelerated mRNA degradation. Our results suggest that the c-Yes 3'-UTR contains at least three newly identified AREs which are bound specifically by ARE-BPs, and provide a structural basis for post-transcriptional regulation of c-Yes expression.

3' Untranslated Regions↗
PubMed2004

Targeted knockdown of the RNA-binding protein CRD-BP promotes cell proliferation via an insulin-like growth factor II-dependent pathway in human K562 leukemia cells.

The c-myc mRNA coding region determinant-binding protein (CRD-BP) was first identified as a masking protein that stabilizes c-myc mRNA in a cell-free mRNA degradation system. Thus, CRD-BP is thought to promote cell proliferation by maintaining c-Myc at critical levels. CRD-BP also appears to be an oncofetal protein, based upon its expression during mammalian development and in some tumors. By using K562 leukemia cells as a model, we show that CRD-BP gene silencing by RNA interference significantly promoted proliferation, indicating an inhibitory effect of CRD-BP on proliferation. Unexpectedly, CRD-BP knockdown had no discernible effect on c-myc mRNA levels. CRD-BP is also known as insulin-like growth factor II (IGF-II) mRNA-binding protein-1. It has been reported to repress translation of a luciferase reporter mRNA containing an IGF-II 5'-untranslated region known as leader 3 but not one containing IGF-II leader 4. CRD-BP knockdown markedly increased IGF-II mRNA and protein levels but did not alter translation of luciferase reporter mRNAs containing 5'-untranslated regions consisting of either IGF-II leader 3 or leader 4. Addition of antibody against IGF-II to cell cultures inhibited the proliferative effect of CRD-BP knockdown, suggesting that regulation of IGF-II gene expression, rather than c-myc mRNA levels, mediates the proliferative effect of CRD-BP knockdown. Thus, we have identified a dominant function for CRD-BP in cell proliferation of human K562 cells, involving a possible IGF-II-dependent mechanism that appears independent of its ability to serve as a c-myc mRNA masking protein.

5' Untranslated Regions↗
PubMed2004

Identification of TINO: a new evolutionarily conserved BCL-2 AU-rich element RNA-binding protein.

Modulation of mRNA stability by regulatory cis-acting AU-rich elements (AREs) and ARE-binding proteins is an important posttranscriptional mechanism of gene expression control. We previously demonstrated that the 3'-untranslated region of BCL-2 mRNA contains an ARE that accounts for rapid BCL-2 down-regulation in response to apoptotic stimuli. We also demonstrated that the BCL-2 ARE core interacts with a number of ARE-binding proteins, one of which is AU-rich factor 1/heterogeneous nuclear ribonucleoprotein D, known for its interaction with mRNA elements of others genes. In an attempt to search for other BCL-2 mRNA-binding proteins, we used the yeast RNA three-hybrid system assay and identified a novel human protein that interacts with BCL-2 ARE. We refer to it as TINO. The predicted protein sequence of TINO reveals two amino-terminal heterogeneous nuclear ribonucleoprotein K homology motifs for nucleic acid binding and a carboxyl-terminal RING domain, endowed with a putative E3 ubiquitin-protein ligase activity. In addition the novel protein is evolutionarily conserved; the two following orthologous proteins have been identified with protein-protein BLAST: posterior end mark-3 (PEM-3) of Ciona savignyi and muscle excess protein-3 (MEX-3) of Caenorhabditis elegans. Upon binding, TINO destabilizes a chimeric reporter construct containing the BCL-2 ARE sequence, revealing a negative regulatory action on BCL-2 gene expression at the posttranscriptional level.

3' Untranslated Regions↗
PubMed2003

Regulation of A + U-rich element-directed mRNA turnover involving reversible phosphorylation of AUF1.

Proteins binding A + U-rich elements (AREs) contribute to the rapid cytoplasmic turnover of mRNAs containing these sequences. However, this process is a regulated event and may be accelerated or inhibited by myriad signal transduction systems. For example, monocyte adherence at sites of inflammation or tissue injury is associated with inhibition of ARE-directed mRNA decay, which contributes to rapid increases in cytokine and inflammatory mediator production. Here, we show that acute exposure of THP-1 monocytic leukemia cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate mimics several features of monocyte adherence, including rapid induction and stabilization of ARE-containing mRNAs encoding interleukin-1 beta and tumor necrosis factor alpha. Additionally, TPA treatment alters the activity of cytoplasmic complexes that bind AREs, including complexes containing the ARE-specific, mRNA-destabilizing factor, AUF1. Analyses of AUF1 from control and TPA-treated cells indicated that post-translational modifications of the major cytoplasmic isoform, p40AUF1, are altered concomitant with changes in RNA binding activity and stabilization of ARE-containing mRNAs. In particular, p40AUF1 recovered from polysomes was phosphorylated on Ser83 and Ser87 in untreated cells but lost these modifications following TPA treatment. We propose that selected signal transduction pathways may regulate ARE-directed mRNA turnover by reversible phosphorylation of polysome-associated p40AUF1.

Amino Acid Sequence↗
PubMed2003

Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure.

Messenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment.

Amino Acid Sequence↗
PubMed2003

Bcl-2 protein is required for the adenine/uridine-rich element (ARE)-dependent degradation of its own messenger.

We have shown previously that the decay of human bcl-2 mRNA is mediated by an adenine/uridine-rich element (ARE) located in the 3'-untranslated region. Here, we have utilized a non-radioactive cell-free mRNA decay system to investigate the biochemical and functional mechanisms regulating the ARE-dependent degradation of bcl-2 mRNA. Using RNA substrates, mutants, and competitors, we found that decay is specific and ARE-dependent, although maximized by the ARE-flanking regions. In unfractionated extracts from different cell types and in whole cells, the relative enzymatic activity was related to the amount of Bcl-2 protein expressed by the cells at steady state. The degradation activity was lost upon Bcl-2 depletion and was reconstituted by adding recombinant Bcl-2. Ineffective extracts from cells that constitutively do not express Bcl-2 acquire full degradation activity by adding recombinant Bcl-2 protein. We conclude that Bcl-2 is necessary to activate the degradation complex on the relevant RNA target.

3' Flanking Region↗
PubMed2003

Characteristics of the interaction of a synthetic human tristetraprolin tandem zinc finger peptide with AU-rich element-containing RNA substrates.

Tristetraprolin (TTP) and its two known mammalian family members are tandem CCCH zinc finger proteins that can bind to AU-rich elements (AREs) in cellular mRNAs and destabilize those transcripts, apparently by initiating their deadenylation. Previous studies have shown that the approximately 70-amino acid tandem zinc finger domain of TTP is required and sufficient for RNA binding, and that the integrity of both zinc fingers is also required. However, little is known about the kinetics or structure of the peptide-RNA interaction, in part because of difficulties in obtaining soluble recombinant protein or peptides. We characterized the binding of a synthetic 73-amino acid peptide from human TTP to the tumor necrosis factor (TNF) ARE by gel mobility shift analyses and fluorescence anisotropy experiments. Both types of studies yielded a peptide-RNA dissociation constant of approximately 10 nM. Surprisingly, we found that the "footprint" from the TNF ARE required for peptide binding was only approximately 9 bases and that two molecules of peptide could bind to probes containing as little as 19 bases. An identical recombinant peptide exhibited gel shift characteristics similar to those of the synthetic peptide. NMR analysis of the 15N-labeled recombinant peptide suggested that its first zinc finger was structured in solution but that the second was not. The titration of oligonucleotides representing 17, 13, and even 9 bases of the TNF ARE caused an essentially identical, dramatic shift of existing resonances, and the appearance of new resonances in the peptide spectra, so that all amino acids could be assigned. These data suggest that this TTP peptide-RNA complex is structured in solution and might be amenable to NMR structure determination.

Amino Acid Sequence↗
PubMed2003

Increased interleukin-10 mRNA stability in melanoma cells is associated with decreased levels of A + U-rich element binding factor AUF1.

Abnormal production of interleukin-10 (IL-10) is observed in some pathologic conditions. For example, compared with normal melanocytes, IL-10 expression is elevated in melanoma cells. IL-10 overexpression could inhibit both immune surveillance and tumor rejection. We investigated a potential posttranscriptional mechanism for IL-10 overexpression in melanoma cells. In normal melanocytes, the half-life of IL-10 mRNA is 7 min, whereas in the melanoma cell line MNT1, the half-life is 75 min. This 10-fold difference could account, at least in part, for IL-10 overexpression in MNT1 cells. Examination of the 3'-untranslated region (3'-UTR) of IL-10 mRNA revealed a suspected A + U-rich element (ARE) that might target the mRNA for rapid degradation. Transfection experiments confirmed that these sequences promote rapid degradation when inserted into a normally stable mRNA, indicating ARE functionality. As AREs act via their interactions with ARE-binding proteins, we examined cytoplasmic proteins from normal melanocytes and MNT1 cells for IL-10 ARE-binding activity. Compared with cytoplasmic extracts of normal melanocytes, cytoplasmic extracts of MNT1 cells possess substantially less ARE-binding activity, consistent with the extended half-life of IL-10 mRNA in MNT1 cells. Finally, we find that the ARE-binding protein AUF1 comprises the major ARE-binding activity in cytoplasmic extracts of normal melanocytes. By contrast, AUF1 is not detectable in cytoplasmic extracts of MNT1 cells but appears restricted to the nuclear fraction. Together, these data suggest a mechanism whereby reduced cytoplasmic levels of AUF1 in MNT1 melanoma cells may lead to IL-10 overexpression, with deleterious consequences for tumor surveillance and rejection.

3' Untranslated Regions↗
PubMed2002

Cell death inhibiting RNA (CDIR) derived from a 3'-untranslated region binds AUF1 and heat shock protein 27.

Regulators of programmed cell death were previously identified using a technical knockout genetic screen. Among the elements that inhibited interferon-gamma-induced apoptosis of HeLa cells was a 441-nucleotide fragment derived from the 3'-untranslated region (UTR) of KIAA0425, a gene of unknown function. This fragment was termed cell death inhibiting RNA (CDIR). Deletion and mutation analyses of CDIR were employed to identify the features required for its anti-apoptotic activity. Single nucleotide alterations within either copy of the duplicated U-rich motif found in the CDIR sequence abolished the anti-apoptotic activity of CDIR and altered its in vitro association with a protein complex. Further analysis of the CDIR-binding complex indicated that it contained heat shock protein 27 (Hsp27) and the regulator of mRNA turnover AUF1 (heterogeneous nuclear ribonucleoprotein D). In addition, recombinant AUF1 bound directly to CDIR. Furthermore, expression of another AUF1-binding RNA element, derived from the 3'-UTR of c-myc, inhibited apoptosis. We also demonstrate that the level and the stability of p21(waf1/Cip1/sdi1) mRNA, a target of AUF1 with anti-apoptotic activity, were increased in CDIR-transfected cells. The level of mRNA and protein of Bcl-2, another anti-apoptotic gene, containing an AUF1 binding site in its 3'-UTR was also increased in CDIR-transfected cells. Our data suggest that AUF1 regulates apoptosis by altering mRNA turnover. We propose that CDIR inhibits apoptosis by acting as a competitive inhibitor of AUF1, preventing AUF1 from binding to its targets.

3' Untranslated Regions↗
PubMed2002

Lactate dehydrogenase is an AU-rich element-binding protein that directly interacts with AUF1.

Post-transcriptional pathways provide a major means of regulating eukaryotic gene expression. Reiterations of the AU-rich element (ARE) within the 3'-untranslated region of many cytokine and proto-oncogene mRNAs serve as signals for rapid degradation and translational repression. The identification of this cis-acting stability determinant has fueled the search for ARE-binding proteins (AUBP) that function as trans-acting factors that transduce this function. Previous work identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a major AUBP capable of binding the ARE of granulocyte-macrophage colony stimulating factor (GM-CSF) RNA in the context of a full-length mRNA. We report here that functional studies failed to indicate a role for hnRNP A1 in ARE-dependent mRNA turnover. In an effort to identify other functionally relevant AUBP, the major GM-CSF ARE-specific binding protein in cells lacking hnRNP A1 was purified from CB3 mouse erythroleukemia cells. Microsequencing identified this protein as the glycolytic enzyme lactate dehydrogenase (LDH) M. RNA binding by LDH was shown to occur in the NAD(+)-binding region (Rossmann fold). Polysome gradient analysis demonstrates that LDH is found in the translationally active fraction. Polysomal localization of LDH was dependent on RNA binding. Moreover, polysomal LDH exists in a complex with AUF1 and hsp-70, which has been implicated previously in the regulation of mRNA turnover. The interaction between LDH and AUF1 is direct as it can be demonstrated in vitro with purified proteins. Collectively these data implicate a role for LDH in the post-transcriptional regulation of gene expression.

Animals↗
PubMed2002

AUF1 Is a bcl-2 A + U-rich element-binding protein involved in bcl-2 mRNA destabilization during apoptosis.

We previously identified a conserved A + U-rich element (ARE) in the 3'-untranslated region of bcl-2 mRNA. We have also recently demonstrated that the bcl-2 ARE interacts with a number of ARE-binding proteins (AUBPs) whose pattern changes during apoptosis in association with bcl-2 mRNA half-life reduction. Here we show that the AUBP AUF1 binds in vitro to bcl-2 mRNA. The results obtained in a yeast RNA three-hybrid system have demonstrated that the 1-257-amino acid portion of p37 AUF1 (conserved in all isoforms), containing the two RNA recognition motifs, also binds to the bcl-2 ARE in vivo. UVC irradiation-induced apoptosis results in an increase of AUF1. Inhibition of apoptosis by a general caspase inhibitor reduces this increase by 2-3-fold. These results indicate involvement of AUF1 in the ARE/AUBP-mediated modulation of bcl-2 mRNA decay during apoptosis.

Apoptosis↗
PubMed2002

Messenger RNA decay during aging and development.

Gene expression is a combination of many processes, including transcription, pre-mRNA processing, nucleocytoplasmic transport of mRNA, translation, mRNA decay, and protein modification and decay. Many changes in the programs of gene expression occur during development, differentiation, and aging. These alterations are reflected at both the mRNA and protein levels. While altered gene expression at the levels of transcription and protein turnover has been appreciated for some time, mRNA decay is now emerging as an important control point and a major contributor to gene expression as well. Continuing identification of the protein factors and cofactors, and mRNA instability elements, responsible for mRNA decay are allowing us to build a comprehensive picture of the highly orchestrated processes involved in mRNA decay and its regulation. Here, I survey mRNA decay processes in eukaryotes and describe some molecular mechanisms that alter the decay rates of specific mRNAs, leading to major changes in gene expression during development and aging.

Aging↗