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G Brewer

Publications and source records attributed to G Brewer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Control of mRNA decay by heat shock-ubiquitin-proteasome pathway.

Cytokine and proto-oncogene messenger RNAs (mRNAs) are rapidly degraded through AU-rich elements in the 3' untranslated region. Rapid decay involves AU-rich binding protein AUF1, which complexes with heat shock proteins hsc70-hsp70, translation initiation factor eIF4G, and poly(A) binding protein. AU-rich mRNA decay is associated with displacement of eIF4G from AUF1, ubiquitination of AUF1, and degradation of AUF1 by proteasomes. Induction of hsp70 by heat shock, down-regulation of the ubiquitin-proteasome network, or inactivation of ubiquitinating enzyme E1 all result in hsp70 sequestration of AUF1 in the perinucleus-nucleus, and all three processes block decay of AU-rich mRNAs and AUF1 protein. These results link the rapid degradation of cytokine mRNAs to the ubiquitin-proteasome pathway.

3' Untranslated Regions↗
PubMed1999

Identification of AUF-1 ligands reveals vast diversity of early response gene mRNAs.

Cell activation is associated with diverse and widespread changes in gene expression at both the transcriptional and post-transcriptional levels. AUF1 is a recently described cytoplasmic protein which likely participates in the post-transcriptional regulation (PTR) of AU-rich (ARE) mRNAs including those coding for cytokines and proto-oncogenes. Individual mRNAs subject to AUF1-mediated PTR can be predicted if AREs are present or the mRNA in question interacts in vitro or in vivo with AUF1. However, there are few, if any, general approaches for characterizing the overall repertoire of mRNAs subject to PTR by AUF1. In an effort to identify these mRNAs, we incubated total mRNA from mitogen-activated peripheral blood mono-nuclear cells (PBMCs) with AUF1 in vitro. AUF1-mRNA complexes were retarded on membranes, bound mRNAs eluted with high salt, and either used to generate a cDNA library or rebound to AUF1 a second or third time prior to elution and cDNA library construction. We have obtained partial nucleotide sequences from 130 clones which shows that the AUF1 selected libraries are rich in mRNAs containing 3' untranslated region AREs including a large number of early response gene cDNAs. As a test of the validity of this method, we also show that a randomly selected, novel mRNA contained in the library is stabilized upon cell activation.

Chromatography, Affinity↗
PubMed1999

Identification and characterization of proteins binding A + U-rich elements.

A + U-Rich elements (AREs) have been extensively investigated as cis-acting determinants of rapid mRNA turnover. Recently, a number of RNA-binding proteins interacting with AREs have been described. This article presents strategies and techniques used by our laboratory to identify and characterize a family of ARE-binding proteins collectively termed AUF1. However, these techniques may be applied to the study of any protein displaying sequence-specific RNA binding activity. The techniques described here include the purification of native AUF1 from cultured cells as well as the preparation of recombinant AUF1 proteins using a bacterial expression system. Analyses of RNA-protein interactions are also described, including the use of gel mobility shift assays with synthetic RNA probes to monitor specific RNA binding activity in cell extracts or with recombinant proteins. Variations of this technique are also described to evaluate the RNA binding affinity of recombinant proteins and the use of specific RNA competitors to assess RNA determinants of protein binding specificity. Other techniques presented include the identification of specific proteins in RNA:protein complexes using antibody supershifts and the estimation of molecular weights of RNA-binding proteins by UV crosslinking. Results of individual experiments are presented as examples of some techniques. Throughout the article, suggestions are included to avoid commonly encountered problems and to assist in the optimization of these techniques for the study of other RNA-binding proteins.

Adenine↗
PubMed1999

The search for trans-acting factors controlling messenger RNA decay.

Control of mRNA turnover is an integral component of regulated gene expression. Individual mRNAs display a wide range of stabilities, which in many cases have been linked to discrete sequence elements. The most extensively characterized determinants of rapid constitutive mRNA turnover in mammalian systems are A + U-rich elements (AREs), first identified in the 3' untranslated regions of many cytokine/lymphokine and protooncogene mRNAs. In this article, we describe recent advances in the characterization of ARE-directed mRNA turnover, including links to deadenylation kinetics and functional heterogeneity among AREs from different mRNAs. We then describe strategies employed in the search for trans-acting factors interacting with these elements. Using such techniques, an ARE-binding activity capable of accelerating c-myc mRNA turnover in vitro was identified, and named AUF1. Subsequent cloning and characterization revealed that AUF1 exists as a family of four proteins formed by alternative splicing of a common pre-mRNA and appears to function as part of a multisubunit trans-acting complex to promote ARE-directed mRNA turnover. Investigations using several systems have demonstrated that AUF1 expression and/or activity correlate with rapid decay of ARE-containing mRNAs, and that both expression and activity of AUF1 are regulated by developmental and signal transduction mechanisms.

Amino Acid Sequence↗
PubMed1999

Regulation of AUF1 expression via conserved alternatively spliced elements in the 3' untranslated region.

The A+U-rich RNA-binding factor AUF1 exhibits characteristics of a trans-acting factor contributing to the rapid turnover of many cellular mRNAs. Structural mapping of the AUF1 gene and its transcribed mRNA has revealed alternative splicing events within the 3' untranslated region (3'-UTR). In K562 erythroleukemia cells, we have identified four alternatively spliced AUF1 3'-UTR variants, including a population of AUF1 mRNA containing a highly conserved 107-nucleotide (nt) 3'-UTR exon (exon 9) and the adjacent downstream intron (intron 9). Functional analyses using luciferase-AUF1 3'-UTR chimeric transcripts demonstrated that the presence of either a spliceable or an unspliceable intron 9 in the 3'-UTR repressed luciferase expression in cis, indicating that intron 9 sequences may down-regulate gene expression by two distinct mechanisms. In the case of the unspliceable intron, repression of luciferase expression likely involved two AUF1-binding sequences, since luciferase expression was increased by deletion of these sites. However, inclusion of the spliceable intron in the luciferase 3'-UTR down-regulated expression independent of the AUF1-binding sequences. This is likely due to nonsense-mediated mRNA decay (NMD) owing to the generation of exon-exon junctions more than 50 nt downstream of the luciferase termination codon. AUF1 mRNA splice variants generated by selective excision of intron 9 are thus also likely to be subject to NMD since intron 9 is always positioned >137 nt downstream of the stop codon. The distribution of alternatively spliced AUF1 transcripts in K562 cells is consistent with this model of regulated AUF1 expression.

Alternative Splicing↗
PubMed1998

Characterization of c-myc 3' to 5' mRNA decay activities in an in vitro system.

The levels of mRNA and protein encoded by the c-myc protooncogene set the balance between proliferation and differentiation of mammalian cells. Thus, it is essential for the cell to tightly control c-myc expression. Indeed, cells utilize many mechanisms to control c-myc expression, including transcription, RNA processing, translation, and protein stability. We have focused on turnover of c-myc mRNA as a key modulator of the timing and level of c-myc expression. c-myc mRNA is labile in cells, and its half-life is controlled by multiple instability elements located within both the coding region and the 3'-untranslated region (3'-UTR). Much work has focused on the protein factors that bind the instability elements, yet little is known about the enzymatic activities that effect the degradation of c-myc mRNA. Here I have utilized a novel cell-free mRNA decay system to characterize the c-myc mRNA decay machinery. This machinery consists of 3' to 5' mRNA decay activities that are Mg2+-dependent, require neither exogenous ATP/GTP nor an ATP-regenerating system, and act independently of a 7mG(5')ppp(5')G cap structure to deadenylate an exogenous mRNA substrate in a c-myc 3'-UTR-dependent fashion. Following deadenylation, nucleolytic decay of the 3'-UTR occurs generating 3' decay intermediates via a ribonucleolytic activity that can assemble on the c-myc 3'-UTR in a poly(A)-independent manner.

3' Untranslated Regions↗
PubMed1998

Developmental expression of AUF1 and HuR, two c-myc mRNA binding proteins.

Several proteins that may regulate c-myc mRNA post-transcriptionally were previously isolated and characterized. Two of them, HuR and AUF1, bind specifically to the 3' untranslated region (UTR) of c-myc mRNA. Because c-myc is regulated post-transcriptionally in various mouse tissues, including quiescent tissues, fetal liver and regenerating liver, we investigated whether HuR and AUF1 expression was also regulated in these tissues. Concerning AUF1, we analysed the expression of various mRNA and protein isoforms. We discovered a new AUF1 mRNA variant with a long AU-rich 3' UTR. We show that AUF1 expression, regardless of the RNA isoform considered, and HuR mRNA expression parallel c-myc expression in quiescent tissues and during liver development; their expression is high in lymphoid tissues and fetal liver and low in adult liver. However, no upregulation of HuR or AUF1 accompanies the upregulation of c-myc mRNA following partial hepatectomy. We discuss our results in relation to the current hypothesis that HuR and AUF1 act as mRNA destabilizing factors.

Animals↗
PubMed1998

Structure and genomic organization of the human AUF1 gene: alternative pre-mRNA splicing generates four protein isoforms.

The steady-state levels of many mRNAs are determined in part by their turnover rates. Turnover rates, in turn, are usually controlled by proteins that bind cis-acting sequence elements in mRNAs. One class of cis-acting instability determinants is composed of A + U-rich elements present in the 3'-UTRs of many labile mRNAs. Many A + U-rich elements are bound by the AUF1 family of RNA-binding proteins, which may target these mRNAs for rapid decay. cDNA cloning and immunoblot analyses suggest that the AUF1 family consists of at least four isoforms. Previous genomic cloning combined with FISH and Southern analyses of a panel of monochromosomal mouse/human or hamster/human somatic cell hybrids localized two AUF1 loci to human 4q21.1-q21.2 and Xq12 (B. Wagner et al., 1996, Genomics 34: 219-222). In the present study AUF1 gene organization was examined. The results suggest that the four known AUF1 isoforms are generated by alternative pre-mRNA splicing of a transcript encoded by the chromosome 4 locus. Functionally, this creates isoforms with different RNA-binding affinities and specificities. Thus, alternative pre-mRNA splicing may serve to create functional versatility within the AUF1 family of proteins.

Alternative Splicing↗
PubMed1997

Structural determinants in AUF1 required for high affinity binding to A + U-rich elements.

AUF1 is an RNA-binding protein that contains two nonidentical RNA recognition motifs (RRMs). AUF1 binds to A + U-rich elements (AREs) with high affinity. The binding of AUF1 to AREs is believed to serve as a signal to an mRNA-processing pathway that degrades mRNAs encoding many cytokines, oncoproteins, and G protein-coupled receptors. Because the ARE binding activity of AUF1 appears central to the regulation of many important genes, we analyzed the domains of the protein that are important for this activity. Examination of the RNA binding affinity of various AUF1 mutants suggests that both RRMs may be required for binding to the human c-fos ARE. However, the two RRMs together are not sufficient. Highest affinity binding of AUF1 to an ARE requires an alanine-rich region of the N terminus and a short glutamine-rich region in the C terminus. In addition, the N terminus is required for dimerization of AUF1. However, AUF1 binds an ARE as a hexameric protein. Thus, protein-protein interactions are important for high affinity ARE binding activity of AUF1.

Adenine↗
PubMed1997

What is a reasonable weight loss? Patients' expectations and evaluations of obesity treatment outcomes.

Expert panels and governmental guidelines now recommend that obese persons seek modest (i.e., "reasonable") reductions in body weight rather than striving for "ideal" weights. Little is known, however, about patients' views of what is a "reasonable" weight loss. This study assessed patients' goals, expectations, and evaluations of various outcomes before, during, and after 48 weeks of treatment. Before treatment, 60 obese women (99.1 +/- 12.3 kg; body mass index of 36.3 +/- 4.3 kg/ m2) defined their goal weight and 4 other weights: "dream weight"; "happy weight"; "acceptable weight"; and "disappointed weight." Goal weight averaged a 32% reduction in body weight. A 17-kg weight loss was defined as disappointed; a 25-kg loss, was acceptable. After 48 weeks of treatment and a 16-kg weight loss, 47% of patients did not achieve even a disappointed weight. These data illustrate the dramatic disparity between patients' expectations and professional recommendations and the need to help patients accept more modest weight loss outcomes.

Adult↗
PubMed1997

Adhesion-dependent regulation of an A+U-rich element-binding activity associated with AUF1.

Monocyte adherence results in the rapid transcriptional activation and mRNA stabilization of numerous mediators of inflammation and tissue repair. While the enhancer and promoter elements associated with transcriptional activation have been studied, mechanisms linking adhesion, mRNA stabilization, and translation are unknown. GROalpha and interleukin-1beta (IL-1beta) mRNAs are highly labile in nonadhered monocytes but stabilize rapidly after adherence. GROalpha and IL-1beta transcripts both contain A+U-rich elements (AREs) in the 3' untranslated region (UTR) which have been directly associated with rapid mRNA turnover. To determine if the GROalpha ARE region was recognized by factors associated with mRNA degradation, we carried out mobility gel shift analyses using a series of RNA probes encompassing the entire GROalpha transcript. Stable complexes were formed only with the proximal 3' UTR which contained the ARE region. The two slower-moving complexes were rapidly depleted following monocyte adherence but not direct integrin engagement. Deadherence reactivated the two largest ARE-binding complexes and destabilized IL-1beta transcripts. Antibody supershift studies demonstrated that both of these ARE RNA-binding complexes contained AUF1. The formation of these complexes and the accelerated mRNA turnover are phosphorylation-dependent events, as both are induced in adherent monocytes by the tyrosine kinase inhibitor genistein and the p38 MAP kinase inhibitor of IL-1beta translation, SK&F 86002. These results demonstrate that cell adhesion and deadhesion rapidly and reversibly modify both cytokine mRNA stability and the RNA-binding complexes associated with AUF1.

Cell Adhesion↗
PubMed1997

Identification of AUF1 (heterogeneous nuclear ribonucleoprotein D) as a component of the alpha-globin mRNA stability complex.

mRNA turnover is an important regulatory component of gene expression and is significantly influenced by ribonucleoprotein (RNP) complexes which form on the mRNA. Studies of human alpha-globin mRNA stability have identified a specific RNP complex (alpha-complex) which forms on the 3' untranslated region (3'UTR) of the mRNA and appears to regulate the erythrocyte-specific accumulation of alpha-globin mRNA. One of the protein activities in this multiprotein complex is a poly(C)-binding activity which consists of two proteins, alphaCP1 and alphaCP2. Neither of these proteins, individually or as a pair, can bind the alpha-globin 3'UTR unless they are complexed with the remaining non-poly(C) binding proteins of the alpha-complex. With the yeast two-hybrid screen, a second alpha-complex protein was identified. This protein is a member of the previously identified A+U-rich (ARE) binding/degradation factor (AUF1) family of proteins, which are also known as the heterogeneous nuclear RNP (hnRNP) D proteins. We refer to these proteins as AUF1/hnRNP-D. Thus, a protein implicated in ARE-mediated mRNA decay is also an integral component of the mRNA stabilizing alpha-complex. The interaction of AUF1/hnRNP-D is more efficient with alphaCP1 relative to alphaCP2 both in vitro and in vivo, suggesting that the alpha-complex might be dynamic rather than a fixed complex. AUF1/hnRNP-D could, therefore, be a general mRNA turnover factor involved in both stabilization and decay of mRNA.

DNA-Binding Proteins↗
PubMed1997

Wilson's disease: presymptomatic patients and Kayser-Fleischer rings.

PURPOSE: We evaluated patients with Wilson's disease to determine (1) whether presymptomatic patients who have Kayser-Fleischer (K F) rings demonstrate a more significant alteration of copper metabolism than those who do not have K F rings, and (2) whether presymptomatic patients have smaller K F rings than symptomatic Wilson's disease patients. METHODS: Thirty-two patients with presymptomatic Wilson's disease were retrospectively analyzed. Sixteen of these had received no prior anti-copper therapy and underwent testing for baseline copper metabolism (24-hour urine copper, liver copper, and plasma ceruloplasmin). Quantitative measurements of K F rings were made for the group of untreated presymptomatic patients and a control group of symptomatic Wilson's disease patients. RESULTS: We hypothesized that the 24-hour urine copper, in particular, would correlate with the presence of a K F ring. However, no significant difference was found between any of the baseline copper variables for presymptomatic patients who had K F rings compared to those who did not. K F rings of presymptomatic patients were found to be significantly smaller than K F rings of patients with symptomatic Wilson's disease (p < 0.05). CONCLUSIONS: While this study does not show any relationship between urinary copper excretion and the presence of K F rings, it suggests that the larger K F ring size correlates with Wilson's disease severity.

Adolescent↗
PubMed1996

Increased granulocyte-macrophage colony-stimulating factor mRNA instability in cord versus adult mononuclear cells is translation-dependent and associated with increased levels of A + U-rich element binding factor.

The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA is fourfold lower in phorbol myristate acetate (PMA) + phytohemagglutinin (PHA)-activated mononuclear cells (MNC) from newborns compared with adults. The GM-CSF transcription rate is similar in umbilical cord and adult MNC, but transcript half-life is threefold lower in cord activated MNC. Interaction of RNA binding proteins, such as the cloned adenosine + uridine-rich element, binding factor, AUF1, with eight AUUUA motifs in the human GM-CSF mRNA 3'-untranslated region (GM-3'-UTR) has been implicated in regulating transcript stability. Translational inhibition by cycloheximide (CHX) significantly increased GM-CSF mRNA accumulation and half-life by three-fold in activated cord MNC, but had a minimal effect in activated adult MNC as compared with PMA + PHA alone. Electrophoretic mobility-shift assays with a 32P-labeled, 305-nucleotide RNA comprising the GM-3'-UTR revealed two RNaseT1-resistant, bound complexes that were almost twice as abundant in cord than in adult MNC extracts. Mobility-shift competition assays and RNaseT1 mapping localized the binding site of both complexes to a 52-nucleotide region containing seven of eight AUUUA motifs. Inclusion of AUF1 antiserum produced a supershifted complex at 35-fold higher levels in cord than in adult MNC extracts. Extracts from the carcinoma cell line 5637, with extended GM-CSF mRNA half-life, also had very low levels of anti-AUF1 supershifted complex. Anti-AUF1 immunoblotting showed significantly higher levels of two AUF1 protein isoforms and lower levels of one in cord than in adult MNC or 5637 extracts. These results suggest that destabilization of GM-CSF mRNA in cord MNC is translation-dependent and that increased levels of specific AUF1 isoforms in cord MNC may target transcripts for increased degradation, which could account in part for dysregulation of neonatal phagocytic immunity.

Adult↗
PubMed1996

Localization and physical mapping of genes encoding the A+U-rich element RNA-binding protein AUF1 to human chromosomes 4 and X.

Messenger RNAs encoding many oncoproteins and cytokines are relatively unstable. Their instability, which ensures appropriate levels and timing of expression, is controlled in part by proteins that bind to A+U-rich instability elements (AREs) present in the 3'-untranslated regions of the mRNAs. cDNAs encoding the AUF1 family of ARE-binding proteins were cloned from human and murine cDNA libraries. In the present study monochromosomal somatic cell hybrids were used to localize two AUF1 loci to human chromosomes 4 and X. In situ hybridization analyses using P1 clones as probes identified the 4q21.1-q21.2 and Xq12 regions as the locations of the AUF1 genes.

Animals↗
PubMed1996

AUF1 binding affinity to A+U-rich elements correlates with rapid mRNA degradation.

Rapid degradation of many labile mRNAs is regulated in part by an A + U-rich element (ARE) in their 3'-untranslated regions. Extensive mutational analyses of various AREs have identified important components of the ARE, such as the nonamer motif UUAUUUAUU, two copies of which serve as a potent mRNA destabilizer. To investigate the roles of trans-acting factors in ARE-directed mRNA degradation, we previously purified and molecularly cloned the RNA-binding protein AUF1 and demonstrated that both cellular and recombinant AUF1 bind specifically to AREs as shown by UV cross-linking assays in vitro. In the present work, we have examined the in vitro RNA-binding properties of AUF1 using gel mobility shift assays with purified recombinant His6-AUF1 fusion protein. We find that ARE binding affinities of AUF1 correlate with the potency of an ARE to direct degradation of a heterologous mRNA. These results support a role for AUF1 in ARE-directed mRNA decay that is based upon its affinity for different AREs.

Adenine↗