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PubMed2005

The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation.

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5'-untranslated region (5'-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5'-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3'-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5'-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5'-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5'-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5'-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5'-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis.

5' Untranslated Regions↗
PubMed2005

Interaction of Staufen1 with the 5' end of mRNA facilitates translation of these RNAs.

Staufen1 is a component of transported ribonucleoprotein complexes. Genetic work in Drosophila has suggested that Staufen plays a role in the de-repression of translation of oskar mRNA following localization. To determine whether Staufen1 can play a similar role in mammals, we studied translation of transcripts in the presence or in the absence of Staufen1. Translationally repressed mRNAs were generated by fusing the structured human immunodeficiency virus type 1 trans-activating response (TAR) element to the 5' end of a reporter transcript. In rabbit reticulocyte lysates and in mammalian cultured cells, the addition of Staufen1 resulted in the up-regulation of reporter activity when translation was driven by the TAR-bearing RNA. In contrast, Staufen1 had no effect on translation of efficiently translated mRNAs lacking an apparent structured 5' end, suggesting that Staufen1-binding to the 5' end is required for enhanced translation. Consistently, Staufen1 RNA-binding activity is necessary for this translational effect. In addition, similar up-regulation of translation was observed when Staufen1 was tethered to the 5' end of mRNAs via other structured RNAs, the highest level of translational increase being obtained with the bona fide Staufen1-binding site of the Arf1 transcript. The expression of Staufen1 promoted polysomal loading of TAR-luciferase transcripts resulting in enhanced translation. Our results support a model in which the expression of Staufen1 and its interaction with the 5' end of RNA and ribosomes facilitate translation initiation.

Animals↗
PubMed1992

Regions of bacteriophage T4 and RB69 RegA translational repressor proteins that determine RNA-binding specificity.

RegA protein of T4 and related bacteriophages is a highly conserved RNA-binding protein that represses the translation of many phage mRNAs that encode enzymes involved in DNA metabolism. RB69, a T4-related bacteriophage, has a unique regA gene, which we have cloned, sequenced, and expressed. The predicted amino acid sequence of RB69 RegA is 78% identical to that of T4 RegA. Plasmid-encoded RB69 RegA expressed in vivo represses the translation of T4 early mRNAs, including those of rIIA, rIIB, 44, 45, rpbA, and regA. Nucleotide sequences were determined for several T4 and RB69 regA mutations, and their corresponding repressor properties were characterized. All of the 10 missense mutations affect residues conserved between RB69 and T4 RegA. Two regions of RegA are especially sensitive to mutation: one between Val-15 and Ala-25 and another between Arg-70 and Ser-73. Sequence alignments and mutational data suggest that the region from Val-15 to Ala-25 is similar to helix-turn-helix domains of DNA-binding proteins and confers RNA-binding specificity upon RegA. The RegA691 protein (Ile-24----Thr) has an in vivo phenotype that appears to distinguish site-specific and cooperative binding modes of hierarchical RegA-mediated translational repression.

Amino Acid Sequence↗
PubMed1997

Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli.

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5' strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3' strand are located 280 nt downstream, immediately upstream of the Shine-Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

Bacterial Proteins↗
PubMed1999

Translational control by an upstream open reading frame in the HER-2/neu transcript.

Overexpression of the HER-2 (neu, erbB-2) receptor results in cellular transformation and is associated with a variety of human cancers. Multiple mechanisms, including gene amplification and transcriptional, post-transcriptional, and translational controls contribute to the regulation of HER-2 expression. One of the components of these regulatory mechanisms is a short upstream open reading frame (uORF) in the HER-2 mRNA that represses downstream translation in a variety of cell types. Here we explore the mechanism by which this uORF exerts its inhibitory effect. As judged by comparisons of protein and mRNA abundance and by polysomal distribution analyses, the uORF represses translation of the HER-2 cistron or of a heterologous reporter gene. Despite its conservation among mammalian species, the peptide sequence of the uORF is not required for this inhibitory effect. Rather, the majority of ribosomes that load on the HER-2 mRNA most likely translate the uORF and are then unable to reinitiate at the downstream AUG codon, in part due to the short intercistronic spacing. A minority of ribosomes gain access to the HER-2 initiation codon either by leaky scanning past the upstream AUG codon or by reinitiating after having translated the uORF despite the short intercistronic region. These results suggest that the HER-2 uORF controls synthesis of this oncoprotein by limiting ribosomal access to downstream initiation sites.

Animals↗
PubMed1990

Endotoxin-responsive sequences control cachectin/tumor necrosis factor biosynthesis at the translational level.

The biosynthesis of cachectin/TNF is largely regulated at a post-transcriptional level. Bacterial endotoxin, which strongly induces cachectin/TNF production, thus seems to elicit at least some of its effects by altering the macrophage cytoplasmic milieu. It has previously been shown that the 3'-untranslated TTATTTAT element present in numerous cytokines and proto-oncogenes is capable of repressing the translation of mRNA molecules in which it is represented. Using constructs in which the CAT coding sequence is followed by varying segments of the cachectin/TNF 3'-untranslated region, we now demonstrate that downstream sequences present in the cachectin/TNF mRNA are sufficient to mediate greater than 200-fold induction of CAT synthesis in response to activation by endotoxin. Induction of CAT activity is not attributable to a change in cytoplasmic mRNA concentration, but to a marked enhancement of translational efficiency. The response to endotoxin represents "derepression," and is conferred chiefly by the translationally repressive TTATTTAT element, acting in concert with essential flanking sequences.

Base Sequence↗
PubMed2001

A conserved role of a DEAD box helicase in mRNA masking.

Clam p82 is a member of the cytoplasmic polyadenylation element-binding protein (CPEB) family of RNA-binding proteins and serves dual functions in regulating gene expression in early development. In the oocyte, p82/CPEB is a translational repressor, whereas in the activated egg, it acts as a polyadenylation factor. Coimmunoprecipitations were performed with p82 antibodies in clam oocyte and egg lysates to identify stage-regulated accessory factors. p47 coprecipitates with p82 from oocyte lysates in an RNA-dependent manner and is absent from egg lysate p92-bound material. Clam p47 is a member of the RCK/p54 family of DEAD box RNA helicases. Xp54, the Xenopus homolog, with bona fide helicase activity, is an abundant and integral component of stored mRNP in oocytes (Ladomery et al., 1997). In oocytes, clam p47 and p82/CPEB are found in large cytoplasmic mRNP complexes. Whereas the helicase level is constant during embryogenesis, in contrast to CPEB, clam p47 translocates to nuclei at the two-cell stage. To address the role of this class of helicase in masking, Xp54 was tethered via 3' UTR MS2-binding sites to firefly luciferase, following microinjection of fusion protein and nonadenylated reporter mRNAs into Xenopus oocytes. Tethered helicase repressed luciferase translation three- to fivefold and, strikingly, mutations in two helicase motifs (DEAD--> DQAD and HRIGR-->HRIGQ), activated translation three- to fourfold, relative to MS2. These data suggest that this helicase family represses translation of maternal mRNA in early development, and that its activity may be attenuated during meiotic maturation, prior to cytoplasmic polyadenylation.

Amino Acid Sequence↗
PubMed2004

Expression of the Alzheimer protease BACE1 is suppressed via its 5'-untranslated region.

The aspartyl protease BACE1 has a pivotal role in the pathogenesis of Alzheimer's disease. Recently, it was shown that in Alzheimer's disease patients, BACE1 levels were elevated although mRNA levels were not changed compared with controls. Here, we demonstrate that the 5'-untranslated region (5'UTR) of BACE1 controls the rate of BACE1 translation. In the presence of the 5'UTR, we observed more than 90% reduction of BACE1 protein levels in HEK293, COS7 and H4 cells, and a similar reduction of BACE1 activity in vitro. mRNA levels were not affected, demonstrating that the 5'UTR repressed the translation but not the transcription of BACE1. The 3'UTR did not affect BACE1 expression. An extensive mutagenesis analysis predicts that the GC-rich region of the 5'UTR forms a constitutive translation barrier, which may prevent the ribosome from efficiently translating the BACE1 mRNA. Our data therefore demonstrate translational repression as a new mechanism controlling BACE1 expression.

5' Untranslated Regions↗
PubMed1987

Regulation of alpha operon gene expression in Escherichia coli. A novel form of translational coupling.

The alpha operon of Escherichia coli contains the genes for ribosomal proteins S13, S11, S4, RNA polymerase subunit alpha, and r-protein L17, in this order. Previous studies have shown that translation of all four ribosomal proteins is regulated by S4, and that binding of S4 to the mRNA at the start site for S13 translation is probably responsible for the regulation of translation of S13, S11 and S4. The alpha gene is "unique" in that it is located between the genes for two ribosomal proteins (S4 and L17) and yet appears to be regulated independently of them. In the present studies, we have measured the synthesis rates of all the alpha operon proteins under a variety of physiological conditions. Our results confirm that alpha gene expression is regulated independently of the co-transcribed ribosomal protein genes and is relatively insensitive to translational feedback repression by S4. S1 nuclease analysis of alpha operon mRNA failed to reveal the presence of any unique transcription start or mRNA cleavage that leads to separation of the alpha cistron from preceding ribosomal protein cistrons. Therefore, it appears that differential regulation of alpha synthesis takes place at the level of mRNA translation. We have also carried out a deletion analysis of the alpha operon leader and identified a region of the alpha operon leader mRNA that is required for regulation by S4. Furthermore, deletion of this region results in increased synthesis of L17 together with S13, S11 and S4, whereas alpha synthesis did not increase significantly. Therefore, we conclude that interaction of S4 with this single target site results in translational repression of not only the proximal three cistrons for S13, S11 and S4 but also that of the last cistron, L17, without affecting the intervening alpha cistron.

Escherichia coli↗
PubMed1995

Repression of cap-dependent translation by 4E-binding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E.

An important aspect of the regulation of gene expression is the modulation of translation rates in response to growth factors, hormones and mitogens. Most of this control is at the level of translation initiation. Recent studies have implicated the MAP kinase pathway in the regulation of translation by insulin and growth factors. MAP kinase phosphorylates a repressor of translation initiation [4E-binding protein (BP) 1] that binds to the mRNA 5' cap binding protein eukaryotic initiation factor (eIF)-4E and inhibits cap-dependent translation. Phosphorylation of the repressor decreases its affinity for eIF-4E, and thus relieves translational inhibition. eIF-4E forms a complex with two other polypeptides, eIF-4A and p220, that promote 40S ribosome binding to mRNA. Here, we have studied the mechanism by which 4E-BP1 inhibits translation. We show that 4E-BP1 inhibits 48S pre-initiation complex formation. Furthermore, we demonstrate that 4E-BP1 competes with p220 for binding to eIF-4E. Mutants of 4E-BP1 that are deficient in their binding to eIF-4E do not inhibit the interaction between p220 and eIF-4E, and do not repress translation. Thus, translational control by growth factors, insulin and mitogens is affected by changes in the relative affinities of 4E-BP1 and p220 for eIF-4E.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Drosophila Brain Tumor is a translational repressor.

The Drosophila brain tumor (brat) gene encodes a member of the conserved NHL family of proteins, which appear to regulate differentiation and growth in a variety of organisms. One of the founding family members, Caenorhabditis elegans LIN-41, is thought to control posttranscriptional gene expression. However, the mechanism by which LIN-41, or any other NHL protein, acts has not been clear. Using a yeast "four-hybrid" interaction assay, we show that Brain Tumor is recruited to hunchback (hb) mRNA through interactions with Nanos and Pumilio, which bind to the RNA to repress its translation. Interaction with the Nanos/Pumilio/RNA complex is mediated by the Brat NHL domain; single amino acid substitutions in this domain compromise quaternary complex assembly in vitro and hb regulation in vivo. Thus, recruitment of Brat is necessary for translational repression and the normal development of posterior embryonic pattern. In addition to regulating abdominal segmentation, previous genetic analysis has shown that Brat, Nanos, and Pumilio govern a variety of developmental processes. We examined the role of Brat in two of these processes-regulation of maternal Cyclin B mRNA in the embryo and regulation of imaginal disc development. The results of these experiments suggest that NHL domain proteins are recruited to various mRNAs by combinatorial protein-protein interactions.

Animals↗
PubMed2006

Genome-wide analysis of the transcriptional response of murine hybridomas to osmotic shock.

Hyperosmotic stress has been shown to increase specific antibody productivity in murine hybridoma systems; however, the mechanisms underlying this phenomenon are still poorly understood. To elucidate the mechanisms for this phenomenon as well as other physiological changes that occur in response to hyperosmotic stress, we performed a genome-wide analysis of the transcriptional response of murine hybridoma OKT3 toward hyperosmotic stress using DNA microarrays. GeneChip MOE430A from Affymetrix was used to determine the differences in transcription patterns between OKT3 in hyperosmotic culture (approximately 100 mOsm above control) and control culture. The chip contains 22,690 probe sets for over 14,000 known genes and more than 4,000 ESTs. Signals were normalized using the GC-RMA algorithm and the effectiveness of hyperosmotic stress in altering the expression of each gene was evaluated using one-way ANOVA. 2,793 probe sets on the chip were differentially expressed with a P < 0.05. Among them, 349 probe sets exhibited a two-fold or greater change (with 202 up-regulated and 147 down-regulated) at one or more time points. Within the 215 characterized, differentially expressed genes, many are involved in metabolism/catabolism (19 induced, 12 repressed), cell-cycle regulation (10 induced, 5 repressed) and apoptosis (8 induced, 2 repressed), regulation of transcription (18 induced, 13 repressed) and translation (2 induced, 2 repressed), transport and signaling pathways (24 induced, 12 repressed). Surprisingly, there were very few changes within the stress-response genes. Interestingly, the transcription levels of both the immunoglobulin kappa and lambda light chains showed a significant change in response to hyperosmotic stress, although there is no detectable lambda chain in the immunoglobulin produced in this cell line. Quantitative PCR assays with TaqMan probes were applied to selected genes to validate the results obtained from microarray analysis.

Animals↗
PubMed2003

Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure.

DsrA RNA regulates the translation of two global regulatory proteins in Escherichia coli. DsrA activates the translation of RpoS while repressing the translation of H-NS. The RNA-binding protein Hfq is necessary for DsrA to function in vivo. Although Hfq binds to DsrA in vitro, the role of Hfq in DsrA-mediated regulation is not known. One hypothesis was that Hfq acts as an RNA chaperone by unfolding DsrA, thereby facilitating interactions with target RNAs. To test this hypothesis, we have examined the structure of DsrA bound to Hfq in vitro. Comparison of free DsrA to DsrA bound to Hfq by RNase footprinting, circular dichroism, and thermal melt profiles shows that Hfq does not alter DsrA secondary structures, but might affect its tertiary conformation. We identify the site on DsrA where Hfq binds, which is a structural element in the middle of DsrA. In addition, we show that although long poly(U) RNAs compete with DsrA for binding to Hfq, a short poly(U) stretch present in DsrA is not necessary for Hfq binding. Finally, unlike other RNAs, DsrA binding to Hfq is not competed with by poly(A) RNA. In fact, DsrA:poly(A):Hfq may form a stable ternary complex, raising the possibility that Hfq has multiple RNA-binding sites.

Bacterial Outer Membrane Proteins↗
PubMed2004

Translational control: a cup half full.

Repression of translation of oskar and nanos mRNAs prior to their posterior localization in the egg and embryo is essential for body patterning in Drosophila. The Cup protein is now found to have an important role in repression of both mRNAs, and apparently does so in a manner similar to the action of the Xenopus Maskin protein.

Animals↗
PubMed1999

Role for mRNA localization in translational activation but not spatial restriction of nanos RNA.

Patterning of the anterior-posterior body axis during Drosophila development depends on the restriction of Nanos protein to the posterior of the early embryo. Synthesis of Nanos occurs only when maternally provided nanos RNA is localized to the posterior pole by a large, cis-acting signal in the nanos 3' untranslated region (3'UTR); translation of unlocalized nanos RNA is repressed by a 90 nucleotide Translational Control Element (TCE), also in the 3'UTR. We now show quantitatively that the majority of nanos RNA in the embryo is not localized to the posterior pole but is distributed throughout the cytoplasm, indicating that translational repression is the primary mechanism for restricting production of Nanos protein to the posterior. Through an analysis of transgenes bearing multiple copies of nanos 3'UTR regulatory sequences, we provide evidence that localization of nanos RNA by components of the posteriorly localized germ plasm activates its translation by preventing interaction of nanos RNA with translational repressors. This mutually exclusive relationship between translational repression and RNA localization is mediated by a 180 nucleotide region of the nanos localization signal, containing the TCE. These studies suggest that the ability of RNA localization to direct wild-type body patterning also requires recognition of multiple, unique elements within the nanos localization signal by novel factors. Finally, we propose that differences in the efficiencies with which different RNAs are localized result from the use of temporally distinct localization pathways during oogenesis.

3' Untranslated Regions↗
PubMed1989

The translation start signal region of TEM beta-lactamase mRNA is responsible for heat shock-induced repression of amp gene expression in Escherichia coli.

pBR322 contains the amp gene encoding beta-lactamase. When Escherichia coli carrying this plasmid is exposed to heat shock, beta-lactamase synthesis is repressed transiently at the translational level. To identify the DNA element responsible for this translational repression, DNA segments containing the translation start region of the amp gene were excised from pAT153 and fused in frame with the lacZ reading frame in the open reading frame vector pORF1. These constructs were introduced into E. coli, and the effect of heat shock of the cells on the synthesis of beta-galactosidase starting from the amp start codon was examined. As is the case for pBR322-encoded synthesis of beta-lactamase, the synthesis of beta-galactosidase encoded by the fused genes also ceased transiently upon heat shock. It is concluded that the heat shock-induced repression of the amp gene occurs at the initiation step of translation. As far as the present study is concerned, the minimum DNA segment responsible for the repression is AT TGA AAA AGG AAG AGT ATG AG, which includes the Shine-Dalgarno sequence (AAGGA) and the initiation codon (ATG).

Base Sequence↗
PubMed2007

L13a blocks 48S assembly: role of a general initiation factor in mRNA-specific translational control.

Transcript-specific translational control restricts macrophage inflammatory gene expression. The proinflammatory cytokine interferon-gamma induces phosphorylation of ribosomal protein L13a and translocation from the 60S ribosomal subunit to the interferon-gamma-activated inhibitor of translation (GAIT) complex. This complex binds the 3'UTR of ceruloplasmin mRNA and blocks its translation. Here, we elucidate the molecular mechanism underlying repression by L13a. Translation of the GAIT element-containing reporter mRNA is sensitive to L13a-mediated silencing when driven by internal ribosome entry sites (IRESs) that require initiation factor eIF4G, but is resistant to silencing when driven by eIF4F-independent IRESs, demonstrating a critical role for eIF4G. Interaction of L13a with eIF4G blocks 43S recruitment without suppressing eIF4F complex formation. eIF4G attack, e.g., by virus, stress, or caspases, is a well-known mechanism of global inhibition of protein synthesis. However, our studies reveal a unique mechanism in which targeting of eIF4G by mRNA-bound L13a elicits transcript-specific translational repression.

Binding Sites↗