Biomedical subjects

N Sonenberg

Publications and source records attributed to N Sonenberg.

At least 145 records · Page 8Linked to original sources

PubMed1995

Overexpression of initiation factor eIF-4E does not relieve the translational repression of ribosomal protein mRNAs in quiescent cells.

Translation of ribosomal protein (rp) mRNA is selectively repressed in mouse erythroleukemia (MEL) cells, which cease to proliferate upon differentiation, and in NIH 3T3 cells, for which growth is arrested by either serum starvation, contact inhibition, or treatment with the DNA polymerase inhibitor, aphidicolin. The efficiency of translation of rp mRNAs correlates with the expression of the gene encoding the cap binding protein, eIF-4E, as indicated by the fact that the abundance of the corresponding mRNA and protein also fluctuates in a growth-dependent manner. To examine the hypothesis that eIF-4E plays a role in regulation of the translation efficiency of rp mRNAs, we utilized an NIH 3T3-derived eIF-4E-overexpressing cell line. These cells overproduce eIF-4E to the extent that even under conditions of growth arrest, the abundance of the respective protein in its active (phosphorylated) form is higher than that found in exponentially growing NIH 3T3 cells. Nevertheless, this surplus amount of eIF-4E does not prevent the translational repression of rp mRNAs when the growth of these cells is arrested by blocking DNA synthesis with aphidicolin or hydroxyurea. In complementary experiments we used an in vitro translation system to compare the competitive potential of mRNAs, containing the translational cis-regulatory element (5' terminal oligopyrimidne tract) and mRNAs lacking such a motif, for the cap binding protein. Our results demonstrate that both types of mRNAs, regardless of their translational response to growth arrest, exhibit similar sensitivity to the cap analogue m7G(5')ppp(5')G. It appears, therefore, that the presence of the regulatory sequence at the 5' terminus of rp mRNAs does not lessen its competitive potential for the cap binding protein and that the growth-dependent decrease in the activity of eIF-4E does not play a key role in the repression of translation of rp mRNAs.

3T3 Cells↗

PubMed1994

PHAS-I as a link between mitogen-activated protein kinase and translation initiation.

PHAS-I is a heat-stable protein (relative molecular mass approximately 12,400) found in many tissues. It is rapidly phosphorylated in rat adipocytes incubated with insulin or growth factors. Nonphosphorylated PHAS-I bound to initiation factor 4E (eIF-4E) and inhibited protein synthesis. Serine-64 in PHAS-I was rapidly phosphorylated by mitogen-activated (MAP) kinase, the major insulin-stimulated PHAS-I kinase in adipocyte extracts. Results obtained with antibodies, immobilized PHAS-I, and a messenger RNA cap affinity resin indicated that PHAS-I did not bind eIF-4E when serine-64 was phosphorylated. Thus, PHAS-I may be a key mediator of the stimulation of protein synthesis by the diverse group of agents and stimuli that activate MAP kinase.

3T3 Cells↗

PubMed1994

Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function.

The cloning is described of two related human complementary DNAs encoding polypeptides that interact specifically with the translation initiation factor eIF-4E, which binds to the messenger RNA 5'-cap structure. Interaction of these proteins with eIF-4E inhibits translation but treatment of cells with insulin causes one of them to become hyperphosphorylated and dissociate from eIF-4E, thereby relieving the translational inhibition. The action of this new regulator of protein synthesis is therefore modulated by insulin, which acts to stimulate the overall rate of translation and promote cell growth.

Acid Phosphatase↗

PubMed1994

Suppression of a temperature-sensitive cdc33 mutation of yeast by a multicopy plasmid expressing a Drosophila ribosomal protein.

The Saccharomyces cerevisiae cdc33ts4-2 mutant produces a temperature-sensitive allele of the cap-binding subunit of eukaryotic initiation factor-4F (also termed eIF-4E). From a Drosophila cDNA library constructed in a multicopy yeast shuttle vector, a clone was isolated which restored the ability to grow at elevated temperature to cdc33ts4-2 cells. The rescuing Drosophila clone encodes a small ribosomal subunit protein, which we name S15a based on its molecular weight and similarity with the Brassica napus S15a ribosomal protein. Transcription of the Drosophila gene, RpS15a, occurs at all developmental stages and is enhanced during oogenesis. The ribosomal protein gene is capable of suppressing other alleles of cdc33 but not an inactivation mutation, suggesting that suppression is dependent upon the presence of the temperature-sensitive eIF-4E protein. Supporting this, Western blot analysis shows that far more eIF-4E protein is present in cdc33 yeast cells expressing the RpS15a gene than lacking it. Levels of other unrelated proteins are unaffected. We propose therefore that the expression of high levels of the Drosophila S15a ribosomal protein in the cdc33 yeast cells leads to a selective stabilization of the temperature-sensitive eIF-4E protein, which accounts for the suppression phenomenon.

Amino Acid Sequence↗

PubMed1994

Angiotensin II induces phosphorylation of eukaryotic protein synthesis initiation factor 4E in vascular smooth muscle cells.

Angiotensin II has been shown to induce hypertrophy of cultured vascular smooth muscle cells (VSMC). To understand the mechanisms of induction of the hypertrophy, we studied its effect on the phosphorylation state of eIF-4E, a rate-limiting eukaryotic protein synthesis initiation factor whose activity has been shown to be regulated by phosphorylation. Angiotensin II induced a 2-3-fold increase in the phosphorylation of eIF-4E in VSMC. The stimulation of phosphorylation was apparent at 20 min and persisted for at least 12 h. Phosphoamino acid analysis revealed that serine is the major residue of eIF-4E phosphorylated by angiotensin II. Staurosporine and calphostin C, two potent inhibitors of the serine/threonine protein kinase, protein kinase C, significantly attenuated the angiotensin II-induced eIF-4E phosphorylation. Staurosporine and calphostin C also blunted the angiotensin II-stimulated protein synthesis. Together, these observations indicate that angiotensin II induces phosphorylation of eIF-4E in a protein kinase C-dependent manner and suggest that this pathway may play an important role in the mechanism by which angiotensin II causes hypertrophy of VSMC.

Alkaloids↗

PubMed1994

Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation.

Eukaryotic translation initiation factor-4A (eIF-4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA-dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA-ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF-4F. To gain further understanding into the mechanism of action of eIF-4A in mRNA-ribosome binding, defective eIF-4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF-4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap-independent internal initiation mechanism. Addition of eIF-4A or eIF-4F relieved inhibition of translation, but eIF-4F was six times more effective than eIF-4A, whereas eIF-4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF-4A is defective in recycling through eIF-4F, thus explaining the dramatic inhibition of translation. Mutant eIF-4A proteins also inhibited eIF-4F-dependent, but not eIF-4A-dependent RNA helicase activity. Taken together these results suggest that eIF-4A functions primarily as a subunit of eIF-4F, and that singular eIF-4A is required to recycle through the complex during translation. Surprisingly, eIF-4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.

Amino Acid Sequence↗

PubMed1994

Distinct structural elements and internal entry of ribosomes in mRNA3 encoded by infectious bronchitis virus.

Infectious bronchitis virus (IBV) mRNA3 encodes three small proteins, 3a, 3b, and 3c, at its 5' end. Recently, it was demonstrated that initiation of protein 3c is dependent on the upstream sequence. Monte Carlo simulations of RNA folding in this tricistronic mRNA3 indicate that a highly significant folding region occurs prior to the initiator AUG of 3c. The unusual folding region (UFR) of 265 nucleotides (nt) contains the coding sequences of proteins 3a and 3b. Details of the structural analyses show that five highly significant RNA stem-loops in the UFR can be modeled into a compact superstructure by the interaction of two predicted pseudoknot structures. The folded superstructure comprising nt 44 to 330, with additional 22 nt downstream from this UFR, is suggested to serve as a ribosome landing pad (or an internal ribosomal entry site) in the cap-independent translation of the 3c of IBV. Intriguingly, the proposed structural motif of this coronavirus shares structural features similar to those proposed in a number of picornavirus mRNAs. Based on the common structural features, a plausible base pairing model between mRNA3 and 18 S rRNA is suggested, which is consistent with a general mechanism for regulation of internal initiation described in many picornaviruses.

Base Sequence↗

PubMed1994

Regulation of translation and cell growth by eIF-4E.

This review discusses the regulation of a key controlling step in the initiation of protein synthesis, the binding of mRNA to ribosomes. Particular focus is given to the phosphorylation of the cap-binding factor, eIF-4E, and the role of this factor in the regulation of cell growth.

Animals↗

PubMed1994

mRNA translation: influence of the 5' and 3' untranslated regions.

Eukaryotic messenger RNA utilization is a tightly controlled process. Control of translation is exerted at several levels, but the predominant step is ribosome binding, which is rate limiting for translation of most mRNAs. There appear to be several alternative modes by which ribosomes bind to the mRNA and initiate translation. Recent data show that both the 5' and 3' untranslated regions of eukaryotic mRNAs play critical roles in mRNA recruitment for translation, and several cis-acting elements have been characterized in detail. In addition, a few trans-acting factors that bind to these elements have been identified. It is possible that the terminal regions of mRNAs interact to enhance translation.

Animals↗

PubMed1994

La autoantigen alleviates translational repression by the 5' leader sequence of the human immunodeficiency virus type 1 mRNA.

The trans-activation response element (TAR) at the 5' end of the human immunodeficiency virus type 1 (HIV-1) mRNAs forms a stable hairpin structure which is a target for binding of the virally encoded protein Tat, which activates viral gene expression, as well as several cellular factors. TAR is also inhibitory to translation. One of several host factors that binds to TAR RNA is the La autoantigen, an RNA-binding protein which functions in RNA polymerase III transcription termination and has also been implicated in cap-independent internal translation initiation on poliovirus RNA. Here we show that La autoantigen alleviates translational repression by the HIV-1 leader RNA. In rabbit reticulocyte lysate, La relieves the cis-inhibitory effect of the TAR RNA on translation of bacterial chloramphenicol acetyltransferase (CAT) mRNA but not inhibition that is mediated by an artificial secondary structure element. Canonical translation factors exhibited slight (eIF-2 and GEF) or no (eIF-4A, eIF-4B, eIF-4E, eIF-4F, eIF-3, and eEF-1 alpha) stimulatory activity on translation of TAR-containing CAT mRNA. In addition, we show that poliovirus RNA, in spite of being an inefficient template in rabbit reticulocyte lysate, is a strong competitive inhibitor of translation of TAR-containing CAT mRNA but not CAT mRNA. This inhibition can be relieved by La but not by any other translation factor. The results suggest a possible involvement of the La autoantigen in HIV-1 gene expression.

Autoantigens↗

PubMed1994

Internal translation initiation on poliovirus RNA: further characterization of La function in poliovirus translation in vitro.

Initiation of poliovirus RNA translation by internal entry of ribosomes is believed to require the participation of trans-acting factors. The mechanism of action of these factors is poorly defined. The limiting amount of one of these factors, La protein, in rabbit reticulocyte lysates (RRL) has been postulated to partially explain the inefficient translation of poliovirus RNA in this system. To further characterize La activity in translation and to identify other potential limiting factors, we assayed the ability of La protein as well as purified initiation factors, eIF-2, guanine nucleotide exchange factor (GEF), eIF-4A, eIF-4B, eIF-4F, and eIF-3, to stimulate the synthesis of P1, the capsid precursor protein, in poliovirus type 1 (Mahoney) RNA-programmed RRL. Of the proteins tested, only La, GEF, and to some extent eIF-2 stimulated the synthesis of P1. The enhanced translation of P1 in response to La occurred concomitantly with the inhibition of synthesis of most aberrant polypeptides, resulting from initiation in the middle of the genome. Deletion of the carboxy-terminal half (214 amino acids) of La did not decrease its binding to the poliovirus 5' untranslated region but abrogated the stimulatory and correcting activity in translation. In contrast to La, GEF and eIF-2 stimulated the overall translation and increased the synthesis of aberrant products as well as P1. Neither La, GEF, nor any other factor stimulated translation of encephalomyocarditis virus RNA in RRL. The implications of these findings for the mechanism of internal translation initiation on picornavirus RNAs are discussed.

Animals↗

PubMed1994

The translation initiation factor eIF-4B contains an RNA-binding region that is distinct and independent from its ribonucleoprotein consensus sequence.

eIF-4B is a eukaryotic translation initiation factor that is required for the binding of ribosomes to mRNAs and the stimulation of the helicase activity of eIF-4A. It is an RNA-binding protein that contains a ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM). We examined the effects of deletions and point mutations on the ability of eIF-4B to bind a random RNA, to cooperate with eIF-4A in RNA binding, and to enhance the helicase activity of eIF-4A. We report here that the RNP-CS/RRM alone is not sufficient for eIF-4B binding to RNA and that an RNA-binding region, located between amino acids 367 and 423, is the major contributor to RNA binding. Deletions which remove this region abolish the ability of eIF-4B to cooperate with eIF-4A in RNA binding and the ability to stimulate the helicase activity of eIF-4A. Point mutations in the RNP-CS/RRM had no effect on the ability of eIF-4B to cooperate with eIF-4A in RNA binding but significantly reduced the stimulation of eIF-4A helicase activity. Our results indicate that the carboxy-terminal RNA-binding region of eIF-4B is essential for eIF-4B function and is distinct from the RNP-CS/RRM.

Amino Acid Sequence↗

PubMed1994

His-154 is involved in the linkage of the Saccharomyces cerevisiae L-A double-stranded RNA virus Gag protein to the cap structure of mRNAs and is essential for M1 satellite virus expression.

The coat protein (Gag) of the double-stranded RNA virus L-A was previously shown to form a covalent bond with the cap structure of eukaryotic mRNAs. Here, we identify the linkage as a phosphoroimidazole bond between the alpha phosphate of the cap structure and a nitrogen in the Gag protein His-154 imidazole side chain. Mutations of His-154 abrogate the ability of Gag to bind to the cap structure, without affecting cap recognition, in vivo virus particle formation from an L-A cDNA clone, or in vitro specific binding and replication of plus-stranded single-stranded RNA. However, genetic analyses demonstrate that His-154 is essential for M1 satellite virus expression.

Amino Acid Sequence↗

PubMed1994

Translational regulation of the mammalian growth-related protein P23: involvement of eIF-4E.

Synthesis of the mammalian growth-related protein P23 is rapidly induced after serum stimulation of mouse fibroblasts and Ehrlich ascites tumour cells. This induction occurs at the translational level. Growth-induction leads also to an increase in phosphorylation of the rate-limiting initiation factor eIF-4E. Here, we present the following evidence indicating the involvement of eIF-4E in the regulation of P23 synthesis: 1) P23 synthesis is induced by the same mitogenic stimuli which lead to enhanced eIF-4E phosphorylation. 2) Upon heat shock treatment of Ehrlich ascites cells (which results in immediate dephosphorylation and concomitant inactivation of eIF-4E), P23 synthesis is rapidly shut off. 3) In control NIH 3T3 cells, P23 synthesis is readily induced by growth stimulation. This response is strongly diminished in cells overexpressing eIF-4E, and the basal level of P23 synthesis is elevated in these cells. Overexpression of a nonfunctional mutant of eIF-4E diminishes the basal level of P23 synthesis as well as the serum-response of the cells with respect to P23 induction. 4) Cells transformed by overexpression of the ras or src genes in which eIF-4E is highly phosphorylated do not show any inducibility of P23 synthesis. 5) HeLa cells expressing antisense RNA of eIF-4E, have reduced levels of eIF-4E/F and show reduced rates of growth and protein synthesis. In these cells the total amount of P23 protein is about 50% compared with control cells. The results suggest that P23 is one of the gene products, the synthesis of which is regulated by eIF-4E activity.

3T3 Cells↗

PubMed1994

Disruption of I kappa B alpha regulation by antisense RNA expression leads to malignant transformation.

NF-kappa B transcription factors regulate the expression of a variety of genes involved in immune regulation and cell growth. In most cell types NF-kappa B proteins are localized in an inactive form in the cytoplasm coupled to the inhibitory I kappa B proteins. Viruses, cytokines, lipopolysaccharides and other stimulating agents promote the dissociation of the cytosolic NF-kappa B/I kappa B complexes, via phosphorylation and degradation of I kappa B, resulting in the translocation of DNA binding, NF-kappa B complexes to the nucleus. To further understand the association of I kappa B with cell growth regulation, the effect of ectopic expression of sense and antisense I kappa B genes was examined in NIH3T3 cells. Overexpression of I kappa B alpha antisense RNA but not I kappa B gamma antisense RNA decreased the steady state levels of I kappa B alpha protein, altered NF-kappa B DNA binding and gene activity and, most importantly, induced malignant transformation as measured by saturation density, growth in soft agar and tumorigenicity in nude mice. In contrast, overexpression of I kappa B alpha resulted in decreased saturation density, a flattened cellular morphology and decreased NF-kappa B dependent reporter gene activity. These results indicate that overexpression of an I kappa B alpha antisense RNA may disrupt the NF-kappa B/I kappa B autoregulatory loop, leading to cellular transformation. Our results raise the interesting possibility that I kappa B alpha represents a potential tumor suppressor activity.

3T3 Cells↗

PubMed1993

Conserved tertiary structural elements in the 5' nontranslated region of cardiovirus, aphthovirus and hepatitis A virus RNAs.

Statistical analyses of RNA folding in 5' nontranslated regions (5'NTR) of encephalomyocarditis virus, Theiler's murine encephalomyelitis virus, foot-and-mouth disease virus, and hepatitis A virus indicate that two highly significant folding regions occur in the 5' and 3' portions of the 5'NTR. The conserved tertiary structural elements are predicted in the unusual folding regions (UFR) for these viral RNAs. The theoretical, common structural elements predicted in the 3' parts of the 5'NTR occur in a cis-acting element that is critical for internal ribosome binding. These structural motifs are expected to be highly significant from extensive Monte Carlo simulations. Nucleotides (nt) in the conserved single-stranded polypyrimidine tract for these RNAs are involved in a distinctively tertiary interaction that is located at about 15 nt prior to the initiator AUG. Intriguingly, the proposed common tertiary structure in this study shares a similar structural feature to that proposed in human enteroviruses and rhinoviruses. Based on these common structural features, plausible base pairing models between these viral RNAs and 18 S rRNA are suggested, which are consistent with a general mechanism for regulation of internal initiation of cap-independent translation.

Aphthovirus↗

PubMed1993

Regulation of eukaryotic translation initiation factor expression during T-cell activation.

Primary T-cells are metabolically quiescent, with little DNA, RNA or protein synthesis. Upon mitogenic stimulation the rate of protein synthesis increases 10-fold. We have studied the role of eIF-2 and eIF-4 alpha (eIF-4E) expression in the mechanism of translational activation. During this period, the levels of eIF-2 alpha and eIF-4 alpha mRNA increase some 50-fold. Similar to the increase in ribosomes and mRNA, the number of eIF-2 alpha, eIF-2 beta, and eIF-4 alpha molecules per cell also increase 2-3-fold. This suggests that in addition to an increase in the pool size of translational components, an additional mechanism exists which results in an increased efficiency of factor utilization. We have looked at initiation factor phosphorylation. We find that eIF-2 alpha does not undergo significant changes in its phosphorylation state nor is there a change in the efficiency of eIF-2 utilization. However, there is a rapid increase in the phosphorylation state of eIF-4 alpha which correlates with the rapid increase in translational activity. It thus appears there are 2 distinct components responsible for the translational activation of quiescent T-cells during mitogenic stimulation. The first is the phosphorylation of eIF-4 alpha, with a concomitant increase in the efficiency of eIF-4 alpha utilization. The second is an increase in the pool sizes of eIF-2 and eIF-4 alpha.

Eukaryotic Initiation Factor-2↗

PubMed1993

The p46 subunit of eukaryotic initiation factor (eIF)-4F exchanges with eIF-4A.

The p46 subunit of eukaryotic initiation factor (eIF)-4F purified from rabbit reticulocyte lysate has previously been found to be composed of eIF-4AI and eIF-4AII in a 4:1 ratio, respectively, whereas the free form of rabbit eIF-4A is composed solely of eIF-4AI. Using sucrose gradient centrifugation and an m7GTP-Sepharose 4B assay, it was shown that eIF-4A exchanges with the p46 subunit of eIF-4F. Incubation of [14C]eIF-4A and eIF-4F resulted in the incorporation of [14C] eIF-4A into the eIF-4F complex. Conversely, the [14C] p46 subunit of [14C]eIF-4F was shown to dissociate from the [14C]eIF-4F complex in the presence of eIF-4A, presumably due to the incorporation of unlabeled eIF-4A. Similar experiments were conducted in which 14C-labeled initiation factors were incubated with rabbit reticulocyte lysate. When [14C]eIF-4A was incubated with lysate, [14C]eIF-4A became incorporated into the eIF-4F complex present in the lysate. Additionally, when [14C]eIF-4F was incubated with lysate, the [14C]p46 subunit of [14C]eIF-4F dissociated from the [14C]eIF-4F complex, most likely due to the exchange of unlabeled eIF-4A (present in the lysate) with the [14C]p46 subunit. The exchange of mouse eIF-4AI and eIF-4AII expressed in Escherichia coli was also investigated in the presence of eIF-4F and rabbit reticulocyte lysate. Both the sucrose gradient experiments and m7GTP-Sepharose 4B assays demonstrated that the [14C]p46 subunit of [14C]eIF-4F was displaced in the presence of eIF-4AI or eIF-4AII and that mouse [14C]eIF-4AI or [14C]eIF-4AII became incorporated into the eIF-4F complex in the same manner as rabbit reticulocyte eIF-4A.

Animals↗