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Biomedical subjects

N Sonenberg

Publications and source records attributed to N Sonenberg.

At least 181 records · Page 10Linked to original sources
PubMed1992

The eukaryotic translation initiation factor 4E is not modified during the course of vaccinia virus replication.

The ability of vaccinia virus to inhibit processes of cap-dependent translational initiation by inactivating the eukaryotic translation initiation factor 4E (eIF-4E) has been examined. Analyses of the quantities of eIF-4E present in either uninfected mouse L929 cells or vaccinia virus-infected cells showed that during the first 12 hr of virus replication, when there is a marked decrease in host gene expression in infected cells, there is no change in the total amount of eIF-4E present. Analyses of eIF-4E that was metabolically labeled with [32P] and then purified by affinity chromatography using m7GTP-Sepharose 4B, indicated that neither the incorporation of radiolabel into eIF-4E nor the amounts of eIF-4E capable of binding to cap structures changed significantly during virus replication. Immunodetection of phosphorylated and unphosphorylated eIF-4E in cell lysates fractionated by two-dimensional gel electrophoresis showed that the steady-state levels of phosphorylated and unphosphorylated forms of eIF-4E were similar in uninfected and virus-infected cells. These results suggest that vaccinia virus does not gain preferential translation of viral mRNAs over other mRNAs in the cell by reducing either eIF-4E phosphorylation or its ability to bind to the cap structure.

Animals↗
PubMed1992

Signal transduction and regulation of translation initiation.

Regulation of the rate of protein synthesis is important in the control of cellular proliferation. Changes in the rate of protein translation are brought about primarily at the level of initiation, which is usually rate limiting. This regulation involves the reversible phosphorylation of key initiation factors. Translation initiation factors eIF-4F, eIF-4B, and ribosomal protein S6 are phosphorylated in response to a wide variety of mitogens, growth factors, and tyrosine kinase oncogenes. Thus, translation initiation factors are important components of signal transduction pathways activated by extracellular factors and oncogenes. Of particular interest is the messenger RNA 5' cap-binding protein, eIF-4E. Overexpression of eIF-4E in fibroblasts results in malignant transformation, suggesting that it is an important transducer of growth signals, and that aberrant expression of a translation factor can cause malignancy. Elucidation of the components of the signalling pathways which regulate initiation factor activity should increase our understanding of how extracellular factors and oncogenes effect cellular proliferation, and the role that translation plays in this process.

Animals↗
PubMed1992

Isolation of a yeast gene encoding a protein homologous to the human Tat-binding protein TBP-1.

We have cloned a putative yeast homolog of the gene encoding the human Tat-binding protein, TBP-1. The gene termed TBPY encodes a 45,243-dalton protein displaying a heptad repeat of hydrophobic amino acids reminiscent of a leucine zipper. Secondary structure predictions suggest the possibility of formation of an amphipathic helix that could further be organized into a coiled-coil. Additionally, the protein product of TBPY shows amino acid signatures characteristic of a large family of RNA and DNA helicases. We propose that the hydrophobic region of yTBP-1 participates in self-dimerization or heterodimerization.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1992

Ras mediates translation initiation factor 4E-induced malignant transformation.

Translation initiation factor eIF-4E binds to the eukaryotic mRNA 5' cap structure (m7 GpppN, where N is any nucleotide). eIF-4E is a limiting factor in translation and plays a key role in regulation of translation. We have shown previously that overexpression of eIF-4E in rodent fibroblasts results in tumorigenic transformation. eIF-4E also exhibits mitogenic activity when microinjected into serum-starved NIH-3T3 cells. To understand the mechanisms by which eIF-4E exerts its mitogenic property, we examined the involvement of the Ras signaling pathway in this activity. Here, we report that Ras is activated in eIF-4E-overexpressing cells, as the proportion of GTP-bound Ras is increased. Overexpression of the negative effector of cellular Ras, GTPase activating protein, causes reversion of the transformed phenotype. Furthermore, we show that neutralizing antibodies to Ras, or a dominant-negative mutant of Ras, inhibit the mitogenic activity of eIF-4E. We conclude that eIF-4E exerts its mitogenic and oncogenic activities by the activation of Ras.

3T3 Cells↗
PubMed1992

Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.

Interaction between the human immunodeficiency virus type 1 (HIV-1) trans-activator Tat and its cis-acting responsive RNA element TAR is necessary for activation of HIV-1 gene expression. We investigated the hypothesis that the essential uridine residue at position 23 in the bulge of TAR RNA is involved in intramolecular hydrogen bonding to stabilize an unique RNA structure required for recognition by Tat. Nucleotide substitutions in the two base pairs of the TAR stem directly above the essential trinucleotide bulge that maintain base pairing but change sequence prevent complex formation with Tat in vitro. Corresponding mutations tested in a trans-activation assay strongly affect the biological activity of TAR in vivo, suggesting an important role for these nucleotides in the Tat-TAR interaction. On the basis of these data, a model is proposed which implicates uridine 23 in a stable tertiary interaction with the GC pair directly above the bulge. This interaction would cause widening of the major groove of the RNA, thereby exposing its hydrogen-bonding surfaces for possible interaction with Tat. The model also predicts a gap between uridine 23 and the first base pair in the stem above, which would require one or more unpaired nucleotides to close, but does not predict any other role for such nucleotides. In accordance with this prediction, synthetic propyl phosphate linkers of equivalent length to 1 or 2 nucleotides, were found to be fully acceptable substitutes in the bulge above uridine 23, demonstrating that neither the bases nor the ribose moieties at these positions are implicated in the recognition of TAR RNA by Tat.

Acquired Immunodeficiency Syndrome↗
PubMed1992

The mRNA 5' cap-binding protein, eIF-4E, cooperates with v-myc or E1A in the transformation of primary rodent fibroblasts.

We present evidence that eIF-4E, the mRNA 5' cap-binding protein, cooperates with two immortalizing oncogenes, v-myc and E1A, to cause transformation of rat embryo fibroblasts. eIF-4E alone can transform rat embryo fibroblasts when selection is applied. The pattern of transformation by eIF-4E is similar to that of p21 Ras, raising the possibility that eIF-4E shares a common signal transduction pathway with p21 Ras.

Adenovirus Early Proteins↗
PubMed1992

Phosphorylation of translation initiation factor eIF-4E is induced in a ras-dependent manner during nerve growth factor-mediated PC12 cell differentiation.

Translation initiation factor eIF-4E, which binds to the 5' cap structure of eukaryotic mRNAs, is believed to play an important role in the control of cell growth. Consistent with this, overexpression of eIF-4E in fibroblasts results in their malignant transformation. The activity of eIF-4E is thought to be regulated by phosphorylation on a single serine residue (Ser-53). Treatment of rat pheochromocytoma (PC12) cells with nerve growth factor (NGF) strongly curtails their growth and causes their differentiation into cells that resemble sympathetic neurons. The present study shows that eIF-4E is rapidly phosphorylated in PC12 cells upon NGF treatment, resulting in a significant increase in the steady-state levels of the phosphorylated protein. In contrast, epidermal growth factor, a factor which elicits a weak mitogenic response in PC12 cells, did not significantly enhance eIF-4E phosphorylation. We also show that although the mitogen and tumor promoter, phorbol 12-myristate-13-acetate, is able to induce phosphorylation of eIF-4E in PC12 cells, the NGF-mediated increase is primarily a protein kinase C-independent response. The NGF-induced enhancement of eIF-4E phosphorylation is abrogated in PC12 cells expressing a dominant inhibitory ras mutant (Ser-17 replaced by Asn), indicating that eIF-4E phosphorylation is dependent on a ras signalling pathway. As phosphorylation of eIF-4E effects translation initiation, these results suggest that NGF-mediated and ras-dependent eIF-4E phosphorylation may play a role in switching the pattern of gene expression during the differentiation of PC12 cells.

Animals↗
PubMed1992

The coat protein of the yeast double-stranded RNA virus L-A attaches covalently to the cap structure of eukaryotic mRNA.

The eukaryotic mRNA 5' cap structure m7GpppX (where X is any nucleotide) interacts with a number of cellular proteins. Several of these proteins were studied in mammalian, yeast, and drosophila cells and found to be involved in translation initiation. Here we describe a novel cap-binding protein, the coat protein of L-A, a double-stranded RNA virus that is persistently maintained in many Saccharomyces cerevisiae strains. The results also suggest that the coat protein of a related double-stranded RNA virus (L-BC) is likewise a cap-binding protein. Strikingly, in contrast to the cellular cap-binding proteins, the interaction between the L-A virus coat protein and the cap structure is through a covalent bond.

Amino Acid Sequence↗
PubMed1991

Tat-dependent adenosine-to-inosine modification of wild-type transactivation response RNA.

Tat is a potent activator of gene expression in human immunodeficiency virus type 1 (HIV-1). Activation by Tat requires a cis-acting element, the transactivation response (TAR) site, located in the viral long terminal repeat and the 5' end of all viral mRNAs. Sequences in TAR RNA can fold into a specific stem-loop structure, and certain features of the stem-loop are essential for Tat-mediated transactivation. In Xenopus oocytes, TAR sequences can inhibit the translation of 3' cis-linked mRNAs. However, coinjection of Tat and the TAR-containing RNA into oocyte nuclei relieves this translational inhibition [Braddock, M., Chambers, A., Wilson, W., Esnout, M. A., Adams, S.E. & Kingsman, S.M. (1989) Cell 58, 269-279]. We report here that the intramolecular TAR stem-loop structure is a substrate for the double-stranded RNA (dsRNA)-modifying activity, which converts adenosines to inosines. This activity is located in the nuclei of Xenopus oocytes. The specificity and extent of modification of adenosines in TAR is dependent on Tat. We propose that the dsRNA-modifying activity may be one of the cellular proteins that interacts with TAR in the nucleus. The possible role of TAR RNA modification in the expression of HIV-1 is discussed.

Adenosine↗
PubMed1991

Translational control of ornithine aminotransferase. Modulation by initiation factor eIF-4E.

Ornithine aminotransferase (OAT) is a mitochondrial enzyme expressed at high levels in liver, kidney, and retina. To characterize OAT regulation in retinal lines, we have been studying OAT synthesis in retinoblastomas RB355 and Y79. Our previous data (Fagan, R.J., Sheffield, W.P., and Rozen, R. (1989) J. Biol. Chem. 264, 20513-20517) indicated similar OAT mRNA levels in the two strains with 3-fold greater immunoreactive OAT protein and enzyme activity in Y79. To examine the regulatory mechanisms in these cell lines, we performed nuclear runoff experiments and characterized polysome-associated OAT mRNAs. The nuclear runoff data did not reveal any differences in transcription between the two strains. However, OAT mRNA of the RB355 strain was present in the lighter polysome fractions as compared with Y79. Treatment with cycloheximide, which slows the rate of elongation, indicated that initiation was decreased in RB355. Eukaryotic initiation factor eIF-4E mRNA and protein were reduced in RB355, suggesting that eIF-4E might be rate-limiting for OAT translation. Overexpression of a wild-type eIF-4E in RB355 shifted the OAT mRNA into denser fractions of the gradient and increased the amount of OAT protein to the level observed in Y79; overexpression of a mutant eIF-4E had no such effect. We previously identified an alternatively spliced OAT mRNA (containing exon 2) in these cells. This mRNA appeared in the lightest fractions of the gradient in both strains and was not affected by eIF-4E overexpression.

Cycloheximide↗
PubMed1991

The effect of poliovirus proteinase 2Apro expression on cellular metabolism. Inhibition of DNA replication, RNA polymerase II transcription, and translation.

Infection of cells with poliovirus results in a rapid inhibition of host RNA and protein synthesis. Concordant with this shutoff, the p220 subunit of the cap-binding protein complex is cleaved, probably indirectly, by the poliovirus proteinase p2A (2Apro). To elucidate the mechanism of action of 2Apro in inhibiting protein synthesis in vivo, we studied the effect of transient expression of 2Apro in COS-1 monkey kidney cells. In cells transfected with a 2Apro expression plasmid, p220 was cleaved and the 2Apro mRNA was reduced 30-fold compared to an identical plasmid containing a translation termination codon within the 2Apro coding region. The reduced expression from the 2Apro vector results from a 4-fold reduction in DNA replication and 22-fold reduction in transcription by RNA polymerase II from the adenovirus major late promoter/SV40 enhancer utilized in this vector. In contrast, no decrease in transcription of the adenovirus virus-associated I RNA gene by RNA polymerase III was observed. The effect of 2Apro expression on cap-dependent mRNA translation was studied by producing a dicistronic beta-globin mRNA harboring the encephalomyocarditis virus leader and 2Apro coding region within the 3' end of the mRNA to mediate cap-independent translation of 2Apro. Expression of this mRNA was also reduced 25-fold compared to an identical plasmid harboring a termination codon within the 2Apro coding region. Translation of the beta-globin marker gene from this mRNA was reduced 3-fold when corrected for mRNA level. These results suggest that p220 cleavage itself is not sufficient for complete inhibition of host translation and that an important effect of 2Apro expression on host protein synthesis is a reduction in RNA polymerase II transcription and to a lesser extent, DNA replication. This reduction could be a primary effect of 2Apro, or a secondary effect caused by the inhibition of translation.

Animals↗
PubMed1991

The number of positively charged amino acids in the basic domain of Tat is critical for trans-activation and complex formation with TAR RNA.

The basic domain of Tat is required for trans-activation of viral gene expression. We have performed scanning peptide studies to demonstrate that only this domain is capable of binding to the TAR RNA stem-loop. Strikingly, the basic domain of the other human immunodeficiency virus trans-acting factor, Rev, but no other region, is also capable of binding to TAR. Peptide derivatives of Tat do not require the highly conserved glutamine residue at position 54 for TAR binding, since it may be substituted or deleted. In addition, the two lysine residues may be replaced by arginines. Analysis of binding and trans-activation demonstrated that homopolymers of arginine can completely substitute for the basic domain. Such homopolymers have high affinity for wild-type TAR RNA and lower affinity for mutant TAR. Homopolymers of six to nine arginines substituting for the basic domain of Tat enable full trans-activation in vivo. Homopolymers of at least seven arginines are required for detectable in vitro complex formation, although approximately 30% trans-activation is achieved with a mutant Tat containing only five arginines.

Amino Acid Sequence↗
PubMed1991

Multiple mRNAs encode the murine translation initiation factor eIF-4E.

All eukaryotic cellular mRNAs (except organellar) possess at their 5' end the structure m7GpppX (where X is any nucleotide) termed the "cap." The cap structure facilitates the melting of mRNA 5' secondary structure through the action of initiation factor-4F (eIF-4F) in conjunction with eIF-4B. eIF-4F consists of three subunits of which one, eIF-4E (eIF-4E has recently been designated eIF-4 alpha according to the Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (Safer, B. (1989) Eur. J. Biochem. 186, 1-3)), contains the cap binding site. Several lines of evidence suggest that eIF-4E regulates the rate of translation initiation. Consequently, changes in cellular eIF-4E levels could control growth and differentiation. To investigate the possibility that eIF-4E expression is regulated, we studied the pattern of eIF-4E expression in several cell lines. Here, we show the existence of multiple mRNAs for eIF-4E that are generated by differential polyadenylation. In addition, we show tissue-specific differences in eIF-4E mRNA expression and utilization of polyadenylation sites.

Animals↗
PubMed1991

Critical chemical features in trans-acting-responsive RNA are required for interaction with human immunodeficiency virus type 1 Tat protein.

The human immunodeficiency virus type 1 Tat protein binds to an RNA stem-loop structure called TAR which is present at the 5' end of all human immunodeficiency virus type 1 transcripts. This binding is centered on a bulge within the stem of TAR and is an essential step in the trans-activation process which results in a dramatic increase in viral gene expression. By analysis of a series of TAR derivatives produced by transcription or direct chemical synthesis, we determined the structural and chemical requirements for Tat binding. Tat binds well to structures which have a bulge of two to at least five unpaired bases bounded on both sides by a double-stranded RNA stem. This apparent flexibility in bulge size is in contrast to an absolute requirement for an unpaired uridine (U) in the 5'-most position of the bulge (+23). Substitution of the U with either natural bases or chemical analogs demonstrated that the imido group at the N-3 position and, possibly, the carbonyl group at the C-4 position of U are critical for Tat binding. Cytosine (C), which differs from U at only these positions, is not an acceptable substitute. Furthermore, methylation at N-3 abolishes binding. While methylation of U at the C-5 position has little effect on binding, fluorination reduces it, possibly because of its effects on relative tautomer stability at the N-3 and C-4 positions. Thus, we have identified key moieties in the U residue that are of importance for the binding of Tat to TAR RNA. We hypothesize that the invariant U is involved in hydrogen bond interactions with either another part of TAR or the TAR-binding domain in Tat.

Base Composition↗
PubMed1991

Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies.

The naturally uncapped genomic and mRNAs of poliovirus initiate translation by an internal ribosome-binding mechanism. The mRNA 5' untranslated region (UTR) of poliovirus is approximately 750 nucleotides in length and has seven to eight (depending on the serotype) AUG codons upstream of the initiator AUG. The sequence required for internal ribosome binding has been termed the ribosome landing pad (RLP). To better understand the mechanisms of internal initiation, we have determined the boundaries and critical elements of the RLP of poliovirus type 2 (Lansing strain) in vivo. By using deletion analysis, we demonstrate the existence of a core RLP in the poliovirus mRNA 5' UTR whose boundaries are between nucleotides 134 and 155 at the 5' end and nucleotides 556 and 585 at the 3' end. Sequences flanking the core RLP affect translational activity. The importance of several stem-loop structures in the RLP for internal initiation has been determined. Mutation of the phylogenetically conserved loop sequences in the proximal stem-loop structure of the RLP (stem-loop structure III; nucleotides 127 to 165) abolished internal translation. However, deletion of the second stem-loop in the RLP (stem-loop structure IV; nucleotides 189 to 223) reduced internal translation by only 50%. Internal deletions encompassing nucleotides 240 to 300, 350 to 380, or 450 to 480, predicted to disrupt stem-loop structure V and possibly VI, also abrogated internal initiation. Small point mutations within a short polypyrimidine sequence, highly conserved among all picornaviruses, abolished translation. A conservation of distance between the conserved polypyrimidine tract and a downstream AUG could play an important role in the mechanism of internal initiation.

Animals↗
PubMed1991

In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5' untranslated region.

Initiation of translation on poliovirus RNA occurs by internal binding of ribosomes to a region within the 5' untranslated region (UTR) of the mRNA. This region has been previously roughly mapped between nucleotides 140 and 631 of the 5' UTR and termed the ribosome landing pad. To identify cis-acting elements in the 5' UTR of poliovirus type 2 (Lansing strain) RNA that confer cap-independent internal initiation, we determined the in vitro translational efficiencies of a series of deletion and point mutations within the 5' UTR of the mRNA. The results demonstrate that the 3' border of the core poliovirus ribosome landing pad is located between nucleotides 556 and 585, whereas a region extending between nucleotides 585 and 612 confers enhanced translation. We studied two cis-acting elements within this region of the 5' UTR: a pyrimidine stretch which is critical for translation and an AUG (number 7 from the 5' end) that is located approximately 20 nucleotides downstream from the pyrimidine stretch and augments translation. We also show that the stem-loop structure which contains this AUG is not required for translation.

Base Composition↗