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Biomedical subjects

N Sonenberg

Publications and source records attributed to N Sonenberg.

At least 253 records · Page 14Linked to original sources
PubMed1985

Differential efficiencies of in vitro translation of mouse c-myc transcripts differing in the 5' untranslated region.

We have studied the in vitro translational efficiencies of two murine c-myc transcripts synthesized in vitro that differ in the lengths of their 5' noncoding regions (448 and 83 nucleotides) and also in their 3' noncoding regions. When translated in a reticulocyte translation system, the shorter transcript was translated 10-fold more efficiently. These results are consistent with the hypothesis of Saito et al. [Saito, H., Hayday, A. C., Wiman, K., Hayward, W. S. & Tonegawa, S. (1983) Proc. Natl. Acad. Sci. USA 80, 7476-7480] that translation of full-length human c-myc mRNA is normally repressed, whereas in several Burkitt lymphomas that have deletions of the mRNA 5' noncoding region (resulting from translocation of the c-myc gene), translation of the c-myc mRNA is more efficient. Our results suggest that activation of murine c-myc expression by production of a more efficient mRNA might in some cases play a role in neoplastic transformation.

Animals↗
PubMed1985

Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure.

We studied the association of several eucaryotic viral and cellular mRNAs with cytoskeletal fractions derived from normal and virus-infected cells. We found that all mRNAs appear to associate with the cytoskeletal structure during protein synthesis, irrespective of their 5' and 3' terminal structures: e.g., poliovirus that lacks a 5' cap structure or reovirus and histone mRNAs that lack a 3' poly A tail associated with the cytoskeletal framework to the same extent as capped, polyadenylated actin mRNA. Cellular (actin) and viral (vesicular stomatitis virus and reovirus) mRNAs were released from the cytoskeletal framework and their translation was inhibited when cells were infected with poliovirus. In contrast, actin mRNA was not released from the cytoskeleton during vesicular stomatitis virus infection although actin synthesis was inhibited. In addition, several other conditions under which protein synthesis is inhibited did not result in the release of mRNAs from the cytoskeletal framework. We conclude that the association of mRNA with the cytoskeletal framework is required but is not sufficient for protein synthesis in eucaryotes. Furthermore, the shut-off of host protein synthesis during poliovirus infection and not vesicular stomatitis virus infection occurs by a unique mechanism that leads to the release of host mRNAs from the cytoskeleton.

Actins↗
PubMed1985

Isolation and structural characterization of cap-binding proteins from poliovirus-infected HeLa cells.

In poliovirus-infected HeLa cells, poliovirus RNA is translated at times when cellular mRNA translation is strongly inhibited. It is thought that this translational control mechanism is mediated by inactivation of a cap-binding protein complex (comprising polypeptides of 24 [24-kilodalton cap-binding protein], 50, and approximately 220 kilodaltons). This complex can restore the translation of capped mRNAs in extracts from poliovirus-infected cells. We have previously shown that the virally induced defect prevents interaction between cap recognition factors and mRNA. Here, we show that the cap-binding protein complex (and not the 24-kilodalton cap-binding protein) has activity that restores the cap-specific mRNA-protein interaction when added to initiation factors from poliovirus-infected cells. Thus, the activity that restores the cap-specific mRNA-protein interaction and that which restores the translation of capped mRNAs in extracts from poliovirus-infected cells, copurify. The results also indicate, by an alternative assay, that the cap-binding protein complex is the only factor inactivated by poliovirus. We also purified cap-binding proteins from uninfected and poliovirus-infected HeLa cells. By various criteria, the 24-kilodalton cap-binding protein is not structurally modified as a result of infection. However, the 220-kilodalton polypeptide of the cap-binding protein complex is apparently cleaved by a putative viral (or induced) protease. By in vivo labeling and m7GDP affinity chromatography, we isolated a modified cap-binding protein complex from poliovirus-infected cells, containing proteolytic cleavage fragments of the 220-kilodalton polypeptide.

Carrier Proteins↗
PubMed1985

Identification of a new polypeptide coded by reovirus gene S1.

The reovirus S1 gene has recently been shown potentially to encode two polypeptides (from two overlapping reading frames) having predicted molecular weights of 49,071 and 16,143 (Nagata et al., Nucleic Acids Res. 12:8699-8710, 1984; Bassel-Duby et al., Nature [London], in press). The larger polypeptide is reovirus protein sigma 1, but synthesis of the smaller polypeptide has not been described to date. A truncated clone of the S1 gene in which the first ATG is deleted was expressed in an in vitro protein synthesis system to yield a approximately 13-kilodalton polypeptide, as determined from migration on sodium dodecyl sulfate-polyacrylamide gels. A polypeptide with a similar migration pattern on sodium dodecyl sulfate-polyacrylamide gels was present in reovirus-infected cells and absent from mock-infected cells. Comparative tryptic peptide analysis of the 13-kilodalton polypeptides produced in vivo and in vitro showed them to be identical. Thus, the s1 mRNA of reovirus type 3 is apparently bicistronic, and we suggest that the approximately 13-kilodalton polypeptide be called sigma s (standing for sigma small).

Genes, Viral↗
PubMed1985

Poliovirus protease 3C (P3-7c) does not cleave P220 of the eucaryotic mRNA cap-binding protein complex.

Infection of HeLa cells by poliovirus results in proteolysis of the large subunit (P220) of the cap-binding protein complex. This is believed to cause the rapid shut-off of host protein synthesis during poliovirus infection. In this communication we examined the possible involvement of poliovirus proteins 3C (a proteinase) and 2C in cleavage of P220. Using antisera against these two viral polypeptides, we were unable to inhibit proteolysis of P220 in an in vitro assay. These results indicate that viral proteins 3C and 2C are not directly involved in cleaving P220 and hence do not cause shut-off of cellular protein synthesis.

Carrier Proteins↗
PubMed1985

Poliovirus mutant that does not selectively inhibit host cell protein synthesis.

A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV-1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus-mediated translational inhibition.

Animals↗
PubMed1985

Photochemical cross-linking of cap binding proteins to eucaryotic mRNAs: effect of mRNA 5' secondary structure.

We used UV light-induced cross-linking to study the interactions of cap binding proteins with the 5' cap structure of eucaryotic mRNAs. Thymidine kinase gene (herpes simplex virus type 1) transcripts prepared in vitro using the SP6 RNA polymerase transcription system were capped and methylated posttranscriptionally with [alpha-32P]GTP and S-adenosyl-L-methionine to yield cap-labeled transcripts. Irradiation of capped transcripts with crude rabbit reticulocyte initiation factors in the presence of ATP-Mg2+ resulted in the cap-specific cross-linking of two polypeptides with molecular masses of 24 and 80 kilodaltons (kDa). The cross-linking characteristics of these polypeptides resemble those of the cap-binding proteins previously detected by a chemical cross-linking assay (N. Sonenberg, D. Guertin, D. Cleveland, and H. Trachsel, Cell 27:563-572, 1981). However, the relative efficiency of the cross-linking of these two polypeptides to the cap structure was different from that in previous studies, and there was no detectable cross-linking of the previously described 50-kDa polypeptide. In addition, we present data indicating that the insertion of secondary structure into the 5' noncoding region of tk mRNA, 6 nucleotides from the cap structure, decreases the cap-specific cross-linking of the 80-kDa but not the 24-kDa polypeptide. In contrast, the insertion of secondary structure 37 nucleotides from the cap structure had no significant effect on the cross-linking of either the 24- or the 80-kDa cap-specific polypeptide. These results demonstrate that the position of mRNA 5'-proximal secondary structure relative to the cap structure can influence the cap-specific interaction between the mRNA and a translation initiation factor.

Animals↗
PubMed1984

Preferential stimulation of rabbit alpha globin mRNA translation by a cap-binding protein complex.

A cap-binding protein complex (Edery et al. (1983) J. Biol. Chem. 258, 11398-11403) is shown here to stimulate preferentially the translation of endogenous alpha versus beta globin mRNA in a rabbit reticulocyte lysate. Several initiation factors (eIF-2, eIF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4E and eIF-5) and elongation factor 1 were found to have no such discriminatory effect. These results are in contrast to several previous reports and demonstrate that the only factor capable of relieving translational competition between alpha and beta globin mRNAs is the cap-binding protein complex.

Animals↗
PubMed1984

Functional characterization of eukaryotic mRNA cap binding protein complex: effects on translation of capped and naturally uncapped RNAs.

We examined the effects of a eukaryotic mRNA cap binding protein (CBP) complex purified by cap analogue affinity chromatography [Edery, I., Humebelin, M., Darveau, A., Lee, K.A. W., Milburn, S., Hershey, J.W.B., Trachsel, H., & Sonenberg, N. (1983) J. Biol. Chem. 258, 11398 11403], on translation of several capped and naturally uncapped mRNAs in extracts prepared from poliovirus-infected or mock-infected HeLa cells. The CBP complex has activity that restores capped mRNA (globin, tobacco mosaic virus, and others) function in extracts from poliovirus-infected HeLa cells. Translation of two naturally uncapped RNAs (poliovirus and mengovirus RNAs), the translation of which is not restricted in extracts from poliovirus-infected cells, is also not stimulated by the CBP complex. Translation of several capped eukaryotic mRNAs (vesicular stomatitis virus, reovirus, and tobacco mosaic virus) in extracts from mock-infected cells is inhibited when the potassium ion concentration is increased. However, translation of capped AMV-4 RNA, which has negligible secondary structure at its 5' end, is resistant to this inhibition. Furthermore, the CBP complex reverses the high salt induced inhibition of translation of the former mRNAs. Since mRNA secondary structure is more stable at elevated salt concentrations, these data are consistent with a model in which the CBP complex has a role in melting mRNA secondary structure involving 5'-proximal sequences, to facilitate ribosome binding.

Avian Myeloblastosis Virus↗
PubMed1984

Demonstration in vitro that eucaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus-infected HeLa cells.

Protein synthesis initiation factor preparations from poliovirus-infected HeLa cells have reduced ability to initiate translation on capped mRNA. The defect in initiation factors has been variously attributed to inactivation of eucaryotic initiation factor 3 (eIF3), eIF4B, or a cap-binding protein (CBP) complex. We have developed a series of in vitro protein synthesis assays to show that eIF3 is active but a CBP complex activity is inactivated after poliovirus infection. eIF3 activity, when determined in the presence of purified CBP complex, is present in sucrose gradients of factors from both infected and uninfected cells. CBP complex activity, determined in the presence of eIF3 from poliovirus-infected cells, is present in uninfected cells only and comigrates on sucrose gradient with an activity which restores the ability of crude initiation factors from infected cells to translate capped globin mRNA. This is the first demonstration by a fractionated translation system that an activity which is attributable to CBP complex is inactivated in poliovirus-infected cells. The results also indicate that eIF3 is undetectable or has greatly reduced activity in the absence of CBP complex.

Carrier Proteins↗
PubMed1983

5'-Conformation of capped alfalfa mosaic virus ribonucleic acid 4 may reflect its independence of the cap structure or of cap-binding protein for efficient translation.

Most eukaryotic mRNAs are characterized by the presence of a 5'-terminal cap structure (m7GpppN), and removal of the cap or translation of capped mRNAs in the presence of cap analogues (m7G) results in most cases in a significant decrease in the translational efficiency of the mRNAs. One way of explaining the importance of the 5'-cap is that cap-binding proteins recognize the cap structure, destabilize the mRNA secondary structure, and thus allow the 40S ribosomal subunit to bind to the mRNA [Sonenberg, N., Guertin, D., Cleveland, D., & Trachsel, H. (1981) Cell (Cambridge, Mass.) 27, 563-572]. Our data and those of others indicate that the translational efficiency of alfalfa mosaic virus RNA 4 (AMV-4 RNA), a naturally capped RNA, is not affected significantly by cap analogues or by removal of the cap. In order to examine the potential relationship between the function of the cap structure and secondary structure at the 5'-mRNA terminus, partial enzymatic digestion of capped AMV-4 RNA with single strand specific and double strand specific nucleases has been performed, and the experimental data have been compared with computer-generated models of AMV-4 secondary structure. In addition, the in vitro translatability of AMV-4 has been examined as a function of increasing potassium concentration, conditions that are likely to increase mRNA secondary structure. The nuclease-digestion results demonstrate that under native ionic conditions, the 5'-terminus of AMV-4 RNA is predominantly single stranded, although computer modeling and double-strand nuclease digestions indicate that the 5'-terminus can form weak base pairs with internal regions of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1983

Involvement of eukaryotic initiation factor 4A in the cap recognition process.

Antibodies against eukaryotic initiation factor 4A (eIF-4A) were used to study the involvement of this factor in recognizing the 5' cap structure of eukaryotic mRNA. We demonstrate that an approximately 50-kilodalton polypeptide present in rabbit reticulocyte ribosomal high salt wash which can be specifically cross-linked to the 5' oxidized cap structure of reovirus mRNA (Sonenberg, N. (1981) Nucleic Acids Res. 9, 1643) reacts with an anti-eIF-4A monoclonal antibody. We also show that antibodies against eIF-4A react with a 50-kilodalton polypeptide present in a cap-binding protein complex obtained by elution from a m7GTP-agarose affinity column. Comparative peptide analysis of eIF-4A and the 50-kilodalton component of the cap-binding protein complex indicates a very strong similarity between the two polypeptides.

Adenosine Triphosphate↗
PubMed1983

mRNA secondary structure as a determinant in cap recognition and initiation complex formation. ATP-Mg2+ independent cross-linking of cap binding proteins to m7I-capped inosine-substituted reovirus mRNA.

Polypeptides of Mr = 50,000 and 80,000 in rabbit reticulocyte initiation factor preparations can be specifically cross-linked to the oxidized t' cap structure of native reovirus mRNA in an ATP-Mg2+-dependent manner (Sonenberg, N., Guertin, D., Cleveland, D., and Trachsel, H. (1981) Cell 27, 563-572). However, specific cross-linking of these polypeptides can occur in the absence of ATP-Mg2+ when m7I-capped inosine substituted mRNA, which contains less secondary structure than native reovirus mRNA, is used. We also found, using wheat germ extract, that inhibition of initiation complex formation by high salt concentrations is directly related to the degree of secondary structure of the mRNA. Binding of ribosomes to bromouridine-substituted reovirus mRNA is severely inhibited at high K+ concentrations, while binding to inosine-substituted mRNA is only slightly inhibited and binding of native reovirus mRNA is inhibited to an intermediate degree. The results are consistent with the hypothesis that cap recognition factors mediate an ATP-dependent melting of secondary structures involving 5' proximal sequences to the initiation codon in order to facilitate binding of ribosomes during translation initiation.

Adenosine Triphosphate↗
PubMed1982

Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex.

Following poliovirus infection of HeLa cells, the synthesis of cellular proteins is inhibited but translation of poliovirus mRNA proceeds. The defect in the recognition of host cell mRNA may be due to a change in a cap recognition complex which, when added to an infected cell lysate, restores the ability to translate capped mRNAs. We employed immunoblotting techniques to examine initiation factors in crude lysates from uninfected and poliovirus-infected HeLa cells. Using an antiserum against eucaryotic initiation factor 3, we detected an antigen of approximate molecular weight 220,000 in uninfected cell lysates but not in infected cell lysates. Antigenically related polypeptides of 100,000 to 130,000 daltons, presumably degradation products, were detected in the infected cell lysate. The time course for degradation of the 220,000-dalton polypeptide correlates with that for inhibition of cellular protein synthesis in vivo. A portion of the population of 220,000-dalton polypeptides apparently associates with initiation factor eIF3 but is readily dissociated in buffers containing high salt. Affinity-purified antibodies against the polypeptide recognize a protein of the same size in a purified preparation of a cap binding protein complex obtained by cap-affinity chromatography. We postulate that the 220,000-dalton polypeptide is an essential component of the cap recognition complex and that its degradation in poliovirus-infected cells results in the inhibition of host cell translation. These results are in the first demonstration of a specific structural defect in an initiation factor resulting from poliovirus infection.

Carrier Proteins↗
PubMed1982

Association of cap binding protein-related polypeptides with cytoplasmic RNP particles of chick embryonic muscle.

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (Mr 78 000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-Mr immunoreactive peptides (Mr 43 000 and Mr 29 000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive Mr 43 000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.

Animals↗
PubMed1982

Inactivation of cap-binding proteins accompanies the shut-off of host protein synthesis by poliovirus.

Infection of HeLa cells with poliovirus results in a rapid shut-off of host protein synthesis. It has been suggested that inactivation of a protein that binds to the cap structure of cellular mRNAs would explain the selective inhibition of host protein synthesis because the naturally uncapped poliovirus RNA can be translated by a cap-independent mechanism. To test directly for the presence of cap-binding proteins in poliovirus-infected and mock-infected cells, we analyzed initiation factor preparations for their ability to specifically crosslink to the 5' cap structure of oxidized reovirus mRNA. The data presented here show that the crosslinking ability of the different cap-binding proteins (24-, 28-, 32-, 50-, and 80-kilodalton polypeptides) is reduced in preparations from poliovirus-infected as compared to mock-infected cells. This reduction correlates with the inability of initiation factor preparations from infected cells to restore translation of capped mRNAs in extracts of poliovirus-infected cells. In addition, initiation factor preparations from poliovirus-infected cells have the ability to rapidly inactivate cap-binding proteins and can also impair the restoring activity of initiation factors from mock-infected cells.

Carrier Proteins↗
PubMed1982

Capped mRNAs with reduced secondary structure can function in extracts from poliovirus-infected cells.

Extracts from poliovirus-infected HeLa cells were used to study ribosome binding of native and denatured reovirus mRNAs and translation of capped mRNAs with different degrees of secondary structure. Here, we demonstrate that ribosomes in extracts from poliovirus-infected cells could form initiation complexes with denatured reovirus mRNA, in contrast to their inability to bind native reovirus mRNA. Furthermore, the capped alfalfa mosaic virus 4 RNA, which is most probably devoid of stable secondary structure at its 5' end, could be translated at much higher efficiency than could other capped mRNAs in extracts from poliovirus-infected cells.

HeLa Cells↗