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Biomedical subjects

N Sonenberg

Publications and source records attributed to N Sonenberg.

At least 271 records · Page 15Linked to original sources
PubMed1982

Association of an Mr 50,000 cap binding protein with the cytoskeleton in BHK cells.

A monoclonal antibody (anti-CBP antibody) is shown to be directed against cap binding protein(s) (CBP) by virtue of its ability to inhibit the translation of capped reovirus mRNA in a cell-free system derived from L-cells and inhibit the specific (cap analogue-inhibited) cross-linking of proteins to the oxidized 5' terminal cap structure of reovirus mRNA. Anti-CBP antibody reacts with an Mr 50,000 polypeptide in rabbit reticulocyte polysomes and this polypeptide appears to be associated with the 5' cap structure of mRNA. In BHK-21 cells immunofluorescence microscopy reveals that the antibody reacts with a fibrous network extending through the cytoplasm in a radial arrangement. The network behaves like intermediate filaments in colchicine-treated cells suggesting a direct or indirect linkage of CBP with intermediate filaments. The association of CBP with a cytoskeletal element is further confirmed by isolation of proteins from Triton X-100-extracted cells and identification of CBP in the cytoskeletal fraction with anti-CBP antibody. The major polypeptide reacting with anti-CBP antibody is an Mr 50,000 component. Tryptic peptide mapping shows that this polypeptide is related to an Mr 24,000 polypeptide identified as cap binding protein in earlier experiments [Sonenberg, Morgan, Merrick & Shatkin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4843-4847].

Animals↗
PubMed1981

Design and preparation of affinity columns for the purification of eukaryotic messenger ribonucleic acid cap binding protein.

2',3'-O-[1-(2-Carboxyethyl) ethylidene]-7-methylguanosine 5'-diphosphate (5) and 7-(5-carboxypentyl) guanosine 5'-diphosphate (13) have been synthesized and immobilized on AH-Sepharose 4B to the extent of 17.4 and 36.6 mumol of ligand/g of gel, respectively. The affinity resins thus derives were employed in columns for the purificaton of 24K cap binding protein (CBP) from rabbit reticulocytes. Each resin was found to retain the protein of interest; elution of 24K CBP could then be effected by washing with 70 microM m7GDP. The 24K CBPs released from both columns were found to be active, both as judged by a cross-linking assay that utilized 10(4)-oxidized methyl-3H-labeled reovirus mRNA as a substrate for the protein and also by the ability of the isolated 24K CBP to stimulate the translocation of capped Sindbis virus mRNA in HeLa cell extracts.

Carrier Proteins↗
PubMed1981

In vitro translation in reovirus- and poliovirus-infected cell extracts. Effects of anti-cap binding protein monoclonal antibody.

Monoclonal antibodies directed against rabbit reticulocyte cap binding proteins were used as probes to study cap-dependent translation initiation in mock-infected and virus (poliovirus or reovirus)-infected cells. The antibodies inhibited capped mRNA translation, but did not have any effect on the translation of naturally uncapped mRNAs in uninfected cell-free extracts prepared from either L-cells or HeLa cells. Cell-free extracts prepared from either reovirus-infected L-cells or poliovirus-infected HeLa cells did not support capped mRNA translation. However, translation of naturally uncapped mRNAs in the latter extracts was comparable to that observed in extracts from uninfected cells. Thus, translation of mRNAs in extracts from either reovirus- or poliovirus-infected cells proceeds most probably via a cap-independent mechanism, since it is not inhibited by antibodies directed against cap binding proteins.

Animals↗
PubMed1981

ATP/Mg++-dependent cross-linking of cap binding proteins to the 5' end of eukaryotic mRNA.

Two proteins of apparent molecular weights of 28,000 and 50,000 daltons were shown to recognize and cross-link specifically to the 5' cap end of oxidized reovirus mRNA. Cross-linking of these proteins to mRNA was ATP/Mg++ dependent, in sharp contrast to cross-linking of a 24K cap binding protein which was purified and characterized previously (Sonenberg, N., Rupprecht, K.M., Hecht, S.M. and Shatkin, A.J. (1979) Proc. Natl. Acad. Sci, USA 76, 4345-4349). Non-hydrolyzable analogues of ATP as well as other nucleotides did not substitute for ATP in the cross-linking reaction and Mg++ was significantly preferred over other divalent cations in cross-linking of the 28K and 50K dalton proteins. A model involving the function of the latter proteins in recognition and unwinding of the 5' end structure of capped eukaryotic mRNAs is suggested.

Adenosine Triphosphate↗
PubMed1981

Probing the function of the eucaryotic 5' cap structure by using a monoclonal antibody directed against cap-binding proteins.

A monoclonal antibody directed against cap-binding proteins was used to elucidate the possible mechanism by which cap-binding proteins function in initiation of eucaryotic translation. The monoclonal antibody preparation employed in this study exhibited a marked differential effect in inhibiting the translation of folded, capped eucaryotic-mRNAs to a far greater extent than naturally uncapped mRNAs or native capped mRNAs that do not possess extensive 5' end secondary structure. These findings were consistent with the effects of the antibody on initiation complex formation with three different types of reovirus mRNA: native reovirus mRNA; inosine-substituted reovirus mRNA, which has a relaxed secondary structure; and bromouridine-substituted reovirus mRNA, in which base pairing is enhanced relative to regular reovirus mRNA. The extent that translation initiation complex formation was inhibited by the monoclonal antibody directly correlated to the degree of secondary structure present in the mRNA. Binding of bromouridine-substituted reovirus mRNA to ribosomes was inhibited to the greatest extent, while binding of inosine-substituted reovirus mRNA was not inhibited at all in the reticulocyte lysate system or was slightly inhibited in a wheat-germ system. These results support the hypothesis that cap-binding proteins are involved in unwinding of the 5' terminal, secondary structure of many eucaryotic mRNAs, thus facilitating the attachment of ribosomes to mRNA.

Animals↗
PubMed1980

Restricted initiation of protein synthesis on the potentially polycistronic Sindbis virus 42 S RNA.

Sindbis 42 S genome RNA was isolated from virions and translated in vitro before and after purification by oligo(dT)-cellulose chromatography and sucrose density gradient centrifugation under denaturing conditions. In intact 42 S RNA, only the 5'-proximal initiation site for the synthesis of nonstructural proteins was used. The internally located start site for viral structural protein formation was active in broken genome RNA molecules where, as a consequence of fragmentation, it was closer to a 5' end. The results of several kinds of experiments indicate that the fragmentation-dependent synthesis of structural proteins directed by virion RNA was not due to the presence of 26 S subgenomic messenger RNA.

Animals↗
PubMed1980

Purification of a factor that restores translation of vesicular stomatitis virus mRNA in extracts from poliovirus-infected HeLa cells.

It was previously shown that the poliovirus-induced inhibition of translation of capped mRNAs can be reversed by a protein found in preparations of the eukaryotic initiation factor eIF-4B [Rose, J. K., Trachsel, H., Leong, K. & Baltimore, D. (1978) Proc. Natl. Acad. Sci. USA 75, 2732--2736]. This "restoring factor" has now been purified from a high-salt wash of rabbit reticulocyte ribosomes by taking advantage of its tight association with factor eIF-3 at low salt concentrations. It did not copurify with the major Mr 80,000 polypeptide of eIF-4B preparations but did copurify with a Mr 24,000 polypeptide previously shown to bind to the cap structures of mRNAs [Sonenberg, N., Rupprecht, K. M., Hecht, S. M. & Shatkin, A. J. (1979) Proc. Natl. Acad. Sci. USA 76, 4345--4349]. Both the electrophoretic mobility and the tryptic peptide pattern of the restoring factor were indistinguishable from those of the cap-binding protein, and the restoring factor could be crosslinked to the 5'-terminal cap on mRNA. Thus, is appears that poliovirus inhibits cellular protein synthesis by inactivation of some crucial property of the cap-binding protein.

Carrier Proteins↗
PubMed1979

Interaction of a limited set of proteins with different mRNAs and protection of 5'-caps against pyrophosphatase digestion in initiation complexes.

A variety of 5'-3H-methyl-labeled, oxidized viral mRNAs were used as probes for detecting in wheat germ initiation complexes proteins that interact with, and can be cross-linked to, the 5'-cap structure. A limited and reproducible set of specific proteins was obtained with the different mRNAs. The binding of these proteins to the 5'-end of mRNA apparently results in protection against nucleotide pyrophosphatase digestion of the cap even in initiation complexes in which the 5'-end is susceptible to pancreatic RNase digestion. Cross-linked proteins from mammalian initiation complexes comigrated with several of the subunits of similarly treated eIF-3. A model for cap binding protein interaction with mRNA cap during initiation of translation is suggested.

Animals↗
PubMed1979

Characterization of rabbit reticulocyte factor(s) that stimulates the translation of mRNAs lacking 5'-terminal 7-methylguanosine.

Purified reticulocyte initiation factors were assayed for their ability to stimulate the translation of uncapped vesicular stomatitis virus (VSV) mRNA relative to capped VSV mRNA in reticulocyte lysates. Both eIF-3 and eIF-4B preparations contained such an activity. However, at least some of the activity was due to the presence of an as yet unidentified factor that contaminated the eIF-3 and eIF-4B preparations. A polypeptide of apparent molecular weight 24,000 that can be specifically cross-linked to the oxidized 5'-terminal cap structures of reovirus and other viral mRNAs co-purifies with this new factor, and may be a component of it. This stimulatory activity present in our eIF-4B preparations was found to be greater when lower concentrations of KCl were present in the reaction mixture, possibly explaining why uncapped VSV mRNA is translated relatively more efficiently at low K+ concentrations.

Animals↗
PubMed1979

Eukaryotic mRNA cap binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.

A 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Natl. Acad. Sci. USA (1978) 75, 4843--4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid O2',3'-acetal of m7GDP to AH-Sepharose 4B. Translation, in HeLa cell extracts, of capped mRNAs including Sindbis virus, reovirus, and rabbit globin mRNAs was stimulated by the cap-binding protein under conditions that did not increase translation of noncapped RNAs of encephalomyocarditis virus and satellite tobacco necrosis virus.

Animals↗
PubMed1978

Nonspecific effect of m7GMP on protein-RNA interactions.

Cap analogs m7GMP and m7GDP inhibit binding of eukaryotic initiation factors to reovirus capped mRNA but also inhibit complex formation involving uncapped mRNA or 18 S rRNA. Furthermore, Escherichia coli DNA-dependent RNA polymerase binds 18 S rRNA and this interaction is also blocked by m7GMP. These results indicate that inhibition by cap analogs is not a stringent test for putative cap-specific binding between proteins and mRNA.

DNA-Directed RNA Polymerases↗
PubMed1978

A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5'-terminal cap in mRNA.

Protein synthesis initiation factors prepared from rabbit reticulocyte and mouse ascites ribosomes were tested for the ability to crosslink to the 5' cap of mRNA. Crosslinking of one polypeptide of apparent molecular weight 24,000 was inhibited by the cap analogs, m7GMP and m7GDP, indicating a specific interaction with the cap. Although specific crosslinking of the 24,000 molecular weight polypeptide was found with eukaryotic initiation factor 3 and to a lesser extent with initiation factor 4B, both of these factors contained less than stoichiometric amounts of this polypeptide. The crosslinking method provides a highly sensitive and specific assay for cap-binding proteins and should facilitate their purification for functional studies.

Chromatography, DEAE-Cellulose↗
PubMed1978

Analysis of terminal structures of RNA from potato virus X.

The 5'-end structure of potato virus X RNA was determined following enzymatic methylation in vitro. A single 3H-methyl group was introduced into the 2'-position of the 5'-penultimate residue and the end structure was determined as m7GpppG(m)pAp(Xp)3G. This part of the RNA apparently is involved in binding to ribosomes since it can be partially protected against RNase digestion by wheat germ 40S ribosomes. PVX RNA was not retained by poly(U)-sepharose, indicating that it does not contain a 3'-terminal poly(A) tract.

Base Sequence↗
PubMed1977

Mapping of 23-S rRNA at the ribosomal peptidyl-transferase center by photo-affinity labeling.

Photo-sensitive peptidyl-tRNA's were used to scan the environment of the peptidyl-transferase center of the ribosome. The specificity of the previously described labeling in the 18-S fragment of 23-S rRNA by Boc-Phe(N3)-Phe-tRNA (4-azido-N-t-butoxycarbonyl-phenylalanyl-phenylalanyl-tRNA) was demonstrated by the ability of the covalently anchored molecule to serve as donor substrate in peptide bond formation. Labeling patterns were also obtained with Boc-Phe(N3)-Phe-Phe-tRNA bound at the acceptor site and with Boc-Phe(N3)-(Gly)n-Phe-tRNA (n = 2,4). The results indicate that subsequences within the 18-S fragment of 23-S rRNA are located close to the acceptor site as well as along the path where the peptide moiety adheres to the ribosome. Identification of the labeled sequences is expected to shed light on the spatial arrangement as well as functional role of rRNA in the peptidyl transferase center.

Acyltransferases↗
PubMed1977

Reovirus mRNA can be covalently crosslinked via the 5' cap to proteins in initiation complexes.

Proteins that are located adjacent to the 5' end of mRNA in initiation complexes have been detected by chemical crosslinking. Reovirus mRNA containing radioactivity exclusively in the [3H]methyl-labeled "cap," m7G(5')ppp(5')-Gm, was oxidized with sodium periodate to convert the 2',3'-cis-diol of the 5'-terminal m7G to a reactive dialdehyde. Oxidized mRNA was incubated in cell-free protein-synthesizing systems derived from wheat germ or mammalian cells, and the resulting mRNA-ribosome initiation complexes were reduced with NaBH3CN. By this chemical procedure, putative Schiff bases between mRNA 5'termini and amino groups of neighboring proteins were stabilized by reduction, yielding covalently linked protein-RNA conjugates. Under conditions of ribosome binding, a limited number of polypeptides associated with the mRNA-ribosome complexes were crosslinked, suggesting that these proteins are positioned near and may interact with the 5' end of mRNA during initiation. This method should also be useful for studying the spatial relationships between molecules in other similar nucleoprotein complexes.

Animals↗