mRNA Cap Structure Research Articles

Darstellung 5′.5″-phosphatverknüpfter Dinucleoside / Preparation of 5′,5″-Phosphate Linked Dinucleosides

Abstract We report on the chemical preparation of 5′,5″-phosphate bridged dinucleosides (1-16) by applying the 1,1′-carbonyl-diimidazole activation method. Nucleotides of this unusual type function as 5′-terminal cap structures of most eukaryotic mRNAs and on the monomer level as regulators in macromolecular biosynthesis.

P1, P3-(5′-Guanosyl-5″-[14C]adenosyl) triphosphat: Darstellung der Kappenmutterverbindung sowie deren Abbauverhalten gegenüber subzellulären Fraktionen der Rattenleberzelle / P1, P3-(5′-Guanosyl-5″-[14C] adenosyl) triphosphate: Preparation of the Cap Parent Compound and Its Catabolic Properties towards Rat Liver Subcellular Fractions

We report on the chemical preparation of 14C-labeled P1, P3-(5'-guanosyl-5''-adenosyl)triphosphate (1) which is the parent compound of 5'-terminal cap structures of most eukaryotic mRNAs. Incubation experiments of cap (1)- and non cap-structured nucleotides as Ap2A (2) as well as their ribose ring opened derivatives (3, 4) with rat liver subcellular fractions show that cap-degrading nucleases being able to unblock 5' termini are present in nuclei but not in mitochondria.

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.

Antigody-nucleic acid complexes. Inhibition of translation of silkmoth chorion messenger ribonucleic acid with antibodies specific for 7-methylguanosine.

Antibodies specific for 7-methylguanosine (m7G) were evaluated for their ability to inhibit the translation of chorion mRNA in a wheat germ, cell-free amino acid incorporating system. Results obtained with antibody concentrations of 0.5--1.5 microM revealed dose-dependent inhibition of [3H]-labeled amino acid incorporation into acid-insoluble radioactivity. Inhibition of translation was attributed to the interaction of anti-m7G antibodies with the 5' termini of chorion mRNAs on the basis that (a) anti-m7G antibodies coupled to Sepharose (anti-m7G-Sepharose) immunospecifically retained 5'-terminal cap structures of chorion mRNAs, i.e., m7G (5')ppp(5')Nm, (b) significant inhibition of translation required a 2-h preincubation of anti-m7G antibodies with mRNA, and (c) similar preincubation periods with anti-m7G antibodies in the presence of the competing nucleoside hapten (m7G) obviated the inhibitory effect of the antibody. The nature of the anti-m7G antibody-mRNA complex was examined by digesting chorion mRNA with nuclease P1 before (predigested) and after (postdigested) immunospecific adsorption to anti-m7G-Sepharose adsorbent. Whereas predigested preparations yielded a single cap structure of the type m7G(5')ppp(5')N, the predominating cap in the postdigested sample was m7G(5')ppp(5')NpNpN. These latter data revealed that the nucleotide sequence adjacent to the cap was not significantly masked by the antibody and suggest the utility of anti-m7G antibody as a site-specific probe.

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.

Interaction between bases involved in the 5′-terminal cap structure of eukaryotic mRNA

Interaction between bases involved in the 5′-terminal cap structure of eukaryotic mRNA

Antibodies distinguishing between intact and alkali-hydrolyzed 7-methylguanosine.

Antibodies specific for intact 7-methylguanosine (m7G) were induced in rabbits and mice by immunization with nucleoside-BSA or nucleoside-hemocyanin conjugates. Since m7G undergoes alkali-catalyzed hydrolytic fission of the purine ring, modifications were made in the procedure for conjugation of m7G to proteins. After periodate oxidation, m7G was incubated with protein at pH 9.1 at 4 degrees C for one hour during which the nucleoside was found to be stable. Reduction of the Schiff base was done with t-butylamine borane for 30 minutes, and the conjugated protein was isolated quickly by gel filtration at pH 7.2. Both rabbits and mice produced antibodies that readily distinguished between the intact and hydrolyzed m7G. Antibody specificity depended largely on the presence of an intact 7-substituted imidazole ring and some cross-reaction occurred with 7-methylinosine. A weaker reaction occurred with ribothymidine and thymidine. Mouse antibodies induced by m7G-hemocyanin showed the highest specificity. They also recognized m7G in the isolated mRNA cap structure m7G(5')ppp(5')A.