Translation of Enzymically Decapped Messenger RNA

Abstract

An enzymic procedure was used to remove the 7-methylguanosine diphosphate moiety at the 5' ends of ratbit hemoglobin mRNA and mouse immunoglobulin light-chain mRNA. Evidence was obtained that the procedure, which involves the use of polynucleotide kinase, does not result in any further degradation of the mRNA.The enzymically decapped mRNA was as effective as untreated mRNA in supporting protein synthesis in a wheat germ system. This was the case over a wide range of mRNA concentrations and over a considerable period of time. The presence in the incubation mixture of S-adenosylhomocysteine, an inhibitor of methylation, did not affect the results.The data indicate that the presence of a 7-methylguanosine diphosphate residue at the 5' end of mRNAs is not an obligatory requirement for translation in eucaryotic systems.It has recently been discovered [1,2] that many eucaryotic mRNAs and heterogeneous nuclear RNAs (hnRNAs) have at the 5' end of the chain certain characteristic features, so-called capped structures. These consist of an 'inverted' 7-methylguanosine, attached to the rest of the messenger through a 5'-5'-triphosphate linkage. Moreover, one or two of the subsequent nucleotides may be methylated in the C-2 positions of the ribose residues.Attempts have been made to elucidate the biological significance of the capped structures by studying the template activity of messengers where the 7-methylguanosine residue has been removed by chemical methods. Thus, Shatkin and coworkers [3] treated rabbit globin messenger and vesicular stomatitis virus mRNA with periodate/aniline, which specifically removes ribose moieties having free hydroxyl groups at the 2 and 3 positions, and hence removes the inverted nucleoside and the 3'-terminal nucleoside. Since the ability of the treated messengers to serve as templates in a protein-synthesizing system was strongly reduced, they concluded that the capped structure is required for normal functioning of the mRNA. It should be realized, however, that periodate/aniline treatment of a capped mRNA leaves abnormal structures at the termini of the chain, viz. a triphosphate group at the 5' end and a phosphate groups at the 3' end of the molecule.Recently we have developed an enzymic procedure which specifically splits the pyrophosphate Abbreviations. m'GDP, 7-methylguanosine diphosphate; Hepes, 4-(2-hydroxyethyl)-l -.piperazineethanesulphonic acid.linkage between m7GDP and the rest of the mRNA [4]. Here we have applied this method to rabbit globin mRNA and mouse immunoglobin light-chain mRNA, and demonstrated that the decapped mRNAs retain their full ability to serve as templates in a protein-synthesizing system. MATERIALS AND METHODS ChemicalsPhosphorylcreatine and creatine kinase were purchased from Calbiochem. Dithiothreitol, ATP, GTP and non-radioactive amino acids were obtained from Sigma Chemical Co. Radioactive amino acids were obtained from the Radiochemical Centre (Amersham, England). 7-Methylguanosine diphosphate was a gift from Dr A. J. Shatkin. Preparation of Crude Immunog...

Effects of Cap Analogue or Cap Removal on the Translation of Rat Brain mRNA In Vitro

“…The use of chemical methods for decapping of mRNA has been criticized on the basis that, in addition to p-elimination of the 5'-terminal m7G, it also produces variable degrees of degradation of mRNA and oxidation of internal nucleosides (Rose and Lodish, 1976;Abraham and Pihl, 1977). In the present experiments, the procedure of Rose and Lodish (1976) was used since it is reported to cause a minimum of mRNA degradation and to lead to a higher recovery of translatable p-eliminated mRNA.…”

“…For cellular mRNA, the 5'-terminal nucleoside is modified or entirely removed by periodate oxidation followed by P-elimination of the nucleoside dialdehyde (Rao et al, 1975;Muthukrishnan et al, 197%;Lodish and Rose, 1977). To avoid degradation and secondary effects of chemical treatments, mRNAs have also been treated with cap-removing enzymes (Abraham and Pihl, 1977;Efstratiadis et al, 1977;Zan-Kowalczewka et al, 1977).…”

Requirement for 7-methylguanosine in translation of globin mRNA in vivo

“…In the absence of appropriate control experiments, results obtained with 6-eliminated RNA prepared by this method must, therefore, be treated with caution. Alternative methods described recently for the removal of the m7G5 ppp X----"CAP" structure involve the use either of polynucleotide kinase (14) or pyrophosphatases isolated from tobacco cells or potato extracts (15,16 5'-terminal labeling with P using ---P-ATP and T4 polynucleotide kinase also showed the 6-eliminated RNA to be intact and usable for nucleotide Figure 1. Polyacrylamide gel electrophoretic analysis in 98% formamide of untreated and treated rabbit globin mRNA.…”

Removal of Wheat‐Germ Agglutinin Increases Protein Synthesis in Wheat‐Germ Extracts

“…Rabbit globin mRNA was isolated by oligo(dT)-cellulose chromatography [7] and purified further by sucrose gradient centrifugation on a 5-20% linear gradient in 20 mM Tris-HCI (pH 7.2) at 40000 rev./min in a SW-40 rotor for 16 h.…”