Inhibitory effect of methylated derivatives of guanylic acid for protein synthesis with reference to the functional structure of the 5′-‘cap’ in viral messenger RNA

Miura, Kiyonori; Kodama, Yaeko; Shimotohna, Kunitada; Fukui, Toshikazu; Ikehara, Morio; Nakagawa, Iwao; Hata, Tsujiaki

doi:10.1016/0005-2787(79)90224-7

Cited by 9 publications

(5 citation statements)

References 29 publications

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“…Effects of Two Methyl Groups at N2 . Previous studies indicated that, while one methyl group on N2 can be tolerated, the presence of two methyl groups greatly diminishes the effectiveness of the cap ( , ). The results of our analysis confirm this observation.…”

Section: Resultsmentioning

confidence: 99%

“…The exocyclic amino group N2 of the 7-methylguanine moiety is known to be important for binding, since cap analogues containing 7-methylinosine or 7-methylxanthosine fail to inhibit mRNA binding to ribosomes (37). Previous studies have shown that a single methyl substituent at N2 is tolerated (37,38,41). Quantitative analysis shows that addition of a single methyl group to N2 increases the effectiveness of cap analogues as translational inhibitors (Table 1).…”

Section: Estimation Of Apparent K I Valuesmentioning

confidence: 99%

“…The direct binding of cap analogues to eIF4E has been assessed by quenching of intrinsic Trp fluorescence in eIF4E ( − ) and enhancement of 7-methylguanosine fluorescence (). Varying the structure of the cap analogue demonstrated that binding to eIF4E (or inhibition of protein synthesis) can still occur if (1) the 7-substituent is an aryl or alkyl group other than methyl, (2) the ribose rings are substituted at the 2‘-position, and (3) the N2 of guanine is substituted with one but not more methyl groups ( − ). Cap analogues with more phosphate groups or that are part of an oligonucleotide bind eIF4E with greater affinity ( , ).…”

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Quantitative Assessment of mRNA Cap Analogues as Inhibitors of in Vitro Translation

et al. 1999

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Fifty-eight analogues of the 5'-terminal 7-methylguanosine-containing cap of eukaryotic messenger RNA were synthesized and tested for their ability to inhibit in vitro protein synthesis. A new algorithm was developed for extracting KI, the dissociation constant for the cap analogue.eIF4E complex, from protein synthesis data. The results indicated that addition of a methyl group to the N2 of guanine produced more inhibitory compounds, but addition of a second methyl group to N2 decreased the level of inhibition dramatically. Aryl substitution at N7 improved the efficacy of guanine nucleoside monophosphate analogues. Substitution of the aromatic ring at the para position with methyl or NO2 groups abolished this effect, but substitution with Cl or F enhanced it. By contrast, aryl substitution at N7 in nucleoside di- or triphosphate analogues produced only minor effects, both positive and negative. By far the strongest determinants of inhibitory activity for cap analogues were phosphate residues. The beneficial effect of more phosphate residues was related more to anionic charge than to the number of phosphate groups per se. The second nucleotide residue in analogues of the form m7GpppN affected inhibitory activity in the order G > C > U > A, but there was no effect of 2'-O-modification. Opening the first ribose ring of m7GpppG analogues dramatically decreased activity, but alterations at the 2'-position of this ribose had no effect. Non-nucleotide-based cap analogues containing benzimidazole derivatives were inhibitory, though less so than those containing 7-methylguanine.

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Section: Resultsmentioning

confidence: 99%

Section: Estimation Of Apparent K I Valuesmentioning

confidence: 99%

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confidence: 99%

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Quantitative Assessment of mRNA Cap Analogues as Inhibitors of in Vitro Translation

et al. 1999

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“…We became interested in the biological activity of diand trimethylated guanine caps through our studies pertaining to ligand binding properties of cap binding proteins as noted above. Previous studies by us (4)(5)(6) and others (3,24) employed nucleotide cap analogs as competitive inhibitors in partial reaction and complete translation assays. However because several cap binding activities are present in partially fractionated cell extracts, competitive inhibition studies in reality characterize the combined cap binding requirements of the translation machinery.…”

Section: Introductionmentioning

confidence: 99%

-globin mRNAs capped with m7G, m22.7G or m32.2.7G differ in intrinsic translation efficiency

Darżynkiewicz¹,

Stępiński²,

Ekiel³

et al. 1988

Nucleic Acids Research

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We examined the intramolecular effect of altered cap structures on translation efficiency of artificial beta-globin mRNAs. For these studies, synthetic dinucleotides of the form X(5')ppp(5')G [X = 7-methyl guanosine (m7G), 2,7-dimethyl guanosine (m2(2,7)G) or 2,2,7-trimethyl guanosine (m3(2,2,7)G)], were transcriptionally incorporated into mRNAs, containing rabbit beta-globin coding sequences, using T7 RNA polymerase and a beta-globin cDNA template. These synthetic mRNAs were assayed in reticulocyte lysate for activity relative to m7G-capped mRNA. m2(2,7)G-Capped mRNA was found to be 1.5-fold more active than m7G-capped mRNA. Messenger RNA capped with m3(2,2,7)G was less active with activity of 0.24 relative to its m7G-capped counterpart (activity = 1.0). These data suggest that m7G-capped mRNAs become more active as translation templates after addition of a single N2 methyl moiety, which is especially pertinent to gene expression in togaviridae. The latter are observed to synthesize m2(2,7)G and m3(2,2,7)G-capped mRNAs in addition to m7G-capped templates during the course of infection in animal cells.

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“…When a cap-deleted mRNA (23,32) or mRNA with an unmethylated-cap (33) was added to the protein-synthesizing system, formation of the 80S initiation complex decreased remarkably. Addition of cap analogue or 7-methylguanylic acid to the in vitro protein-synthesizing system also inhibits initiation-complex formation ofan intact mRNA competitively (34,35). These datasuggest that the cap structure participates in initiation-complex formation but not obligatorily.…”

Section: Discussionmentioning

confidence: 99%

Relationship between structure of the 5' noncoding region of viral mRNA and efficiency in the initiation step of protein synthesis in a eukaryotic system.

Yamaguchi¹,

Hidaka²,

Miura³

1982

Proc. Natl. Acad. Sci. U.S.A.

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To determine whether the rate of protein synthesis is controlled by the structure ofmRNA near its 5' terminus, protein-synthesizing ability, especially in its initial stage, was compared among RNAs of plant viruses. Those viruses used here contain several definite pieces of single-stranded RNA. Each of these RNAs acts as a messenger. Cucumber mosaic virus (CMV) RNA 5 synthesizes a small amount of a protein, Mr 7000, in an in vitro protein-synthesizing system from wheat germ or reticulocyte. Brome mosaic virus (BMV) RNA 4 synthesizes a large amount of a coat protein under the same conditions. Both RNAs have the same 5'-cap structure and a short noncoding region (10 nucleotides in CMV RNA 5 and 9 in BMV RNA 4) between the 5' terminus and the initiation codon AUG. A sequence complementary to the 3' terminal of 18S ribosomal RNA is contained in BMV RNA 4 but is not apparent in CMV RNA 5. Formation of the initiation complex for protein synthesis by the 5'-terminal-labeled mRNA of cytoplasmic polyhedrosis virus was inhibited by the addition of unlabeled BMV RNA 4 whereas it was only slightly inhibited by unlabeled CMV RNA 5. BMV RNA 4, which has a sequence complementary to rRNA, can form the initiation complex more easily than CMV RNA 5. It is concluded that an apparent complementary sequence to the 3' terminal of 18S rRNA in the 5' noncoding region of eukaryotic mRNA and the 5'-cap structure enhance the rate of initiation complex formation in protein synthesis.Usually, prokaryotic mRNA is polycistronic whereas eukaryotic mRNA is monocistronic. The latter carries the cap structure at the 5' terminal and protein synthesis begins from the initiation codon AUG (1). In prokaryotic mRNA, a sequence complementary to the 3'-terminal part of 16S rRNA is located in front of the initiation codon of protein synthesis, and it is considered to bind to ribosomes to form an initiation complex as the first step in protein synthesis (2-5).The nucleotide sequences of the 3'-terminal region of 18S eukaryotic rRNA have been determined for mouse, rat, rabbit, hen, silkworm, wheat, and barley (6-8). These are almost identical to one another and similar to prokaryotic 16S rRNA sequences. Thus, this part of eukaryotic rRNA should have a secondary structure similar to the prokaryotic one, taking a hairpin structure containing the m2A-m2A sequence in the loop part (6-8). According to the psolaren crosslinking experiment of Nakashima et al. (9), the 5'-terminal part of eukaryotic mRNA interacts with the 3'-terminal part of 18S rRNA as seen in prokaryotes. But the eukaryotic sequence of 18S rRNA near the 3' terminus lacks the sequence C-C-U-C-C, which is complementary to mRNA in prokaryotes. Therefore, the mechanism of initiation interaction of mRNA with ribosomes should be different in prokaryotes and in eukaryotes. The 5'-terminal noncoding regions ofthe different eukaryotic mRNAs vary not only in length and secondary structure but also in nucleotide sequence. Some contain a nucleotide sequence complementary to the 3'-terminal part of...

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Inhibitory effect of methylated derivatives of guanylic acid for protein synthesis with reference to the functional structure of the 5′-‘cap’ in viral messenger RNA

Cited by 9 publications

References 29 publications

Quantitative Assessment of mRNA Cap Analogues as Inhibitors of in Vitro Translation

Quantitative Assessment of mRNA Cap Analogues as Inhibitors of in Vitro Translation

-globin mRNAs capped with m7G, m22.7G or m32.2.7G differ in intrinsic translation efficiency

Relationship between structure of the 5' noncoding region of viral mRNA and efficiency in the initiation step of protein synthesis in a eukaryotic system.

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