Discovery of m(7)G-cap in eukaryotic mRNAs

Abstract

Terminal structure analysis of an insect cytoplasmic polyhedrosis virus (CPV) genome RNA in the early 1970s at the National Institute of Genetics in Japan yielded a 2'-O-methylated nucleotide in the 5' end of double-stranded RNA genome. This finding prompted me to add S-adenosyl-L-methionine, a natural methylation donor, to the in vitro transcription reaction of viruses that contain RNA polymerase. This effort resulted in unprecedented mRNA synthesis that generates a unique blocked and methylated 5' terminal structure (referred later to as "cap" or "m(7)G-cap") in the transcription of silkworm CPV and human reovirus and vaccinia viruses that contain RNA polymerase in virus particles. Initial studies with viruses paved the way to discover the 5'-cap m(7)GpppNm structure present generally in cellular mRNAs of eukaryotes. I participated in those studies and was able to explain the pathway of cap synthesis and the significance of the 5' cap (and capping) in gene expression processes, including transcription and protein synthesis. In this review article I concentrate on the description of these initial studies that eventually led us to a new paradigm of mRNA capping.

Figure 1.

Cap structure. m⁷GpppNm pN^m- (Cap 2) representing the m⁷G linked to the 5′-end of the primary transcript via a 5′-5′ triphosphate.

Figure 2.

CPV dsRNA genome segments and the 3′-terminal analysis. A: Polyacrylamide gel electrophoresis of CPV genome dsRNA segments. B: 3′ ³H-labeling of CPV genome dsRNAs by periodate oxidation followed by [³H]-NaBH₄. C: Distribution of ³H-radioactivity in 3′-terminal nucleosides and NNM (non-nucleoside material). Parts of the data are reprinted from the paper by Furuichi and Miura (1973).¹³⁾

Figure 3.

Reaction scheme employed for analysis of 5′ and 3′ terminal structures of CPV genome RNA and retrospective identification of NNM and cap structure. (I): CPV genome RNA. (II): Periodate oxidized RNA, 2′ and 3′ OH were oxidized to aldehydes. (III): [³H]-NaBH₄ reduced RNA which contains ³H labeled-CH₂OH (shown by blue). The shadowed structure was turned out to be NNM later. (IV): CPV RNA produced from (III) by aniline treatment which removes the ribose-oxidized nucleoside. (V): Alkaline phosphatase-treated CPV RNA (IV), which is devoid of sensitive phosphates. (VI): 5′-³²P-labeled CPV RNA which was phosphorylated by polynucleotide kinase and [γ-³²P]ATP (the radioactive phosphates are shown by red).

Figure 4.

Stimulation of CPV mRNA synthesis by S-adenosyl-L-methionine. Data with slight modifications are obtained from the paper by Furuichi (1974).¹⁹⁾

Figure 5.

Mechanism of cap formation.

Figure 6.

Two important biological functions of m⁷G-cap: mRNA stabilization and stimulation of protein synthesis at the initiation. The ³²P-labelled reovirus mRNAs having the three different types of 5′ structures, namely m⁷GpppG^m-, GpppG- and ppG-, were prepared by the method of Furuichi and Shatkin (1976)⁵¹⁾ and were either microinjected to frog oocytes or incubated in the wheat germ protein synthesizing extract and examined mRNA stability and translational activity. Data are obtained from the paper by Furuichi et al. (1977).⁵⁾