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Biomedical subjects

Y Furuichi

Publications and source records attributed to Y Furuichi.

At least 217 records · Page 12Linked to original sources
PubMed1976

Methyl labeling of HeLa cell hnRNA: a comparison with mRNA.

The majority of the mRNA molecules in HeLa cells contain 1-2 residue(s) of m6Ap and one blocked, methylated 5' terminal "cap" structure. The hnRNA, which is longer than mRNA, contains both m6Ap and caps but 4-6 times as many m6Ap residues per chain. In addition, nuclear molecules contain T2 RNA ase-resistant, methyl-labeled oligonucleotides ("di-" and "tri-" nucleotides) which are not found in mRNA. Some of the dinucleotides may be precursors to the 2'-0-methylated nucleotides in the cap structures. These results are compatible with internal methylation of hnRNA molecules (both m6Ap and 2'-0-methyl) followed by hnRNA cleavage and the addition of the cap structure to generate at least some of the HeLa cell mRNA. It also appears that some hnRNA molecules, which are longer than most mRNA molecules, contain cap structures suggesting the derivation of some mRNA molecules from the 5' regions of hnRNA.

Cell Nucleus↗
PubMed1976

Differential synthesis of blocked and unblocked 5'-termini in reovirus mRNA: effect of pyrophosphate and pyrophosphatase.

Viral mRNA synthesized in vitro by reovirusassociated enzymes contained 5'-terminal ppG, GpppG, or m7GpppGm. Selective synthesis of one type of 5'-end was obtained by adjusting the composition of the transcription reaction mixture based on a series of reactions proposed for formation of blocked, methylated 5'-terminal sequences of mRNA (capformation). The presence of inorganic pyrophosphate increased the proportion of ppG 5'-ends, while addition of pyrophosphatase yielded mRNA with 5'-terminal GpppG, which in the presence of a methyl donor was converted to m7GpppGm.

Diphosphates↗
PubMed1976

A protein binding the methylated 5'-terminal sequence, m7GpppN, of eukaryotic messenger RNA.

Ribosomal salt washes of Artemia salina embryos contain a protein(s) that binds [3H]m7GpppGpC and [3H]m7GpppGmpC, as measured by retention on nitrocellulose membrane filters. These oligonucleotides correspond in structure to the methylated 5'-terminal sequences (caps) present in many eukaryotic mRNAs. The cap binding protein does not bind the unmethylated counterparts of caps, e.g., [32P]GpppGpCp, or a derivative of m7GpppGmpC containing ring-opened m7G. None of the purified initiation factors IF-MP, IF-M2A, IF-M2B, IF-M3, or IF-MI binds the m7G-containing oligonucleotides.

Base Sequence↗
PubMed1976

The methylation of adenovirus-specific nuclear and cytoplasmic RNA.

Each poly(A) containing cytoplasmic AD-2 MRNA contains at its 5' terminus the general structure m7 GpppN1 pN2p or m7 GpppN1mpN2mpNp as well as an average of 4 m6A and 0.5-1 m5C residues per molecule. Almost all of the N1m residues are adenine derivatives including Am, m6Am and probably m26,6Am. The N2m is mostly Cm but small amounts of the other three methylated bases are also present. All the methylated constitutents of mRNA are distant from the 3' terminal poly(A). The amount of m6A appears to be greater in larger mRNA than in smaller mRNA. Nuclear Ad-2 specific RNA also contains caps, m6A, and m5C with about twice as much m6A relative to caps as cytoplasmic mRNA. The similarity of Ad-2 nuclear and mRNA to HeLa hnRNA and mRNA suggests that adenovirus mRNA production is a good model for eukaryotic mRNA production.

Adenoviridae↗
PubMed1976

Dependence of translation on 5'-terminal methylation of mRNA.

Many eukaryotic cell and viral mRNAs contain 5'-terminal m7G(5')ppp(5')N. Methylation is important for the in vitro translation of vesicular stomatitis virus and reovirus mRNAs. Unmethylated viral mRNAs are methylated by cell-free extrascts of wheat germ and L cells to form 5'-terminal structures of the type, m7GpppN and m7GpppNm, respectively. Rabbit globin mRNA also contains 5'-terminal 7-methylguanosine (m7G). Removal of the m7G by beta-elimination decreases translation. The efficient binding of mRNA to ribosomes is dependent on the presence of 5'-terminal m7G.

Animals↗
PubMed1975

5'-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation.

Unmethylated reovirus and VSV mRNAs are specifically methylated to form 5'-terminal structures of the type, m-7-G(5')ppp(5')N by protein synthesising extracts prepared from wheat germ and mouse L cells. Reticulocyte mRNA also contains 5'-terminal m-7-G. MRNAs having 5'-terminal m-7-G stimulate protein synthesis in vitro. Removal of m-7-G by beta-elimination abolishes translation of the mRNAs.

Animals↗
PubMed1975

Ribosome binding to reovirus mRNA in protein synthesis requires 5' terminal 7-methylguanosine.

Purified reovirus synthesizes in vitro a mixture of mRNA molecules that contain 5' terminal structures of the type ppG..., GpppG..., and m7GpppGm, the relative amount of each depending upon the presence of S-adenosylmethionine (SAM) or S-adenosylhomocysteine (SAH) in the transcription incubation mixture. Reovirus mRNAs containing 5' termini of the type ppG... and GpppG... can be specifically modified by wheat germ extracts in the presence of SAM to yield the structure m7GpppG...(Muthukrishnan et al., 1975). The mRNA methylase activity is ribosome-independent and is recovered almost entirely in the high speed supernatant fraction of wheat germ extracts. Its activity is inhibited by aurintricarboxylic acid. Ribosome binding experiments with reovirus mRNA and wheat germ extract indicate that only those mRNA molecules containing 5' terminal m7G are capable of participating in the initiation of protein synthesis and subsequent polysome formation. mRNA with unmethylated 5' termini do not form a complex with 40S ribosomal subunits. Discrimination between unmethylated and methylated reovirus mRNA, active in protein synthesis, apparently occurs at or before the formation of 40S-mRNA complexes. T1 or pancreatic RNAase digestion of methylated mRNA bound to 80S ribosomes yields 5' terminal fragments of apparent chain lengths about 32 and 36 nucleotides, respectively. A portion of the T1 RNAase-resistant fragments rebinds to ribosomes to form a nuclease-resistant complex. In contrast, the shorter 5' terminal oligonucleotide m7GpppGmpCpUp(Np)3Gp derived by RNAase T1 digestion of purified reovirus mRNA does not bind to ribosomes. The results suggest that 5' terminal m7G may function as the primary recognition signal for ribosome binding and that wheat germ ribosomes bind very close to the 5' termini of some of the species of methylated reovirus mRNAs.

Aurintricarboxylic Acid↗
PubMed1975

Detection in HeLa cell extracts of a 7-methyl guanosine specific enzyme activity that cleaves m7GpppNm.

Extracts prepared from HeLa cells contain an enzymatic activity which cleaves m7G(5')ppp(5')Gm to m7pG and ppGm. The activity exhibits a high degree of substrate specificity and does not cleave G(5')ppp(5')G or the ring opened derivative of m7GpppGm which has lost the positive charge from the N7 position of m7G. m7GpppGm as the 5' terminal structure of intact reovirus mRNA is resistant to attack by the pyrophosphatase activity, but becomes partially sensitive in the 5' terminal fragment consisting of 7-10 nucleotides derived from the same mRNA by T1 RNAase digestion. m7G(5')ppp(5')GmpCp is completely sensitive to cleavage resulting in the release of m7pG without generation of m7GpppGm as an intermediate. These results establish the existence of a 7-methyl guanosine specific pyrophosphatase activity in HeLa cells.

Coliphages↗
PubMed1975

5'-Terminal m-7G(5')ppp(5')G-m-p in vivo: identification in reovirus genome RNA.

Methylated reovirus mRNA was synthesized in vitro in the presence of S-adenosyl-L-[methyl-3H]-methionine. Viral genome double-stranded RNA that was uniformly labeled with 32-P was isolated from purified virions. The RNAs were mixed and their 5'-terminal structures compared by electrophoretic and chromatographic analyses after enzymatic digestion. Both the mRNA and the corresponding strand in the genome RNA contain m-7G(5')ppp(5')G-m-pCp, indicating that infected cells synthesize viral RNA with blocked, methylated 5' termini.

Base Sequence↗
PubMed1975

Methylated, blocked 5 termini in HeLa cell mRNA.

Poly(A)-containing HeLa cell mRNA prepared from cells labeled with [methyl-(3)H]methionine or [(32)P]phosphate was found to contain a variety of methylated, blocked 5'-terminal structures of two general types: m(7)GpppN(7)-Np and m(7)GpppN(m)-N(m)-Np. In addition, about one-third of the [(3)H]methyl label was present in the N(6)-methyladenosine; this labeled nucleoside was not found in the 3'-terminal one-third of the mRNA chain and thus may also be in the 5' portion of the mRNA.

Base Sequence↗