Cell, Vol. 4, 379-386,
April
1975,
Copyright0
1975
by MIT
Methylated Nucleotides Block 5’ Terminus of HeLa Cell Messenger RNA Cha-Mer Wei, Alan Gershowitz, and Bernard Moss Laboratory of Biology of Viruses National Institute of Allergy and Infectious Diseases Bethesda, Maryland 20014
Poiyadenyiylated [poiy(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poiy(A)+ RNA was calculated from the 3H/l4C ratios and known degrees of methyiation of 18s and 28s rlbosomal RNAs. Ail four 2’-Omethyiribonucieosides, but only two basemethylated derivatives, 7-methylguanosine (7MeG) and 6-methyiadenosine (GMeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyllabeled rlbonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5’ terminus of the poiy(A)+ RNA and could be removed by periodate oxidation and /3 elimination. Evidence for a 5’ to 5’ linkage of 7MeG to adjacent 2’-0-methyiribonucieosldes through at least two and probably three phosphates to give structures of the type 7MeGs’ppps’NMepand 7MeG5’ ppps’NMepNmepwas presented. The previous finding of similar sequences of methyiated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5’ terminl may be a characteristic feature of mRNAs that function in eucaryotic ceils. Introduction Methylated ribonucleosides, thought until recently to be unique to ribosomal and transfer RNAs, have been detected in both eucaryotic (Perry and Kelley, 1974) and viral (Furuichi, 1974; Wei and Moss, 1974; Shatkin, 1974) mRNAs. The methylated nucleosides of mRNAs, synthesized in vitro by enzymes packaged within virus cores, are located exclusively at the 5’ termini (Furuichi, 1974; Wei and Moss, 1974; Shatkin, 1974). Vaccinia virus mRNAs were found to have the unusual sequences 7MeGs’ppps’GMepand 7MeGs’ppps’AMep(Wei 1975). A similar sequence and Moss, 7MeGs’ppps’GMepCpwas found at the termini of reovirus mRNAs (Furuichi et al., 1975). Less is known about the arrangement of methylated nucleotides in eucaryotic cell mRNA. Perry and Kelley (1974) reported the presence of 2.2
methyl groups per 1000 nucleotides of L cell mRNA, corresponding to 6 or 7 methylated nucleotides per RNA molecule, and provided evidence for both base and ribose methylation. The additional finding of a significant amount of methyl label that eluted from DEAE-cellulose as alkali-resistant oligonucleotides was consistent with the presence of either adjacent ribose-methylated nucleotides or methylation of the 5’ terminus. Desrosiers, Friderici, and Rottman (1974) identified all four 2’-O-methylribonucleosides and one predominant base methylated ribonucleoside, 6-methyladenosine, in mRNA from Novikoff hepatoma cells. The primary intention of the present study was to determine whether the 5’ termini of eucaryotic cell mRNAs, like those of viral mRNAs, are blocked by methylated nucleotides. The absence of blocked termini would indicate an important structural difference that might well provide the basis for selective inhibition of host protein synthesis during virus infection and/or interferon-mediated inhibition of viral protein synthesis. Alternatively, the presence of a similar 5’ terminal sequence in mRNAs from double-stranded DNA viruses, double-stranded RNA viruses, and cells would point to a common function in protein synthesis.
Results Number RNA
of Methylated
Nucleotides
in Poly(A)+
HeLa cells were labeled for 4 hr with [3H-methyl]methionine and r4C-uridine, and the cytoplasmic RNA was separated into poly(A)and poly(A)+ fractions by chromatography on poly(U)-Sepharose. The 3H/% ratios of the heterogeneous size poly(A)+ RNA and the 4S, 18S, and 28s components of the poly(A)RNA, are indicated in Table 1. The number of methyl groups in the poly(A)+ RNA was then calculated from the known degree of methylation of tRNA and rRNA (Brown and Attardi, 1965; lwanimi and Brown, 1968). The values, 1.9-2.1 methyl groups per 1000 nucleotides, calculated using 18s and 28s rRNAs as references (Table l), was similar to the 2.2 methyl groups per 1000 nucleotides obtained by a similar analysis of L cell poly(A)+ RNA (Perry and Kelley, 1974).
Evidence for Base-Methylated and RlbossMethylated Nucleotldes Phosphodiester bonds linking adjacent ribonucleotides are hydrolyzed by alkali unless there is a substitution at the 2’-0 position. Accordingly, alkali hydrolysis releases base-methylated ribonucleotides as mononucleotides, 2’-0-methylribonucleotides as dinucleotides, and adjacent ribose-methylated nucleotides as oligonucleotides. Oligonucleotides
Cell 380
Table
1. Number
of Methyl
RNA Species0 4s
Groups
in Poly(A)+
SH/“Cb
‘H/W ‘H/W
15.4
mRNA Poly(A)+ RNA
April
1975,
Copyright0
1975
by MIT
Methylated Nucleotides Block 5’ Terminus of HeLa Cell Messenger RNA Cha-Mer Wei, Alan Gershowitz, and Bernard Moss Laboratory of Biology of Viruses National Institute of Allergy and Infectious Diseases Bethesda, Maryland 20014
Poiyadenyiylated [poiy(A)+] mRNA from HeLa cells that were labeled with [3H-methyl]-methionine and 14C-uridine was isolated by poly(U)-Sepharose chromatography. The presence of approximately two methyl groups per 1000 nucleotides of poiy(A)+ RNA was calculated from the 3H/l4C ratios and known degrees of methyiation of 18s and 28s rlbosomal RNAs. Ail four 2’-Omethyiribonucieosides, but only two basemethylated derivatives, 7-methylguanosine (7MeG) and 6-methyiadenosine (GMeA), were identified. 6MeA was the major component accounting for approximately 50% of the total methyllabeled rlbonucleosides. 7MeG, comprising about 10% of the total, was present exclusively at the 5’ terminus of the poiy(A)+ RNA and could be removed by periodate oxidation and /3 elimination. Evidence for a 5’ to 5’ linkage of 7MeG to adjacent 2’-0-methyiribonucieosldes through at least two and probably three phosphates to give structures of the type 7MeGs’ppps’NMepand 7MeG5’ ppps’NMepNmepwas presented. The previous finding of similar sequences of methyiated nucleotides in mRNA synthesized in vitro by enzymes associated with virus cores indicates that blocked 5’ terminl may be a characteristic feature of mRNAs that function in eucaryotic ceils. Introduction Methylated ribonucleosides, thought until recently to be unique to ribosomal and transfer RNAs, have been detected in both eucaryotic (Perry and Kelley, 1974) and viral (Furuichi, 1974; Wei and Moss, 1974; Shatkin, 1974) mRNAs. The methylated nucleosides of mRNAs, synthesized in vitro by enzymes packaged within virus cores, are located exclusively at the 5’ termini (Furuichi, 1974; Wei and Moss, 1974; Shatkin, 1974). Vaccinia virus mRNAs were found to have the unusual sequences 7MeGs’ppps’GMepand 7MeGs’ppps’AMep(Wei 1975). A similar sequence and Moss, 7MeGs’ppps’GMepCpwas found at the termini of reovirus mRNAs (Furuichi et al., 1975). Less is known about the arrangement of methylated nucleotides in eucaryotic cell mRNA. Perry and Kelley (1974) reported the presence of 2.2
methyl groups per 1000 nucleotides of L cell mRNA, corresponding to 6 or 7 methylated nucleotides per RNA molecule, and provided evidence for both base and ribose methylation. The additional finding of a significant amount of methyl label that eluted from DEAE-cellulose as alkali-resistant oligonucleotides was consistent with the presence of either adjacent ribose-methylated nucleotides or methylation of the 5’ terminus. Desrosiers, Friderici, and Rottman (1974) identified all four 2’-O-methylribonucleosides and one predominant base methylated ribonucleoside, 6-methyladenosine, in mRNA from Novikoff hepatoma cells. The primary intention of the present study was to determine whether the 5’ termini of eucaryotic cell mRNAs, like those of viral mRNAs, are blocked by methylated nucleotides. The absence of blocked termini would indicate an important structural difference that might well provide the basis for selective inhibition of host protein synthesis during virus infection and/or interferon-mediated inhibition of viral protein synthesis. Alternatively, the presence of a similar 5’ terminal sequence in mRNAs from double-stranded DNA viruses, double-stranded RNA viruses, and cells would point to a common function in protein synthesis.
Results Number RNA
of Methylated
Nucleotides
in Poly(A)+
HeLa cells were labeled for 4 hr with [3H-methyl]methionine and r4C-uridine, and the cytoplasmic RNA was separated into poly(A)and poly(A)+ fractions by chromatography on poly(U)-Sepharose. The 3H/% ratios of the heterogeneous size poly(A)+ RNA and the 4S, 18S, and 28s components of the poly(A)RNA, are indicated in Table 1. The number of methyl groups in the poly(A)+ RNA was then calculated from the known degree of methylation of tRNA and rRNA (Brown and Attardi, 1965; lwanimi and Brown, 1968). The values, 1.9-2.1 methyl groups per 1000 nucleotides, calculated using 18s and 28s rRNAs as references (Table l), was similar to the 2.2 methyl groups per 1000 nucleotides obtained by a similar analysis of L cell poly(A)+ RNA (Perry and Kelley, 1974).
Evidence for Base-Methylated and RlbossMethylated Nucleotldes Phosphodiester bonds linking adjacent ribonucleotides are hydrolyzed by alkali unless there is a substitution at the 2’-0 position. Accordingly, alkali hydrolysis releases base-methylated ribonucleotides as mononucleotides, 2’-0-methylribonucleotides as dinucleotides, and adjacent ribose-methylated nucleotides as oligonucleotides. Oligonucleotides
Cell 380
Table
1. Number
of Methyl
RNA Species0 4s
Groups
in Poly(A)+
SH/“Cb
‘H/W ‘H/W
15.4
mRNA Poly(A)+ RNA
