Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.     

A viral sequence in the 3''-untranslated region mimics a 5'' cap in facilitating translation of uncapped mRNA.

For recognition by the translational machinery, most eukaryotic cellular mRNAs have a 5' cap structure [e.g. m7G(5')ppp(5')N]. We describe a translation enhancer sequence (3'TE) located in the 3'-untranslated region (UTR) of the genome of the PAV barley yellow dwarf virus (BYDV-PAV) which stimulates translation from uncapped mRNA by 30- to 100-fold in vitro and in vivo to a level equal to that of efficient capped mRNAs. A four base duplication within the 3'TE destroyed the stimulatory activity. Efficient translation was recovered by addition of a 5' cap to this mRNA. Translation of both uncapped mRNA containing the 3'TE in cis and capped mRNA lacking any BYDV-PAV sequence was inhibited specifically by added 3'TE RNA in trans. This inhibition was reversed by adding initiation factor 4F (eIF4F), suggesting that the 3'TE, like the 5' cap, mediates eIF4F-dependent translation initiation. The BYDV-PAV 5'UTR was necessary for the 3'TE to function, except when the 3'TE itself was moved to the 5'UTR. Thus, the 3'TE is sufficient for recruiting the translation factors and ribosomes, while the viral 5'UTR may serve only for the long distance 3'-5' communication. Models are proposed to explain this novel mechanism of cap-independent translation initiation facilitated by the 3'UTR.     

Translational control in heat-shocked Drosophila embryos. Evidence for the inactivation of initiation factor(s) involved in the recognition of mRNA cap structure

Lysates from normally growing (25 degrees C) or heat shocked (37 degrees C, 45 min) Drosophila melanogaster embryos were obtained and the effect of analogues of the mRNA 5'-terminal cap, m7G(5')ppp(5')N structure and of potassium ions on their endogenous protein synthesis activity was studied. At optimal concentration of KCH3COO (75-80 mM), protein synthesis in normal lysates is strongly inhibited by cap analogues (m7GpppG, m7GDP, and m7GMP). At the same ionic conditions, heat shock lysates translate preferentially the heat shock messengers, and this translation is almost unaffected by the cap analogues. In contrast, residual synthesis of normal proteins in heat shock lysates was reduced by these compounds. By lowering the concentration of potassium ions it was possible to gradually reverse the inhibitory effect of the cap analogues in normal lysates and also to increase specifically the translation of normal mRNAs in heat shock lysates. Translation of normal mRNAs is also partial but specifically rescued by supplementing heat shock lysates with polypeptide chain initiation factors partially purified from rabbit reticulocytes. These data are consistent with the notion that the failure of normal mRNAs to be translated under heat shock conditions might be due, at least to some extent, to the inactivation of polypeptide chain initiation factor(s) involved in the recognition of the mRNA 5'-terminal cap structure.     

mRNA methylation and protein synthesis in extracts from embryos of brine shrimp, Artemia salina.

Cell-free protein-synthesizing extracts prepared from the brine shrimp, Artemia salina, translate methylated mRNAs. Reovirus unmethylated mRNA is inactive as a template when methylation is prevented by the inhibitor, S-adenosylhomocysteine. A salina mRNAs from both undeveloped and developed embryos contain 5'-terminal 7-methylguanosine in an inverted 5'-5' linkage through three phosphate groups to the rest of the polynucleotide chain. Removal of the 7-methylguanosine by beta elimination converts the mRNA from an active form to one inactive in protein synthesis in extracts of A. salina or wheat germ. Extracts of undeveloped and developed embryos methylate reovirus unmethylated mRNA at the 5' ends to form 5'-terminal structures of the type, m7G(5')ppp(5')G and m7G(5')ppp(5')Gm.     

"Cap" sequences in poly(A)-rich RNA from dry wheat embryos

Although template-active RNA in dry seeds and embryos has attracted widespread interest, there have been no published reports about 5'-terminal "capping" sequences in such RNA. Boro[3H]hydride labeling of periodate-oxidized termini and high performance liquid chromatography of cap oligonucleotides have been used to compare terminal sequences in poly(A)-rich RNA from dry and germinating embryos. As is the case in germinating embryos, poly(A)-rich RNA from dry embryos contains only "type 0" cap sequences, i.e., m7G(5')ppp(5')N, in which m7G is the 7-methylguanosine cap and N is any of the classical ribonucleosides: adenosine (A), guanosine (G), cytidine (C),a nd uridine (U). Striking differences between the cell-free translational capacities of bulk messenger RNA (mRNA) populations from dry and germinating embryos are not reflected in signal differences in their proportions of "type 0" cap structures: in general, there is approximately 40% m7G(5')ppp(5')A, with roughly equivalent amounts of m7G(5')ppp(5')G and m7G(5')ppp(5')C accounting for most of the remaining sequences. The findings with mRNA from dry plant embryos serve to emphasize interesting differences between patterns of methylation in the capped and uncapped RNA molecules in higher plants and animals; the differences have not been previously noted in the literature and are the subject of brief comment in this paper.     

Hydrolysis of some mRNA 5'-cap analogs catalyzed by the human Fhit protein--and lupin ApppA hydrolases.

Hydrolysis of the following four cap analogs: m7G(5')ppp(5')A, m7G(5')ppp(5')m6A, m7G(5')ppp(5')m2'OG and m7G(5')ppp(5')2'dG catalyzed by homogeneous human Fhit protein and yellow lupin Ap3A hydrolase has been investigated. The hydrolysis products were identified by HPLC analysis and the K(m) and Vmax values calculated based on the data obtained by the fluorimetric method.     

Multiple methylated cap sequences in adenovirus type 2 early mRNA.

The methylated constituents of early adenovirus 2 mRNA were studied. RNA was isolated from polyribosomes of cells double labeled with [methyl-3H]methionine and 32PO4 from 2 to 7 g postinfection in the presence of cycloheximide. Cycloheximide ensures that methylation and processing are performed by preexisting host cell enzymes. RNA was fractionated into polyadenylic [poly(A)]+ and poly(A)- molecules using poly(U)-Sepharose, and undergraded virus-specific RNA was isolated by hybridization to viral DNA in 50% formamide at 37 degrees C. Viral mRNA was digested with RNase T2 and chromatographed on DEAE-Sephadex in 7 M urea. Two 3H-labeled RNase T2-resistant oligonucleotide fractions with charges between -5 and -6 were obtained, consistent with two classes of 5' terminal methyl "cap" structures, m7G(5')ppp(5')NmpNp (cap 1) and m7G(5')ppp(5')NmNmpNp (cap 2) (Nm is a ribose 2'-O-methylation). The putative cap 1 contains all the methylated constituents of cap 1 plus Cm. The molar ratios of m7G to 2'-O-methylnucleosides is about 1.0 for cap 1 and 0.5 for cap 2, consistent with the proposed cap structures. Most significant, compositional analysis indicates four different cap 1 structures and at least three different cap 2 structures. Thus there is a minimum of seven early viral mRNA species with different cap structures, unless each type of mRNA can have more than one 5' terminus. In addition to methylated caps, early mRNA contains internal base methylations, exclusively as m6A, as shown by analyses of the mononucleotide (-2 charge) fraction. m6A was present in the ratio of 1 mol of m6Ap per 450 nucleotides. Thus viral mRNA molecules contain two to three internal m6A residues per methyl cap, since there is on the average 1 cap per 1,250 nucleotides.     

Characterization of 5'-terminal methylation of human alpha- and beta-globin mRNAs

After incubating peripheral blood of various anemic individuals with [methyl-3H]methionine, 5'-terminal cap structures and the sequential methylation of human globin mRNAs were determined. Human alpha- and beta-globin mRNAs have the same methylated nucleotides at their 5' ends as the alpha- and beta-globin mRNAs of mouse and rabbit: m7G(5')ppp(5')[m6Am/Am]pCmp. The order of methyl group addition to human globin mRNA is also similar to that of mouse globin mRNA in that methylation of the 5'-terminal guanosine and the ribose of adenosine occurs earlier than that of the base of adenosine and the ribose of cytidine. Thus, both the cap structures and the order of methylation are conserved in the globin mRNAs of these species, suggesting that variation in the structure of the 5' terminus of globin mRNAs may alter the function of these molecules.     

Relative importance of 7-methylguanosine in ribosome binding and translation of vesicular stomatitis virus mRNA in wheat germ and reticulocyte cell-free systems.

Vesicular stomatitis virus mRNAs with these four types of 5'-termini, (a) m7G5'ppp5'(m)Am, (b) ppp5'(m)Am, (c) m7G5'-ppp5' Am, and (d) G5'ppp5'A, were prepared and their translation and ribosome binding analyzed in wheat germ and reticulocyte cell-free protein synthesis systems. The relative efficiencies of translation of individual vesicular stomatitis virus (VSV) mRNAs having type 2 termini ranged from 23 to 29% of the control (type 1) RNA in the reticulocyte system and 6 to 7% of control RNA in the wheat germ system. A similar difference between the two systems was seen in ribosome-binding experiments in which type 2 RNA formed an 80 S initiation complex with high efficiency (70% of control type 1 RNA) in the reticulocyte system, but with low efficiency (17% of control RNA) in the wheat germ system. Similar differences in the importance of m7G in translation in the two systems were seen when VSV mRNAs synthesized in vitro with type 3 and type 4 termini were analyzed. However, the analysis of type 4 RNA (which was synthesized in vitro in the presence of S-adenosylhomocysteine) was complicated by the presence of abnormally large poly(A) at its 3'-end. Another series of experiments showed that compounds such as 5'pm7G and m7G5'ppp5'Np are potent and specific inhibitors of translation of all types of VSV mRNAs in the wheat germ system (greater than 98% inhibition) but cause less than 20% inhibition of translation in the reticulocyte system. Taken together, all of the results indicate that a 5'-terminal m7G is far more important in translation of VSV mRNAs in the heterologous plant cell-free system than in the reticulocyte lysate system.     

The effect of the messenger RNA concentration on the competitive inhibition of translation by cap-analogues

Inhibition of translation of several mRNA species in a micrococcal nuclease treated reticulocyte lysate by cap analogues was compared with the competition between two mRNAs. Inhibition characteristics were very similar, only complete mRNA molecules inhibited at concentrations 150 times lower than m⁷ G5ppp5G. The inhibition of mRNA translation by cap analogues could be neutralized by the addition of extra mRNA in a manner predicted from the competitive nature of the inhibition by cap analogues.     

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

The 7-methylguanosine (m7G) residue present in the m7G5' ppp5'X-"CAP" structure of rabbit globin mRNA was removed quantitatively by periodate oxidation followed by beta-elimination in the presence of cyclohexylamine. The RNA thus treated was intact and exhibited no signs of degradation as examined by polyacrylamide gel electrophoresis in formamide. Assay for protein synthesis using a wheat germ cell-free system showed that the globin mRNA lacking m7G had lost most of its messenger activity. Identical treatment, of satellite tobacco necrosis virus (STNV) RNA, which does not contain the 5'-terminal "CAP" structure, resulted in no loss of its mRNA activity. Since the importance of the m7G residue in eukaryotic mRNA has not yet been shown essential for translation in vivo, both untreated and treated globin mRNAs were injected into frog oocytes and their translation into globin was measured at intervals over a ninety-six hour period. Globin mRNA either treated with periodate alone or lacking in m7g altogether were both found to have lost more than 90% of their activity in vivo.     

Impairment of reovirus mRNA 'cap' methylation in interferon-treated mouse L929 cells.

Reovirus mRNAs synthesized in vitro by the virionassociated enzyme have a 5' 'cap 1' structure (m7G(5')ppp(5')GmpCp...). However, about one third to one half of the reovirus mRNAs formed in mouse L929 cells have a 5' 'cap 2' structure (m7G(5')ppp(5')GmpCmp...) and the rest have a 5' 'cap 1' structure. The finding that virus mRNA 'cap' methylation is impaired in extracts of interferon-treated cells prompted us to study the effect of interferon on virus mRNA 'cap' methylation in vivo. Using labeling with [3H]-guanosine and dual labeling with [3H]methionine and [14C]uridine we compared the 5' structures of reovirus mRNAs accumulating between 5 and 11 h after infection in: L929 cells treated with 390 to 2600 U/ml of a partially purified mouse interferon preparation and untreated L929 cells. The treatment resulted in a 70 to 98% decrease in the 24 h virus yield and in a 50 to 55% decrease in the label accumulated in virus mRNAs. The 'capping' of virus mRNAs and the methylation of their 5' terminal and adjacent G residues were not diminished in interferon-treated cells. However, the percent of 'cap 2' termini was 36 to 47% lower in virus mRNAs from interferon-treated cells than in virus mRNAs from control cells. The interferon treatment did not result in the appearance of additional methylated nucleotides in the virus mRNAs.     

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.     

Translation of capped and uncapped vesicular stomatitis virus and reovirus mRNA'S. Sensitivity to m7GpppAm and ionic conditions.

In an attempt to elucidate the role of the 5'-terminal 7-methylguanosine residue in translation of mammalian mRNAs, vesicular stomatitis virus (VS virus), and reovirus mRNAs containing and lacking this residue, and also Qbeta RNA, were translated in cell-free extracts from reticulocytes and wheat germ under a variety of ionic conditions. Optimal translation of mRNAs lacking a 5' m7G occurred at concentrations of KOAc or KCl which were lower than those optimal for normal "capped" mRNAs. However, this salt dependence was much less marked in the mammalian reticulocyte extract and, at salt concentrations optimal for translation of normal capped mRNAs, reticulocyte lysates translated uncapped with mRNAs at 30 to 60% the normal efficiency. At low K+ concentrations, wheat germ ribosomes bound and translated appreciable amounts of uncapped VS virus mRNA; controls showed that no m7G residue is added to the 5' end of the bound RNA. Analogues of the 5' end, such as m7GpppAm, inhibited translation of both normal and uncapped VS virus RNAs in wheat germ extracts to about the same extent, but the efficiency of its action was reduced at low K+ concentrations. We conclude that there is a reduced importance of the 5' m7G residue in ribosome-mRNA recognition at low K+ concentrations, and that translation of mRNAs in reticulocyte extract is, under any reaction conditions, less dependent on the presence of a 5' "cap" than in wheat germ extracts.