"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.     

Antigody-nucleic acid complexes. Inhibition of translation of silkmoth chorion messenger ribonucleic acid with antibodies specific for 7-methylguanosine.

Antibodies specific for 7-methylguanosine (m7G) were evaluated for their ability to inhibit the translation of chorion mRNA in a wheat germ, cell-free amino acid incorporating system. Results obtained with antibody concentrations of 0.5--1.5 microM revealed dose-dependent inhibition of [3H]-labeled amino acid incorporation into acid-insoluble radioactivity. Inhibition of translation was attributed to the interaction of anti-m7G antibodies with the 5' termini of chorion mRNAs on the basis that (a) anti-m7G antibodies coupled to Sepharose (anti-m7G-Sepharose) immunospecifically retained 5'-terminal cap structures of chorion mRNAs, i.e., m7G (5')ppp(5')Nm, (b) significant inhibition of translation required a 2-h preincubation of anti-m7G antibodies with mRNA, and (c) similar preincubation periods with anti-m7G antibodies in the presence of the competing nucleoside hapten (m7G) obviated the inhibitory effect of the antibody. The nature of the anti-m7G antibody-mRNA complex was examined by digesting chorion mRNA with nuclease P1 before (predigested) and after (postdigested) immunospecific adsorption to anti-m7G-Sepharose adsorbent. Whereas predigested preparations yielded a single cap structure of the type m7G(5')ppp(5')N, the predominating cap in the postdigested sample was m7G(5')ppp(5')NpNpN. These latter data revealed that the nucleotide sequence adjacent to the cap was not significantly masked by the antibody and suggest the utility of anti-m7G antibody as a site-specific probe.     

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.     

Methyl group analysis of virion-associated high-molecular-weight RNA synthesized in vitro by purified vaccinia virus.

The methylation pattern of virion-associated high-molecular-weight RNA synthesized in vitro by purified vaccinia virus has been determined. Analysis of purified high-molecular-weight RNA synthesized with S-[methyl-3H]-adenosylmethionine and alpha[32P]UTP as precursors gave the following results. (i) Eessentially all molecules contained blocked and methylated structures of the type m7G(5')ppp(5')Gm and m7G(5')ppp(5')Am. (ii) There was no detectable methylation at internal sites. (iii) Under several different conditions of synthesis, the ratio of molecules containing m7G(5')ppp(5')Gm to those containing m7G(5')ppp(5')Am was imilar for both the virion-associated high-molecular-weight RNA and the virion-released 8-12S mRNA.     

A 54-kDa fragment of the Poly(A)-specific ribonuclease is an oligomeric, processive, and cap-interacting Poly(A)-specific 3' exonuclease

We have previously identified a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mRNAs. Here we report on the purification and identification of a calf thymus 54-kDa polypeptide associated with a similar 3' exonuclease activity. The 54-kDa polypeptide was shown to be a fragment of the poly(A)-specific ribonuclease 74-kDa polypeptide. The native molecular mass of the nuclease activity was estimated to be 180-220 kDa. Protein/protein cross-linking revealed an oligomeric structure, most likely consisting of three subunits. The purified nuclease activity released 5'-AMP as the reaction product and degraded poly(A) in a highly processive fashion. The activity required monovalent cations and was dependent on divalent metal ions. The RNA substrate requirement was investigated, and it was found that the nuclease was highly poly(A)-specific and that only 3' end-located poly(A) was degraded by the activity. RNA substrates capped with m(7)G(5')ppp(5')G were more efficiently degraded than noncapped RNA substrates. Addition of free m(7)G(5')ppp(5')G cap analogue inhibited poly(A) degradation in vitro, suggesting a functional link between the RNA 5' end cap structure and poly(A) degradation at the 3' end of the RNA.     

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.     

Differential inhibition of mRNA degradation pathways by novel cap analogs

mRNA degradation predominantly proceeds through two alternative routes: the 5'-->3' pathway, which requires deadenylation followed by decapping and 5'-->3' hydrolysis; and the 3'-->5' pathway, which involves deadenylation followed by 3'-->5' hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp(3)G, m(7)Gp(3)G, or m(2)(7,3'-O) Gp(3)G) and 3' termini (A(31),A(60), or G(16)) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5' terminus to bind eIF4E, not merely the presence of a blocking group at the 5'-end. Introducing a stem-loop in the 5'-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. To test the relative contributions of the 5'-->3' versus 3'-->5' pathways, we designed and synthesized two new cap analogs, in which a methylene group was substituted between the alpha- and beta-phosphate moieties, m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG, that are predicted to be resistant to cleavage by Dcp1/Dcp2 and DcpS, respectively. These cap analogs were recognized by eIF4E and conferred cap-dependent translation to mRNA both in vitro and in vivo. Oligonucleotides capped with m(2)(7,3'-O)Gpp(CH2)pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m(2)(7,3'-O)Gpp(CH2)pG, but not m(2)(7,3'-O)Gp(CH2)ppG, were more stable in vivo, indicating that the 5'-->3' pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5'-terminal m(2)(7,3'-O)Gpp(CH2)pG and 3'-terminal poly(G) had the greatest stability of all mRNAs tested.     

Sequence of methylated nucleotides at the 5''-terminus of adenovirus-specific RNA.

RNA labeled with [methyl-3H] methionine and [14C]uridine was isolated from the cytoplasm of adenovirus-infected cells and purified by poly(U)-Sepharose chromatography and hybridization to filters containing immobilized adeovirus DNA. Analysis by dimethyl sulfoxide-sucrose gradient sedimentation suggested that the major mRNA species were methylated. 7-Methylguanosine was identified at the 5'-terminus of the advenovirus-specific RNA and could be removed by periodate oxidation and beta-elimination. Structures of the type m7G(5')ppp(5')Nm containing the unusual nucleoside N6, O2'-dimethyladenosine, and smaller amounts of 2'-O-methyladenosine were isolated by DEAE-cellulose chromatography after P1 nuclease digestion of the RNA. Evidence for some 5'-terminal sequences, m7G(5')ppp(5')m6AmpNm, with additional 2'-O-methylribonucleosides was also obtained. A base-methylated nucleoside, N6-methyladenosine, is located within the RNA chain and is released as a mononucleotide by alkali hydrolysis.     

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.