Characterization of distinct 5'-terminal cap structures of adenovirus type 2 early messenger ribonucleic acid and KB cell messenger ribonucleic acid

Blocked and Methylated 5′‐Terminal Cap Structures of Rat Brain Messenger Ribonucleic Acids

“…Some of these nicks could generate internal 5'-OH terminated mRNA fragments that would be accessible to labeling by the action of polynucleotide kinase. Calculation based on the relative proportions of radioactivity in the peaks from the DEAE-Sephadex column indicated that approximately 15-20% of 3zP incorporation occurred into bonafide mRNA termini under our experimental conditions, a situation comparable to the results obtained by other workers using this technique (Lockard and RajBhandary, 1976;Hashimoto et al, 1980).…”

“…This fact as well as the large amount of radioactivity found in this peak and the occurrence of all four normal nucleotides in the nuclease PI digest would indicate that this was due to incorporation of "'P into internal sites of mRNA. Formation of "nicks" in mRNA during the procedures used for 5 'terminal "P-labeling has been noted by a number of other workers using mRNA from different sources (Miura et al, 1974;Ziff and Evans, 1978;Lockard et al, 1979;Hashimoto et al, 1980). Some of these nicks could generate internal 5'-OH terminated mRNA fragments that would be accessible to labeling by the action of polynucleotide kinase.…”

“…The occurrence of purine in the 5 'penultimate position could imply capping of the RNA at the site of initiation of the primary transcript by a mechanism similar to that shown to be used by such eukaryote systems as reovirus or vaccinia virus (Furuichi et a1.,1976;Moss et al, 1976). On the other hand, the presence of a pyrimidine in this position may suggest either that certain eukaryotic mRNAs may initiate transcription in a pyrimidine residue (Hashimoto et al, 1980) or that the mRNA was derived from a larger transcript that was cleaved and then subsequently capped at an internal site of the transcript. An extremely interesting elaboration of the latter possibility was discovered recently in the maturation of adenovirus late mRNAs in which the 5'-terminal capped leader sequence was shown to be constructed by "splicing" together RNA sequences originally transcribed from distant noncontiguous regions of the adenovirus chromosome (Berget et al, 1977;Klessig, 1977).…”

“…The 5 'termini of more than 10 purified eukaryotic mRNA species have to date been characterized and all have been reported to have purines in the penultimate nucleosides of the cap structure (Revel and Groner, 1978), with the exception of one Ad2 early mRNA that was recently shown to contain U(m) in this position (Hashimoto et al, 1980). In cases where more than one cap structure has been detected, the most abundant have been those containing the highest number of methyl groups Hashimoto et al, 1980), indicating that the undermethylated caps were probably precursors of the fully methylated caps. The presence of adenosine in the 5'-penultimate position is reported to confer a greater affinity of binding of mRNA to ribosomes, as compared with guanosine (Muthukrishnan et al, 1978).…”

“…Our results do not permit us to estimate the proportion of mRNA in rat brain that is capped or the number of different types of cap structures that may exist in these mRNAs. Assuming that the less methylated nucleotides in the cap represent precursors of more methylated nucleosides (Lockard and RajBhandary, 1976;Hashimoto, 1980), there appear to be four cap 1 structures. If the diversity of methylated bases in the third position from the 5'terminal is similar to that in the second position, at least 16 cap 2 structures could theoretically be present.…”