Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells |
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Authors: | U Schibler D E Kelley R P Perry |
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Affiliation: | The Institute for Cancer Research, Fox Chase Cancer Center Philadelphia, Penn. 19111, U.S.A. |
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Abstract: | A variety of methylated oligonucleotides were derived from mouse L cell messenger RNA and heterogeneous nuclear RNA by digestion with specific ribonucleases, and the cap-containing oligonucleotides separated from those containing internal m6A by chromatography on diborylaminoethyl-cellulose. Cap-containing sequences of the type m7GpppXmpG, m7GpppXmpY(m)pG, m7GpppXmpY(m) pNpG and m7GpppXmpY(m)p(Np)> 1G have distinctive non-random compositions of the 2′-O-methylated constituent Xm; yet sequences of a particular type and composition occur with a remarkably similar frequency in mRNA and hnRNA2. For example, approximately 20% of the cap sequences in both hnRNA and mRNA are m7Gppp(m6)AmG, whereas less than 1% are m7GpppUmpG. The high degree of similarity in cap sequences is consistent with the previously postulated precursor-product relationship between hnRNA caps and mRNA caps.The composition of the Y position in capped hnRNA molecules was determined to be (29% G, 20% A, 51% Py), which differs considerably from the composition of Ym in the cap II forms of mRNA (8% Gm, 11% Am, 81% Py). Given the precursor-product relationship between hnRNA caps and mRNA caps, this result provides strong evidence that only a restricted subclass of mRNA molecules receive the secondary methylation at position Y.In both hnRNA and mRNA the internal m6A occurs in well-defined sequences of the type: , the 5′ nearest-neighbor of m6A being G in about three-quarters of the molecules and A in about one-quarter of the molecules. The nucleotide N1 is a purine about 90% of the time and the nucleotide N2 is rarely a G. These same sequences are present in large (> 50 S), as well as small (14 S to 50 S) hnRNA. These results raise the possibility that the internal m6A, like caps, may be conserved during the processing of large hnRNA into mRNA. Two models based on this idea are discussed. |
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