Desrosiers R C, Friderici K H, Rottman F M
Biochemistry. 1975 Oct 7;14(20):4367-74. doi: 10.1021/bi00691a004.
KOH digestion of methyl-labeled poly(A)+ mRNA purified by (dT)-cellulose chromatography produced mononucleotide and multiple peaks of a large oligonucleotide (-6 to -8 charge) when separated on the basis of charge by Pellionex-WAX high-speed liquid chromatography in 7 M urea. Heat denaturation of the RNA before application to (dT)-cellulose was required to release contaminants (mostly 18S rRNA) that persisted even after repeated binding to (dT)-cellulose at room temperature. Analysis of the purified poly(A)+ mRNA by enzyme digestion, acid hydrolysis, and a variety of chromatographic techniques has shown that the monucleotide (53%) is due entirely to N6-methyladenosine. The large oligonucleotides (47%) were found to contain 7-methylguanosine and the 2'-0-methyl derivatives of all four nucleosides. No radioactivity was found associated with the poly(A) segment. Periodate oxidation of the mRNA followed by beta elimination released only labeled 7-methylguanine consistent with a blocked 5' terminus containing an unusual 5'-5' bond. Alkaline phosphatase treatment of intact mRNA had no effect on the migration of the KOH produced oligonucleotides on Pellionex-WAX. When RNA from which 7-methylguanine was removed by beta elimination was used for the phosphatase treatment, distinct dinucleotides (NmpNp) and trinucleotides (NmpNmpNp) occurred after KOH hydrolysis and Pellionex-WAX chromatography. Thus Novikoff hepatoma poly(A)+ mRNA molecules can contain either one or two 2'-0-methylnucleotides linked by a 5'-5' bond to a terminal 7-methylguanosine and the 2'-0-methylation can occur with any of the four nucleotides. The 5' terminus may be represented by m7G5'ppp5' (Nmp)lor2Np, a general structure proposed earlier as a possible 5' terminus for all eucaryotic mRNA molecules (Rottman, F., Shatkin, A., and Perry, R. (1974), Cell 3, 197). The composition analyses indicate that there are 3.0 N6-methyladenosine residues, 1.0 7-methylguanosine residue, and 1.7 2'-0-methylnucleoside residues per average mRNA molecule.
对通过（dT）-纤维素柱层析纯化的甲基标记的聚腺苷酸（poly(A)+）mRNA进行氢氧化钾（KOH）消化，然后在7M尿素中通过Pellionex-WAX高速液相色谱根据电荷进行分离时，产生了单核苷酸和一个大的寡核苷酸（-6至-8电荷）的多个峰。在将RNA应用于（dT）-纤维素之前进行热变性，以释放即使在室温下反复与（dT）-纤维素结合后仍残留的污染物（主要是18S核糖体RNA）。通过酶消化、酸水解和各种色谱技术对纯化的聚腺苷酸（poly(A)+）mRNA进行分析表明，单核苷酸（53%）完全是由于N6-甲基腺苷。发现大的寡核苷酸（47%）含有7-甲基鸟苷和所有四种核苷的2'-O-甲基衍生物。未发现与聚腺苷酸（poly(A)）片段相关的放射性。mRNA经高碘酸盐氧化后进行β消除，仅释放出标记的7-甲基鸟嘌呤，这与含有异常5'-5'键的封闭5'末端一致。完整mRNA经碱性磷酸酶处理后，对KOH产生的寡核苷酸在Pellionex-WAX上的迁移没有影响。当使用经β消除去除7-甲基鸟嘌呤的RNA进行磷酸酶处理时，KOH水解和Pellionex-WAX色谱后出现了明显的二核苷酸（NmpNp）和三核苷酸（NmpNmpNp）。因此，诺维科夫肝癌细胞的聚腺苷酸（poly(A)+）mRNA分子可以包含一个或两个通过5'-5'键与末端7-甲基鸟苷相连的2'-O-甲基核苷酸，并且2'-O-甲基化可以发生在四种核苷酸中的任何一种上。5'末端可能由m7G5'ppp5'（Nmp）lor2Np表示，这是先前提出的所有真核mRNA分子可能的5'末端的一般结构（罗特曼，F.，沙特金，A.，和佩里，R.（1974年），《细胞》3，197）。组成分析表明，每个平均mRNA分子有3.0个N6-甲基腺苷残基、1.0个7-甲基鸟苷残基和1.7个2'-O-甲基核苷残基。
