7-Methylguanosine 5' cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different 5'-terminal nucleotides and additional methylations (2'-O-methylation and m6A). Currently available 5'-capping methods do not address this diversity. We report trinucleotide 5' cap analogs (m7GpppN(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m6A, G, C, U) and its methylation status (±2'-O-methylation). HPLC-purified mRNAs carrying these 5' caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5'-terminal A/Am and m6Am, whereas the lowest was observed for G and Gm. The mRNAs carrying 2'-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.

中文翻译：

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真核mRNA 5'帽中第一个转录的核苷酸的身份和甲基化状态调节活细胞中的蛋白质表达。

mRNA上的7-甲基鸟苷5'帽对于在体内和体外有效表达蛋白质是必需的。最近的研究揭示了内源性mRNA帽的结构多样性，其带有不同的5'-末端核苷酸和其他甲基化（2'-O-甲基化和m6A）。当前可用的5'封端方法不能解决这种多样性。我们报告了三核苷酸5'帽类似物（m7GpppN（m）pG），其被RNA聚合酶T7用来从携带Φ6.5启动子的模板开始转录，并能够产生与第一个转录核苷酸不同的身份的mRNA（N = A ，m6A，G，C，U）及其甲基化状态（±2'-O-甲基化）。带有这些5'帽的HPLC纯化的mRNA用于研究三种哺乳动物细胞系（3T3-L1，HeLa和JAWS II）中的蛋白质表达。对于携带5'-末端A / Am和m6Am的mRNA，观察到最高的表达，而对于G和Gm，则观察到最低的表达。仅在JAWS II树突状细胞中，在第一个转录的核苷酸（第1章）上带有2'-O-甲基的mRNAs的表达水平明显高于未甲基化的对应物（第0章）。进一步的实验表明，mRNA的表达特征与翻译起始因子4E的亲和力或体外脱盖的敏感性不相关，而是取决于mRNA的纯度和细胞的免疫状态。