Abstract

The N⁴-methylation of cytidine (m⁴C and m⁴₂C) in RNA plays important roles in both bacterial and eukaryotic cells. In this work, we synthesized a series of m⁴C and m⁴₂C modified RNA oligonucleotides, conducted their base pairing and bioactivity studies, and solved three new crystal structures of the RNA duplexes containing these two modifications. Our thermostability and X-ray crystallography studies, together with the molecular dynamic simulation studies, demonstrated that m⁴C retains a regular C:G base pairing pattern in RNA duplex and has a relatively small effect on its base pairing stability and specificity. By contrast, the m⁴₂C modification disrupts the C:G pair and significantly decreases the duplex stability through a conformational shift of native Watson-Crick pair to a wobble-like pattern with the formation of two hydrogen bonds. This double-methylated m⁴₂C also results in the loss of base pairing discrimination between C:G and other mismatched pairs like C:A, C:T and C:C. The biochemical investigation of these two modified residues in the reverse transcription model shows that both mono- or di-methylated cytosine bases could specify the C:T pair and induce the G to T mutation using HIV-1 RT. In the presence of other reverse transcriptases with higher fidelity like AMV-RT, the methylation could either retain the normal nucleotide incorporation or completely inhibit the DNA synthesis. These results indicate the methylation at N⁴-position of cytidine is a molecular mechanism to fine tune base pairing specificity and affect the coding efficiency and fidelity during gene replication.

中文翻译：

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对N4-甲基胞苷（m4C）和N4，N4-二甲基胞苷（m42C）修饰的RNA进行碱基配对，结构和功能分析。

摘要

RNA中胞苷的N ^4-甲基化（m ⁴ C和m ⁴ ₂ C）在细菌和真核细胞中均起重要作用。在这项工作中，我们合成了一系列m ⁴ C和m ⁴ ₂ C修饰的RNA寡核苷酸，进行了碱基配对和生物活性研究，并解析了包含这两个修饰的RNA双链体的三个新晶体结构。我们的热稳定性和X射线晶体学研究以及分子动力学模拟研究表明，m ⁴ C在RNA双链体中保留了规则的C：G碱基配对模式，并且对其碱基配对的稳定性和特异性影响相对较小。相比之下，⁴ ₂ C修饰会破坏C：G对，并通过将天然Watson-Crick对构象转变为带有两个氢键的摆动状模式而显着降低双链体稳定性。该双甲基化的m ⁴ ₂C还导致C：G与其他错配对（例如C：A，C：T和C：C）之间碱基配对的区分性丧失。在逆转录模型中对这两个修饰残基的生化研究表明，单甲基化或二甲基化的胞嘧啶碱基均可指定C：T对，并使用HIV-1 RT诱导G至T突变。在其他具有更高保真度的逆转录酶（如AMV-RT）存在下，甲基化可能保留正常的核苷酸掺入或完全抑制DNA合成。这些结果表明胞苷在N ⁴位的甲基化是微调碱基配对特异性并影响基因复制过程中编码效率和保真度的分子机制。