Molecular Genetics Division, Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, NSW, 2010, Australia.
Nucleic Acids Res. 2012 Jun;40(11):5023-33. doi: 10.1093/nar/gks144. Epub 2012 Feb 16.
The modified base 5-methylcytosine (m(5)C) is well studied in DNA, but investigations of its prevalence in cellular RNA have been largely confined to tRNA and rRNA. In animals, the two m(5)C methyltransferases NSUN2 and TRDMT1 are known to modify specific tRNAs and have roles in the control of cell growth and differentiation. To map modified cytosine sites across a human transcriptome, we coupled bisulfite conversion of cellular RNA with next-generation sequencing. We confirmed 21 of the 28 previously known m(5)C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10,275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions. We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs. Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function.
经修饰的碱基 5-甲基胞嘧啶（m(5)C）在 DNA 中研究得比较透彻，但对其在细胞 RNA 中的普遍性的研究在很大程度上仅限于 tRNA 和 rRNA。在动物中，已知两种 m(5)C 甲基转移酶 NSUN2 和 TRDMT1 修饰特定的 tRNA，并在控制细胞生长和分化中发挥作用。为了在人类转录组中绘制修饰的胞嘧啶位点图，我们将细胞 RNA 的亚硫酸氢盐转化与下一代测序相结合。我们证实了人类 tRNA 中 28 个已知 m(5)C 位点中的 21 个，并鉴定了 234 个新的 tRNA 候选位点，这些位点主要位于预期的结构位置。令人惊讶的是，我们在 mRNAs 和其他非编码 RNA 中发现了 10275 个位点。我们观察到，修饰的胞嘧啶在不同 RNA 类型之间的分布并非随机的；在 mRNAs 中，它们在非翻译区和 Argonaute 结合区附近富集。我们还鉴定了五个由 NSUN2 修饰的新位点，将其已知的底物范围扩展到另一个 tRNA、RNase P 的 RPPH1 亚基和两个 mRNAs。我们的数据表明，修饰的胞嘧啶在转录组中的编码和非编码序列中广泛存在，这表明这种修饰在细胞 RNA 功能的转录后调控中可能具有更广泛的作用。
