Center for Medical Bioinformatics, Escola Paulista de Medicina, Federal University of São Paulo (UNIFESP), São Paulo 04039032, Brazil.
Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Federal University of São Paulo (UNIFESP), São Paulo 04023062, Brazil.
Viruses. 2021 Oct 20;13(11):2108. doi: 10.3390/v13112108.
The causative agent of COVID-19 pandemic, SARS-CoV-2, has a 29,903 bases positive-sense single-stranded RNA genome. RNAs exhibit about 150 modified bases that are essential for proper function. Among internal modified bases, the -methyladenosine, or m6A, is the most frequent, and is implicated in SARS-CoV-2 immune response evasion. Although the SARS-CoV-2 genome is RNA, almost all genomes sequenced thus far are, in fact, reverse transcribed complementary DNAs. This process reduces the true complexity of these viral genomes because the incorporation of dNTPs hides RNA base modifications. Here, we present an initial exploration of Nanopore direct RNA sequencing to assess the m6A residues in the SARS-CoV-2 sequences of ORF3a, E, M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10 and the 3'-untranslated region. We identified fifteen m6A methylated positions, of which, six are in ORF N. Additionally, because m6A is associated with the DRACH motif, we compared its distribution in major SARS-CoV-2 variants. Although DRACH is highly conserved among variants, we show that variants Beta and Eta have a fourth position C > U change in DRACH at 28,884b that could affect methylation. This is the first report of direct RNA sequencing of a Brazilian SARS-CoV-2 sample coupled with the identification of modified bases.
引发 COVID-19 大流行的病原体 SARS-CoV-2 拥有一条 29903 个碱基的正链单链 RNA 基因组。RNA 中大约有 150 种修饰碱基，这些碱基对于其正常功能至关重要。在内部修饰碱基中，最常见的是 -甲基腺苷（m6A），它与 SARS-CoV-2 的免疫逃逸反应有关。尽管 SARS-CoV-2 的基因组是 RNA，但迄今为止测序的几乎所有基因组实际上都是逆转录互补 DNA（cDNA）。这一过程降低了这些病毒基因组的真实复杂度，因为 dNTP 的掺入掩盖了 RNA 碱基修饰。在这里，我们首次探索使用 Nanopore 直接 RNA 测序来评估 ORF3a、E、M、ORF6、ORF7a、ORF7b、ORF8、N、ORF10 和 3’非翻译区中 SARS-CoV-2 序列的 m6A 残基。我们鉴定出了 15 个 m6A 甲基化位置，其中 6 个位于 ORF N 中。此外，因为 m6A 与 DRACH 基序相关，我们比较了其在主要 SARS-CoV-2 变体中的分布。尽管 DRACH 在变体中高度保守，但我们表明变体 Beta 和 Eta 在 28884b 处的 DRACH 第四位 C > U 发生了变化，这可能会影响甲基化。这是首次对巴西 SARS-CoV-2 样本进行直接 RNA 测序并鉴定修饰碱基的报告。
