Epitranscriptomic N6-Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells.

N6-methyladenosine (m6A) is a dynamic posttranscriptional RNA modification that plays an important role in determining transcript fate. The functional consequence of m6A deposition is dictated by a group of host proteins that specifically recognize and bind the m6A modification, leading to changes in RNA stability, transport, splicing, or translation. The cellular m6A methylome undergoes changes during certain pathogenic conditions such as viral infections. However, how m6A modification of host cell transcripts and noncoding RNAs change during severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection has not been reported. Here, we define the epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells compared to uninfected controls. We identified mRNA and long and small noncoding RNA species that are differentially m6A modified in response to SARS-CoV-2 infection. The most significantly differentially methylated transcript was the precursor of microRNA-4486 (miRNA-4486), which showed significant increases in abundance and percentage of methylated transcripts in infected cells. Pathway analyses revealed that differentially methylated transcripts were significantly associated with several cancer-related pathways, protein processing in the endoplasmic reticulum, cell death, and proliferation. Upstream regulators predicted to be associated with the proteins encoded by differentially methylated mRNAs include several proteins involved in the type-I interferon response, inflammation, and cytokine signaling. IMPORTANCE Posttranscriptional modification of viral and cellular RNA by N6-methyladenosine (m6A) plays an important role in regulating the replication of many viruses and the cellular immune response to infection. We therefore sought to define the epitranscriptomic m6A profile of human lung epithelial cells infected with SARS-CoV-2. Our analyses demonstrate the differential methylation of both coding and noncoding cellular RNAs in SARS-CoV-2-infected cells compared to uninfected controls. Pathway analyses revealed that several of these RNAs may be involved in the cellular response to infection, such as type-I interferon. Our study implicates m6A modification of infected-cell RNA as a mechanism of posttranscriptional gene regulation during SARS-CoV-2 infection.

Effects of Epitranscriptomic RNA Modifications on the Catalytic Activity of the SARS-CoV-2 Replication Complex.

SARS-CoV-2 causes individualized symptoms. Many reasons have been given. We propose that an individual's epitranscriptomic system could be responsible as well. The viral RNA genome can be subject to epitranscriptomic modifications, which can be different for different individuals, and thus epitranscriptomics can affect many events including RNA replication differently. In this context, we studied the effects of modifications including pseudouridine (Ψ), 5-methylcytosine (m5 C), N6-methyladenosine (m6 A), N1-methyladenosine (m1 A) and N3-methylcytosine (m3 C) on the activity of SARS-CoV-2 replication complex (SC2RC). We found that Ψ, m5 C, m6 A and m3 C had little effect, whereas m1 A inhibited the enzyme. Both m1 A and m3 C disrupt canonical base pairing, but they had different effects. The fact that m1 A inhibits SC2RC implies that the modification can be difficult to detect. This fact also implies that individuals with upregulated m1 A including cancer, obesity and diabetes patients might have milder symptoms. However, this contradicts clinical observations. Relevant discussions are provided.

Lessons Learned and Yet-to-Be Learned on the Importance of RNA Structure in SARS-CoV-2 Replication.

SARS-CoV-2, the etiological agent responsible for the COVID-19 pandemic, is a member of the virus family Coronaviridae, known for relatively extensive (~30-kb) RNA genomes that not only encode for numerous proteins but are also capable of forming elaborate structures. As highlighted in this review, these structures perform critical functions in various steps of the viral life cycle, ultimately impacting pathogenesis and transmissibility. We examine these elements in the context of coronavirus evolutionary history and future directions for curbing the spread of SARS-CoV-2 and other potential human coronaviruses. While we focus on structures supported by a variety of biochemical, biophysical, and/or computational methods, we also touch here on recent evidence for novel structures in both protein-coding and noncoding regions of the genome, including an assessment of the potential role for RNA structure in the controversial finding of SARS-CoV-2 integration in "long COVID" patients. This review aims to serve as a consolidation of previous works on coronavirus and more recent investigation of SARS-CoV-2, emphasizing the need for improved understanding of the role of RNA structure in the evolution and adaptation of these human viruses.

Detection techniques for epitranscriptomic marks.

Similar to epigenetic DNA modification, RNA can be methylated and altered for stability and processing. RNA modifications, namely, epitranscriptomes, involve the following three functions: writing, erasing, and reading of marks. Methods for measurement and position detection are useful for the assessment of cellular function and human disease biomarkers. After pyrimidine 5-methylcytosine was reported for the first time a hundred years ago, numerous techniques have been developed for studying nucleotide modifications, including RNAs. Recent studies have focused on high-throughput and direct measurements for investigating the precise function of epitranscriptomes, including the characterization of severe acute respiratory syndrome coronavirus 2. The current study presents an overview of the development of detection techniques for epitranscriptomic marks and briefs about the recent progress in this field.

Direct RNA Sequencing Reveals SARS-CoV-2 m6A Sites and Possible Differential DRACH Motif Methylation among Variants.

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 N6-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.