Journal: bioRxiv

Article Title: The RNA demethylase FTO controls m 6 A marking on SARS-CoV-2 and classifies COVID-19 severity in patients

doi: 10.1101/2022.06.27.497749

Figure Lengend Snippet: a Scheme of m 6 A-seq in SARS-CoV-2-infected cells. Total RNA was extracted from SARS-CoV-2-infected Vero76 cells 24 h post-inoculation. Reads from Input and m 6 A RIP were aligned with STAR on both the Chlorocebus sabaeus and SARS-CoV-2 genomes. Peaks were then identified with m6aViewer. m 6 A-seq was performed in two independent experiments with technical duplicates. b m 6 A landscape in SARS-CoV-2 RNA. Integrative Genomics Viewer (IGV) tracks display reads from input (gray) and m 6 A IP (green) libraries along the SARS-CoV-2 RNA genome from two biological replicates. Three m 6 A peaks were identified from replicate 1 and two from replicate 2 (black squares). SARS-CoV-2 genome organization is indicated below. Enlarged views of the IGV tracks show the enrichment in reads corresponding to the SARS-CoV-2 TRS_L and nucleocapsid regions. c Bar plot shows m 6 A peaks in the TRS_L (gray) and nucleocapsid (blue) regions. The peaks detected exhibit a p value lower than 1E-14. d Density plot (left panel) displaying the distribution of known m 6 A motifs (RRACH, UGAC, and MGACK) across the m 6 A peaks of the nucleocapsid region. The stacked bar chart (right panel) shows motif frequencies within m 6 A peaks in the SARS-CoV-2 nucleocapsid region. e Scheme of m 6 A RIP-qPCR in infected hamsters. Ten hamsters were infected with SARS-CoV-2. Four days post-inoculation, the hamsters were sacrificed, their lungs collected, and total RNA extracted. RIP-qPCR was performed in three independent experiments to validate m 6 A peaks on SARS-CoV-2 RNA in vivo . f In vivo m 6 A peaks on SARS-CoV-2 viral RNA as determined by m 6 A RIP-qPCR. Lysates containing equal amounts of RNA extracted from infected hamster lungs were immunoprecipitated with either control IgG or m 6 A antibody. Primers amplifying the viral m 6 A-marked regions identified in monkey cells were used to assess immuno-precipitate enrichment in viral sequences. An m 6 A-marked oligo was used as positive control and regions found not to be marked in monkey cells were used as negative controls. Data represent means ± SEM. P -values were determined with a two-tailed unpaired Student’s t-test.

Article Snippet: Additionally, a positive control consisting of a 1089-nucleotide methylated transcript (Renilla luciferase) was in vitro transcribed with the MEGAscript T7 Transcription Kit (Invitrogen, AM1334) according to the manufacturer’s instructions, with addition of m 6 ATP (TriLink, N-1013) instead of ATP nucleotides in the reaction mix.

Techniques: Infection, In Vivo, Immunoprecipitation, Positive Control, Two Tailed Test

Journal: bioRxiv

Article Title: The RNA demethylase FTO controls m 6 A marking on SARS-CoV-2 and classifies COVID-19 severity in patients

doi: 10.1101/2022.06.27.497749

Figure Lengend Snippet: a IGV tracks display an exemplative m 6 A-seq profile of the orthologous gene SLC39A14 as a positive control for enrichment through m 6 A-IP. Input and m 6 A IP from two biological replicates are labeled in gray and green, respectively. Black squares represent m 6 A peaks. Gene architecture is shown beneath. b Venn diagram (upper panel) showing the between-replicate overlap of m 6 A peaks identified by m 6 A-seq in Vero76 cells. Peaks are listed below the diagram. Rows colored in gray highlight m 6 A peaks shared by both biological replicates. c Pie chart illustrating the number and distribution of all m 6 A peaks identified along SARS-CoV-2 RNA. d Density plot (left panel) showing known m 6 A motifs (RRACH, UGAC) across the m 6 A peak in the SARS-CoV-2 TRS_L region. Stacked bar chart (right panel) showing motif frequencies within the m 6 A peak in the SARS-CoV-2 TRS_L region.

Article Snippet: Additionally, a positive control consisting of a 1089-nucleotide methylated transcript (Renilla luciferase) was in vitro transcribed with the MEGAscript T7 Transcription Kit (Invitrogen, AM1334) according to the manufacturer’s instructions, with addition of m 6 ATP (TriLink, N-1013) instead of ATP nucleotides in the reaction mix.

Techniques: Positive Control, Labeling

Journal: bioRxiv

Article Title: The RNA demethylase FTO controls m 6 A marking on SARS-CoV-2 and classifies COVID-19 severity in patients

doi: 10.1101/2022.06.27.497749

Figure Lengend Snippet: a , b Percent identity matrix of nucleocapsid (a) and TRS_L (b) sequences of human coronaviruses (HCoV), created with Geneious Prime, using the Muscle Multiple Sequence Alignment (MSA) tool and default parameters. Three groups are indicated: pandemic β-CoV (SARS-CoV-2, SARS-CoV, and MERS-CoV), β-CoV (HCoV-HKU1, HCoV-OC43), and α-CoV (HCoV-NL63, HCoV-229E). c , d Maximum likelihood phylogenetic tree of nucleocapsid (c) and TRS_L (d) sequences of human coronaviruses, generated using Geneious Prime with the Unweighted Pair Group Method with Arithmatic Mean (UPGMA) model and 1,000 bootstrap replicates. Numbers on the branches indicate bootstrap support values. Branch scale bars are shown as 0.2 substitutions per site. e TRS_L-region sequence similarity among human coronaviruses. This plot is based on the full-length TRS_L sequence, derived through MSA of all human coronaviruses with the following parameter settings: window 60 bp, step 20 bp, GapStrip: on, Kimura model. f MSA describing m 6 A consensus motif variations across human coronavirus TRS_L sequences within the peaks identified in SARS-CoV-2. m 6 A consensus sequences conserved across HCoVs are colored in yellow. g Pie chart showing the number of SARS-CoV-2 sequences available from the NGDC-CNCB as of April 25 th 2022 for six different regions: Europe (blue, 54%), North America (red, 34.7%), Asia (yellow, 7.7%), South America (green, 2.1%), Africa (purple, 0.8%) and Oceania (gray, 0.7%). h Pie chart displaying the number of SARS-CoV-2 sequences from different lineages available from the NGDC-CNCB as of April 25 th 2022. Only variants with a frequency higher than 1% are displayed. Variants with lower frequency are either grouped into the “Other Variants Being Monitored” category (Iota, Beta, Epsilon, Mu, Eta, Kappa, Zeta, and Theta) or the “Other Variants” category. i Bar chart displaying, for a total of 9,557,377 SARS-CoV-2 complete isolates, the mutation frequency in TRS_L regions across SARS-CoV-2 variants as of April 25 th 2022. j Bar chart (red) and density plot (gray) displaying, across SARS-CoV-2 variants, the mutation frequency and distribution of known m 6 A motifs (RRACH and UGAC) across m 6 A peaks identified within the TRS_L.

Article Snippet: Additionally, a positive control consisting of a 1089-nucleotide methylated transcript (Renilla luciferase) was in vitro transcribed with the MEGAscript T7 Transcription Kit (Invitrogen, AM1334) according to the manufacturer’s instructions, with addition of m 6 ATP (TriLink, N-1013) instead of ATP nucleotides in the reaction mix.

Techniques: Sequencing, Generated, Derivative Assay, Mutagenesis

Journal: bioRxiv

Article Title: The RNA demethylase FTO controls m 6 A marking on SARS-CoV-2 and classifies COVID-19 severity in patients

doi: 10.1101/2022.06.27.497749

Figure Lengend Snippet: a Sequence similarity of nucleocapsid-protein-encoding regions of human coronaviruses. The upper diagram shows the genomic organization of the SARS-CoV-2 open reading frames (ORFs) and the domain organization of the nucleocapsid protein gene, based on NCBI sequence NC_045512.2. The SimPlot (lower panel) displays nucleotide similarities between SARS-CoV-2 and other human coronaviruses. It is based on the full-length sequence of the nucleocapsid gene derived from the multiple sequence alignment (MSA) of all human coronaviruses, with the following parameter settings: window 200 bp, step 20 bp, GapStrip: on, Kimura model. b MSA describing m 6 A consensus motif variations within peaks identified in SARS-CoV-2 across human coronavirus nucleocapsid sequences. The m 6 A consensus sequences conserved across HCoVs are colored in yellow. c Bar chart displaying mutation frequencies in nucleocapsid regions across SARS-CoV-2 variants as of April 25, 2022 from a total of 9,557,377 SARS-CoV-2 isolates with complete sequences. d Pie chart showing the distribution of mutations across the nucleocapsid domains of SARS-CoV-2 variants. e Bar chart (red) and density plot (blue) displaying, across SARS-CoV-2 variants, the frequencies and distribution of mutations in known m 6 A motifs (RRACH, UGAC and MGACK) associated with m 6 A peaks identified in the sequence encoding the nucleocapsid RNA binding domain.

Article Snippet: Additionally, a positive control consisting of a 1089-nucleotide methylated transcript (Renilla luciferase) was in vitro transcribed with the MEGAscript T7 Transcription Kit (Invitrogen, AM1334) according to the manufacturer’s instructions, with addition of m 6 ATP (TriLink, N-1013) instead of ATP nucleotides in the reaction mix.

Techniques: Sequencing, Derivative Assay, Mutagenesis, RNA Binding Assay

Journal: bioRxiv

Article Title: The RNA demethylase FTO controls m 6 A marking on SARS-CoV-2 and classifies COVID-19 severity in patients

doi: 10.1101/2022.06.27.497749

Figure Lengend Snippet: a FTO gene expression upon SARS-CoV-2 infection in monkey cells. RT-qPCR assays show reduced levels of FTO mRNA in Vero76 cells after SARS-CoV-2 infection. Dots represent mean expression in independent experiments conducted with technical triplicates. b Scheme of m 6 A-seq in infected FTO-depleted monkey cells. Total RNA was extracted from SARS-CoV-2-infected control (Ctrl) and FTO-depleted Vero76 cells 24 h post-inoculation. Then m 6 A-seq was performed in two independent experiments with technical duplicates. c m 6 A landscape of SARS-CoV-2 RNA upon FTO depletion. IGV tracks display input reads (blue) and m 6 A IP reads from control (gray) and FTO-depleted (red) libraries along the SARS-CoV-2 RNA genome. Three m 6 A peaks were identified on the genome of Ctrl cells and five on that of FTO-depleted cells (black squares). The SARS-CoV-2 genome organization is indicated below. Enlarged views of the IGV tracks show the m 6 A peaks within the SARS-CoV-2 TRS_L and nucleocapsid regions. d Scheme of viral titer assays on infected FTO-depleted monkey cells. SARS-CoV-2 replication was assessed by viral plaque assay on infected control and FTO-depleted Vero76 cells and performed in three independent experiments with technical triplicates. e Bar chart showing increased SARS-CoV-2 replication in Vero76 cells upon FTO depletion 24 h post-inoculation. Plaque-forming units (PFU) of infectious SARS-CoV-2 particles in culture supernatants were determined by plaque titration with Vero-TMPRSS2 cells. Dots represent mean titers for three independent experiments conducted with technical triplicates. Data represent means ± SEM. P -values were determined with a two-tailed unpaired Student’s t-test.

Article Snippet: Additionally, a positive control consisting of a 1089-nucleotide methylated transcript (Renilla luciferase) was in vitro transcribed with the MEGAscript T7 Transcription Kit (Invitrogen, AM1334) according to the manufacturer’s instructions, with addition of m 6 ATP (TriLink, N-1013) instead of ATP nucleotides in the reaction mix.

Techniques: Expressing, Infection, Quantitative RT-PCR, Viral Plaque Assay, Titration, Two Tailed Test

Journal: bioRxiv

Article Title: The RNA demethylase FTO controls m 6 A marking on SARS-CoV-2 and classifies COVID-19 severity in patients

doi: 10.1101/2022.06.27.497749

Figure Lengend Snippet: a Host m 6 A machinery gene expression upon SARS-CoV-2 infection in monkey cells. RT-qPCR assays displaying altered levels of mRNAs encoding host m 6 A machinery components after SARS-CoV-2 infection of Vero76 cells. Dots represent mean expression for independent experiments conducted with technical triplicates. b RT-qPCR assay validating decreased mRNA-level FTO expression upon FTO depletion by RNAi in Vero76 cells. Dots represent mean expression for three independent experiments conducted with technical triplicates. c Western blot assays confirm a decreased FTO protein level upon FTO depletion by RNAi in Vero76 cells. Left panel: representative western blot, β-actin serving as a protein loading control. Right panel: western blot quantification from three biological replicates. d The Venn diagram (upper panel) shows the overlap between m 6 A peaks identified by m 6 A-seq in infected controls (gray) and FTO-depleted Vero76 cells (red). All m 6 A peaks identified in both control and FTO-depleted Vero76 cells are listed below the diagram. Rows colored in gray highlight m 6 A peaks shared by both controls and FTO-depleted cells. e Bar chart showing the SARS-CoV-2 inoculum used to infect control (gray) and FTO-depleted (red) Vero76 cells. Plaque-forming units (PFU) of infectious SARS-CoV-2 particles in culture supernatants were determined by plaque titration with Vero-TMPRSS2 cells. Dots represent mean expression for three independent experiments conducted with technical triplicates. Data represent means ± SEM. P -values were determined with a two-tailed unpaired Student’s t test.

Article Snippet: Additionally, a positive control consisting of a 1089-nucleotide methylated transcript (Renilla luciferase) was in vitro transcribed with the MEGAscript T7 Transcription Kit (Invitrogen, AM1334) according to the manufacturer’s instructions, with addition of m 6 ATP (TriLink, N-1013) instead of ATP nucleotides in the reaction mix.

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Titration, Two Tailed Test