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LEAD-m⁶ A-seq for Locus-Specific Detection of N⁶ -Methyladenosine and Quantification of Differential Methylation.

ABSTRACT: N⁶ -methyladenosine (m⁶ A) is a crucial RNA chemical mark which plays important roles in various biological processes. The development of highly multiplexed, cost-effective, and easy-to-operate methodologies for locus-specific analysis of m⁶ A is critical for advancing our understanding of the roles of this modification. Herein, we report a method which builds upon the principle of the previously reported SELECT approach by significantly improving its efficiency and coupling it to next generation sequencing technology for high-throughput validation and detection of m⁶ A modification at selected sites (LEAD-m⁶ A-seq). Through probing cDNA extension mediated by Bst DNA polymerase at and near target cellular sites by sequencing, we evaluated m⁶ A modification at these sites, and estimated differential methylation levels (0-84 %) upon in vitro demethylation by the m⁶ A demethylase FTO with high reproducibility. We envision that this strategy can be readily used for testing a greater number of sites with a broad dynamic range and modified to study other RNA modifications.

SUBMITTER: Wang Y

PROVIDER: S-EPMC7902341 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCucumber green mottle mosaic virus (CGMMV) causes substantial global losses in cucurbit crops, especially watermelon. N6-methyladenosine (m6A) methylation in RNA is one of the most important post-transcriptional modification mechanisms in eukaryotes. It has been shown to have important regulatory functions in some model plants, but there has been no research regarding m6A modifications in watermelon.ResultsWe measured the global m6A level in resistant watermelon after CGMMV infection using a colorimetric method. And the results found that the global m6A level significantly decreased in resistant watermelon after CGMMV infection. Specifically, m6A libraries were constructed for the resistant watermelon leaves collected 48 h after CGMMV infection and the whole-genome m6A-seq were carried out. Numerous m6A modified peaks were identified from CGMMV-infected and control (uninfected) samples. The modification distributions and motifs of these m6A peaks were highly conserved in watermelon transcripts but the modification was more abundant than in other reported crop plants. In early response to CGMMV infection, 422 differentially methylated genes (DMGs) were identified, most of which were hypomethylated, and probably associated with the increased expression of watermelon m6A demethylase gene ClALKBH4B. Gene Ontology (GO) analysis indicated quite a few DMGs were involved in RNA biology and stress responsive pathways. Combined with RNA-seq analysis, there was generally a negative correlation between m6A RNA methylation and transcript level in the watermelon transcriptome. Both the m6A methylation and transcript levels of 59 modified genes significantly changed in response to CGMMV infection and some were involved in plant immunity.ConclusionsOur study represents the first comprehensive characterization of m6A patterns in the watermelon transcriptome and helps to clarify the roles and regulatory mechanisms of m6A modification in watermelon in early responses to CGMMV.

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LEAD-m⁶ A-seq for Locus-Specific Detection of N⁶ -Methyladenosine and Quantification of Differential Methylation.

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OmicsDI is part of the ELIXIR infrastructure

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LEAD-m6 A-seq for Locus-Specific Detection of N6 -Methyladenosine and Quantification of Differential Methylation.

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OmicsDI is part of the ELIXIR infrastructure

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LEAD-m⁶ A-seq for Locus-Specific Detection of N⁶ -Methyladenosine and Quantification of Differential Methylation.