The epitranscriptomic mark -methyladenosine (mA) can be written, read, and erased via the action of a complex network of proteins. mA binding proteins read mA marks and transduce their downstream regulatory effects by altering RNA metabolic processes. The characterization of mA readers is an essential prerequisite for understanding the roles of mA in plants, but the identities of mA readers have been unclear. Here, we characterized the YTH-domain family protein ECT2 as an mA reader whose mA binding function is required for normal trichome morphology. We developed the formaldehyde cross-linking and immunoprecipitation method to identify ECT2-RNA interaction sites at the transcriptome-wide level. This analysis demonstrated that ECT2 binding sites are strongly enriched in the 3' untranslated regions (3' UTRs) of target genes and led to the identification of a plant-specific mA motif. Sequencing analysis suggested that ECT2 plays dual roles in regulating 3' UTR processing in the nucleus and facilitating mRNA stability in the cytoplasm. Disruption of accelerated the degradation of three ECT2 binding transcripts related to trichome morphogenesis, thereby affecting trichome branching. The results shed light on the underlying mechanisms of the roles of mA in RNA metabolism, as well as plant development and physiology.

The epitranscriptomic mark N6‐methyladenosine (m6A) is the most abundant RNA modification in eukaryotic mRNA, but various limitations in currently available m6A detection methods have precluded routine identification of m6A marks at the single‐site level in mRNA transcripts. Herein, we report a single‐base elongation‐ and ligation‐based qPCR amplification method (termed “SELECT”) that exploits the ability of m6A to hinder 1) the single‐base elongation activity of DNA polymerases and 2) the nick ligation efficiency of ligases; SELECT employs qPCR for quantitation. Following optimization and validation, SELECT was applied on three highly relevant proof‐of‐concept cases: determining 1) if a putative m6A site is m6A‐modified in mRNAs and lncRNAs from biological samples, 2) the m6A fraction at biological sites, and 3) if a particular m6A modification enzyme functions on a specific target site. In summary, the rapid and flexible SELECT method facilitates the identification and verification of m6A marks with unprecedented ease.

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