Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification, yet little is known about its prevalence, mechanism and function in mRNA. Here, we performed quantitative MS analysis and show that Ψ is much more prevalent (Ψ/U ratio ∼0.2-0.6%) in mammalian mRNA than previously believed. We developed N3-CMC-enriched pseudouridine sequencing (CeU-Seq), a selective chemical labeling and pulldown method, to identify 2,084 Ψ sites within 1,929 human transcripts, of which four (in ribosomal RNA and EEF1A1 mRNA) are biochemically verified. We show that hPUS1, a known Ψ synthase, acts on human mRNA; under stress, CeU-Seq demonstrates inducible and stress-specific mRNA pseudouridylation. Applying CeU-Seq to the mouse transcriptome revealed conserved and tissue-specific pseudouridylation. Collectively, our approaches allow comprehensive analysis of transcriptome-wide pseudouridylation and provide tools for functional studies of Ψ-mediated epigenetic regulation.

Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

The apical hook is an essential structure that enables epigeal plants to protrude through the soil. Arabidopsis thaliana HOOKLESS1 (HLS1) is reported to be a key regulator of hook development and a direct target gene of the ethylene (ET)-activated transcription factors ETHYLENE INSENSITIVE3 (EIN3) and its close homolog EIN3-Like1. Previous research has shown that the phytohormones jasmonate (JA) and ET antagonistically regulate apical hook development, although the underlying molecular mechanism is largely unknown. Here, we report that JA represses hook formation by reducing HLS1 expression. Our results further reveal that the JAactivated transcription factor MYC2 represses EIN3 function to reduce HLS1 expression through at least the following two layers of regulation: (1) MYC2 binds to the promoter of an F-box gene, EIN3 BINDING F-BOX PROTEIN1, to induce its expression and thus promote EIN3 degradation; and (2) MYC2 physically interacts with EIN3 and inhibits its DNA binding activity. Collectively, our findings shed light on the molecular mechanism underlying the antagonism between JA and ET during apical hook development and provide insight into the coaction of multiple phytohormones in the regulation of plant growth and development.