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N1-methyladenosine methylome in messenger RNA in petunia

ABSTRACT: N1-methyladenosine is a unique base methylation because it blocks Watson-Crick base paring and introduces a positive charge. Previous studies showed that m1A is prevalent in yeast and mammals mRNA and has a functional role in promoting translation of methylated mRNA. However, little is known about its abundance, topology and dynamics in plant mRNA. In this study, dot blotting and LC–MS/MS analyses reveal a dynamic pattern of m1A mRNA modification in various tissues and at different developmental stages in petunia (Petunia hybrida). Transcriptome-wide profiling of mRNA m1A in petunia was reported by applying m1A mRNA immunoprecipitation followed by a deep-sequencing approach (m1A-seq). m1A-seq analysis identified 4993 m1A peaks in 3231 expressed genes in petunia corollas. Each methylated gene averagely carries 1.55 peaks. Among the identified m1A peaks, there are 251 m1A peaks in which the adenines was partly replaced by thymine (T) and/or the reverse transcription stops happened in adenine site, in 199 expressed genes. We found that m1A is enriched in coding sequences with one peaks located immediately after start codons, and a slight negative correlation between methylated genes and gene expression was observed. Totally, ethylene treatment reduced the m1A level of mRNA in petunia corollas. We show that a RNA m1A-methyltransferase, tRNA specific methyltransferase 61A (PhTRMT61A), is an m1A mRNA methyltransferase. PhTRMT61A silencing results in decreased m1A peaks in mRNA in leaves and abnormal leaf development. PhTRMT61A is located to the nucleus. Our results suggest that m1A in mRNA is an important epitranscriptome marker and plays a role in plant development.

ORGANISM(S): Petunia x hybrida

PROVIDER: GSE136921 | GEO | 2020/08/12

REPOSITORIES: GEO

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Project description:Following synthesis, RNA can be modified with over 100 chemically distinct modifications, and in recent years it was shown that processing, localization, stability and translation of mRNAs can be impacted by an increasing number of these modifications. An emerging modification, present across all three domains of life, is N1-methyladenosine (m1A). m1A is of particular interest, as its methyl group disrupts Watson-Crick base pairing and it thus harbors substantial regulatory potential. Recent studies have suggested this modification to be relatively common in mammalian mRNA, but lacked the resolution of identifying individual m1A containing bases, and did not identify specific sequence motifs required for their modification or the enzymatic machinery catalyzing them. This rendered it challenging to validate the putative substrates and to address their function and mechanisms of action. Here we develop a highly sensitive and specific approach allowing the semi-quantitative mapping of m1A at single nucleotide resolution and in a transcriptome-wide manner. We found m1A to be present in cytosolic and mitochondrial mRNAs. In the cytosol m1A is present in a low number of mRNAs, typically at very low stoichiometry, almost invariably in the loop of hairpin structures, where it is introduced by the TRMT6/TRMT61A complex. In contrast, we found a single site in the mitochondrial ND5 mRNA, catalyzed by TRMT10C, with tightly regulated methylation levels in human development, ranging from nearly 100% up to the 8-cell stage and down to ~20% at the late blastocyst stage. We find that methylation at this site is the hallmark of hyper-stable transcripts, which are maternally transmitted and persist until the onset of zygotic mitochondrial transcription. Finally, we found that polysomes are strongly depleted from methylated mRNAs and that reporters harboring m1A in regions encountered by ribosomes (5’ UTR and CDS), but not in 3’ UTRs, are translated at reduced rates. Our findings suggest that m1A on mRNA, likely due to its dramatic impact on base pairing, lead to repression of translation which is by and large avoided by cells, while revealing one case - in mitochondria - where tight spatiotemporal control over m1A levels was adopted by cells as a potential means of post-transcriptional regulation.

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N1-methyladenosine methylome in messenger RNA in petunia

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