Guan-Zheng Luo

N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas.

N(6)-methyldeoxyadenosine (6mA or m(6)A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here, we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms.

High-resolution N(6) -methyladenosine (m(6) A) map using photo-crosslinking-assisted m(6) A sequencing.

N(6) -methyladenosine (m(6) A) is an abundant internal modification in eukaryotic mRNA and plays regulatory roles in mRNA metabolism. However, methods to precisely locate the m(6) A modification remain limited. We present here a photo-crosslinking-assisted m(6) A sequencing strategy (PA-m(6) A-seq) to more accurately define sites with m(6) A modification. Using this strategy, we obtained a high-resolution map of m(6) A in a human transcriptome. The map resembles the general distribution pattern observed previously, and reveals new m(6) A sites at base resolution. Our results provide insight into the relationship between the methylation regions and the binding sites of RNA-binding proteins.

Unique features of the m6A methylome in Arabidopsis thaliana

Recent discoveries of reversible N6-methyladenosine (m6A) methylation on messenger RNA (mRNA) and mapping of m6A methylomes in mammals and yeast have revealed potential regulatory functions of this RNA modification. In plants, defects in m6A methyltransferase cause an embryo-lethal phenotype, suggesting a critical role of m6A in plant development. Here, we profile m6A transcriptome-wide in two accessions of Arabidopsis thaliana and reveal that m6A is a highly conserved modification of mRNA in plants. Distinct from mammals, m6A in A. thaliana is enriched not only around the stop codon and within 3′ untranslated regions (3′ UTRs), but also around the start codon. Gene ontology analysis indicates that the unique distribution pattern of m6A in A. thaliana is associated with plant-specific pathways involving the chloroplast. We also discover a positive correlation between m6A deposition and the mRNA abundance, suggesting a regulatory role of m6A in plant gene expression.

Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis

N6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA. It is dynamically installed and removed, and acts as a new layer of mRNA metabolism, regulating biological processes including stem cell pluripotency, cell differentiation, and energy homeostasis. m6A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to affect the translation of m6A-containing mRNAs, YTHDF2 expedites mRNA decay, and YTHDC1 affects the nuclear processing of its targets. The biological function of YTHDC2, the final member of the YTH protein family, remains unknown. We report that YTHDC2 selectively binds m6A at its consensus motif. YTHDC2 enhances the translation efficiency of its targets and also decreases their mRNA abundance. Ythdc2 knockout mice are infertile; males have significantly smaller testes and females have significantly smaller ovaries compared to those of littermates. The germ cells of Ythdc2 knockout mice do not develop past the zygotene stage and accordingly, Ythdc2 is upregulated in the testes as meiosis begins. Thus, YTHDC2 is an m6A-binding protein that plays critical roles during spermatogenesis.

DNA N 6 -methyladenine: a new epigenetic mark in eukaryotes?

DNA N6-adenine methylation (N6-methyladenine; 6mA) in prokaryotes functions primarily in the host defence system. The prevalence and significance of this modification in eukaryotes had been unclear until recently. Here, we discuss recent publications documenting the presence of 6mA in Chlamydomonas reinhardtii, Drosophila melanogaster and Caenorhabditis elegans; consider possible roles for this DNA modification in regulating transcription, the activity of transposable elements and transgenerational epigenetic inheritance; and propose 6mA as a new epigenetic mark in eukaryotes.

Widespread occurrence of N6-methyladenosine in bacterial mRNA

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m6A as well as m6A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m6A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m6A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m6A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m6A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m6A pattern that is distinct from those in eukaryotes. Most m6A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m6A peaks indicates that the majority of m6A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m6A in these pathways.

Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig.

DNA N6-methyldeoxyadenosine (6mA) is a well-known prokaryotic DNA modification that has been shown to exist and play epigenetic roles in eukaryotic DNA. Here we report that 6mA accumulates up to ∼0.1–0.2% of total deoxyadenosine during early embryogenesis of vertebrates, but diminishes to the background level with the progression of the embryo development. During this process a large fraction of 6mAs locate in repetitive regions of the genome.

YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs

N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m6A-selective ‘reader’ proteins of the YTH family, which incorporate m6A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m6A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m6A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m6A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

Characterization of eukaryotic DNA N 6 -methyladenine by a highly sensitive restriction enzyme-assisted sequencing

Although extensively studied in prokaryotes, the prevalence and significance of DNA N6-methyladenine (6mA or m6dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark. To interrogate 6mA sites at single-base resolution, we report DA-6mA-seq (DpnI-Assisted N6-methylAdenine sequencing), an approach that uses DpnI to cleave methylated adenine sites in duplex DNA. We find that DpnI cuts other sequence motifs besides the canonical GATC restriction sites, thereby expanding the utility of this method. DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches. We study 6mA at base resolution in the Chlamydomonas genome and apply the new method to two other eukaryotic organisms, Plasmodium and Penicillium. Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

DNA N6-methyladenine in metazoans: functional epigenetic mark or bystander?

The DNA-adenine modification N6-methyladenine (6mA), initially thought to be mainly restricted to prokaryotes and certain unicellular eukaryotes, has recently been found in metazoans. Proposed functions vary from gene activation to transposon suppression. However, since most metazoan genomes possess 5-methylcytosine (5mC) as a dominant epigenetic mark, it raises the question of why 6mA is required. This Perspective summarizes the latest discoveries and suggests potential functional roles for 6mA in metazoan genomes.