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Chemical manipulation of m1A mediates its detection in human tRNA

ABSTRACT: N1-methyl adenosine (m1A) is a wide-spread RNA modification present in tRNA, rRNA and mRNA. m1A modification sites in tRNAs are evolutionary conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick base pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red‑m1A-seq to sequence human small RNA and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-ATT and 5.8S rRNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE253657 | GEO | 2024/04/24

REPOSITORIES: GEO

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N1-methyladenosine methylations in tRNA drive liver tumorigenesis by PPARδ-mediated cholesterol synthesis

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2021-03-01 | GSE147840 | GEO

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2022-07-23 | GSE184909 | GEO

m1A inhibition fuels oncolytic virus-elicited antitumor immunity via downregulating PD-L1 expression

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The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates metabolism and development via translation (Identification of m1A mRNA methyltransferase in nucleus by TMT11-LC-MS/MS)

Project description:m1A has long been known to be important in tRNA and rRNA, but we recently mapped many m1A sites in mRNA as well. m1A is found on the 5’ ends of genes, usually around the first splice site, but we have yet to understand how it effects transcript fate. Identifying the methyltransferase would be a major first step towards understanding m1A function.

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human liver CSCs LC-MSMS in sgTRMT6/61A and control

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2021-09-15 | PXD019576 | Pride

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Project description:N1-methyladenosine (m1A) is an abundant post-transcriptional RNA modification, yet little is known about its prevalence, topology and dynamics in mRNA. Here, we show that m1A is abundant in human mRNA, with an m1A/A ratio of ~0.02%. We develop m1A-ID-Seq, based on m1A immunoprecipitation and the inherent property of m1A to stall reverse transcription, for the transcriptome-wide m1A analysis. m1A-ID-Seq identifies 901 m1A peaks (from 583 genes) in mRNA and ncRNA, and reveals a prominent feature of enrichment in the 5â-untranslated region of mRNA transcripts, distinct from that of N6-methyladenosine, the most abundant internal mammalian mRNA modification. m1A in mRNA is also reversible by ALKBH3, a known DNA/RNA demethylase. Lastly, m1A responds dynamically to stimuli and hundreds of stress-induced m1A sites are identified. Collectively, our approaches allow comprehensive analysis of m1A methylation and provide an important tool for functional studies of potential epigenetic regulation via the reversible and dynamic m1A methylation. Identification of m1A sites in human embryonic kidney cells. Comparisons of m1A profiles of wild type HEK293T with ALKBH3 knock out cell line reveals the ALKBH3 specific sites. Stress inducible m1A sites are also identified by comparing the profiles of untreated cells with stress treated cells.

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Base-resolution mapping reveals distinct classes of N1-methyladenosine methylome in nuclear- and mitochondrial-encoded transcripts

Project description:Gene expression can be post-transcriptionally regulated via dynamic and reversible RNA modifications. N1-methyladenosine (m1A) is a recently identified mRNA modification; however, little is known about its precise location, regulation and function. Here, we develop a single-nucleotide resolution m1A profiling method, based on m1A-induced misincorporation during reverse transcription, and report distinct classes of m1A methylome in the human transcriptome. m1A in the 5’-untranslated region, particularly those located exactly at the first nucleotide of mRNA transcripts, associates with increased translation efficiency. A different subset of m1A sites exhibit a GUUCRA tRNA-like motif, are evenly distributed in the transcriptome and are dependent on the methyltransferase complex TRMT6/61A. Additionally, we show for the first time that m1A is prevalent in the mitochondrial-encoded transcripts. Manipulation of m1A level via TRMT61B, a mitochondria-localizing m1A methyltransferase, demonstrates that m1A in mitochondrial mRNA interferes with translation. Collectively, our approaches reveal distinct classes of m1A methylome and provide a resource for functional studies of m1A-mediated epitranscriptomic regulation.

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Project description:We reported the tRNA-m1A sequencing results performed in young and aged hematopoietic stem and progenitor cells. Our results defined aging-associated alterations of tRF in hematopoietic stem and progenitor cells and identifed a subset of tRF as hallmark of HSC aging.RNA-seq data analysis of WT v.s. Trmt6/61a-TG hematopoietic stem cell (lineage-, c-kit+ Sca-1+ CD135-, CD34-), the two population of hematopoietic stem cell (HSC) were purified from the bone marrow of WT and Trmt6/61a-TG mice, revealed that differnent expression of WT v.s. Trmt6/61a-TG hematopoietic stem cell.Results provide insight into the role of TRMT6/61A complex in HSC.

2024-01-18 | GSE163301 | GEO

Antibody crossreactivity with mRNA cap accounts for widespread appearance of m1A throughout the transcriptome

Project description:N1-methyladenosine (m1A) was recently identified as a new mRNA modification based on its mapping to the 5’UTRs of thousands of mRNAs with an m1A-binding antibody. More recent studies have questioned the prevalence of m1A. To address this discrepancy, we mapped m1A using ultra-deep RNA-Seq datasets based on m1A-induced misincorporations during reverse transcription. Using this approach, we find m1A only in the mitochondrial MT-ND5 transcript. In contrast, m1A mapping using an m1A-binding antibody showed binding to transcription start nucleotides in mRNA 5’UTRs. Biochemical measurements indicate that m1A is not present at these sites. Instead, we find that the m1A antibody exhibits m1A-independent binding to mRNA cap structures. Our data demonstrate that high-stoichiometry m1A sites are rare in the transcriptome and that previous mapping of m1A to mRNA 5’UTRs reflect an unintended binding to mRNA caps.

2019-06-23 | GSE97909 | GEO

Gene-silencing activity of small RNAs controlled by TRMT6/61A-dependent base methylation

Project description:We identified human small RNAs containing m1A (N1-methyladenosine) by m1A RIP and TGIRT-seq. Small RNA-seq and long RNA-seq was performed after TRMT6/61A knock-down.

2022-02-09 | GSE171040 | GEO

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