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Sequencing of polyA+ RNA from shCtrl and shMETTL8 HeLa for m3C site identification

ABSTRACT: N3-methylcytidine (m3C), an RNA modification involving methylation of cytidine at the Watson-Crick face, has been identified in human tRNA, mitochondrial tRNA (mt-tRNA), and mRNA. In tRNA, it has been shown to play important roles in tRNA structure and translation. m3C remains understudied in mRNA due to its markedly lower levels, the lack of high-resolution techniques to reproducibly map its location, and under-characterization of its effector proteins on this RNA type. Additionally, METTL8 has been identified as a writer of m3C on mt-tRNA, but has also been shown to have isoforms with nuclear localization. Here, we apply a new approach which utilizes the high readthrough and mutation rates of Protoscript II (PSII) at m3C sites in combination with AlkB demethylation to map m3C at single-base resolution in polyA-purified RNA from HeLa cells either with METTL8-targeting shRNA or control shRNA to assess how the method works on mRNA and to investigate which, if any, of the sites change with METTL8 knockdown.

ORGANISM(S): Homo sapiens

PROVIDER: GSE269470 | GEO | 2026/07/17

REPOSITORIES: GEO

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Detection of N3-methylcytidine in RNA at single base resolution using Protoscript II to read through and generate high mutation rates [m3C_screen]

Project description:N3-methylcytidine (m3C), an RNA modification involving methylation of cytidine at the Watson-Crick face, has been identified in human tRNA, mitochondrial tRNA (mt-tRNA), and mRNA. In tRNA and mt-tRNA, it has been shown to play important roles in tRNA structure and translation. m3C remains understudied in mRNA and other low-abundance RNAs due to its markedly lower levels, the lack of high-resolution techniques to reproducibly map its location, and under-characterization of its effector proteins on these RNA types. Here we describe a new approach which utilizes the high readthrough and mutation rates of Protoscript II (PSII) at m3C sites in combination with AlkB demethylation to detect m3C at single-base resolution. We first validate the method on synthetic probes. Then, using this method, we successfully identify the known m3C sites on tRNA and mt-tRNA and show that none of the m3C sites identified in cytosolic tRNA are likely METTL8 substrates. In contrast, the two m3C sites detected in mt-tRNA are installed by METTL8 since they are sensitive to METTL8 knockdown (KD) and over expression (OE). These results demonstrate that the method can be used to investigate changes in the m3C level in many sites using an unbiased approach. Overall, this study introduces a convenient high-resolution method for m3C mapping with high readthrough rates, paving the way for future mechanistic studies on the biological function of m3C and its effector proteins.

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Project description:Increasing evidence implicates critical roles of various epitranscriptomic RNA modifications in different biological processes. Methyltransferase METTL8 installs 3-methylcytosine modification of mitochondrial tRNAs in vitro, however, its role in intact biological systems is completely unknown. Here we show that Mettl8 is localized in the mitochondria and installs m3C in mitochondrial tRNASer(UCN)/Thr in mouse embryonic cortical neural stem cells. At molecular and cellular levels, Mettl8 deletion in cortical neural stem cells leads to reduced mitochondrial protein translation and attenuated respiration activity. At the functional level, conditional Mettl8 deletion in mice results in impaired embryonic cortical neural stem cell maintenance in vivo, which can be rescued by pharmacologically enhancing mitochondrial functions. Similarly, METTL8 regulates mitochondrial protein expression and neural stem cell maintenance in human forebrain cortical organoids. Together, our study reveals a conserved epitranscriptomic mechanism of Mettl8 and mitochondrial tRNA m3C modification in maintaining embryonic cortical neural stem cells in mice and humans.

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Project description:We report a m3C-specific high-throughput sequencing techinque, Hydrazine-Aniline Cleavage sequencing (HAC-seq) to profile the m3C methylome at single-nucleotide resolution. We apply HAC-seq to analyze ribosomal RNA-depleted total RNA from MCF7 cells. We find that tRNA are the predominant m3C-modified RNA species. We find no evidence of m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNA in MCF7 cells.

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