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Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 in the mitochondrial genome

ABSTRACT: Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 in the mitochondrial genome

PROVIDER: PRJNA488087 | ENA |

REPOSITORIES: ENA

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Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 in the mitochondrial genome

Project description:N6-methyldeoxyadenosine (6mA) is a well-characterized DNA modification in prokaryotes but reports on its presence and function in mammals have been controversial. To address this issue, we established the capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes. Using 6mACE-seq, we generated a human-genome-wide 6mA map that accurately reproduced known 6mA enrichment at active retrotransposons and revealed mitochondrial chromosome-wide 6mA clusters asymmetrically enriched on the heavy-strand. We identified a novel putative 6mA-binding protein in single stranded DNA binding protein 1 (SSBP1), a mitochondrial DNA (mtDNA) replication factor known to coat the heavy-strand, linking 6mA with the regulation of mtDNA replication. Finally, we characterized AlkB homolog 1 (ALKBH1) as a mitochondrial protein with 6mA demethylase activity and showed that its loss decreases mitochondrial oxidative phosphorylation. Our results show that 6mA clusters play a previously unappreciated role in regulating human mitochondrial function, despite 6mA being an uncommon DNA modification in the human genome.

2018-11-09 | GSE119094 | GEO

Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 on the mitochondrial genome.

Project description: Not available

| S-EPMC6294517 | biostudies-literature

Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity.

Project description:Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity. Keywords: siRNA, transfection, chemical modification, off-targets

2008-04-02 | GSE10960 | GEO

siRNA profiling of HeLa cells reveals strand-specific 5-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity.

Project description:Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity. Experiment Overall Design: For details please see: Chen PY, Weinmann L, Gaidatzis D, Pei Y, Zavolan M, Tuschl T, Meister G. RNA (2008) 14(2):263-74.

2008-06-11 | E-GEOD-10960 | biostudies-arrayexpress

Quantitative and whole-transcriptome N6-Methyladenosine profiling at single-nucleotide resolution

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single-nucleotide resolution sequencing of N6-methyladenine in E. coli genomes and mammalian mitochondrial DNA

Project description:N6-methyladenine (6mA) is a natural DNA modification and functions primarily in restriction-modification (R-M) systems in prokaryotes. Recent studies uncovered the existence and revealed the genome-wide distribution of 6mA in eukaryotes. Specifically, it was reported that 6mA was mainly enriched in mammalian mitochondrial DNA (mtDNA) and could regulate mitochondrial activity. we achieved the genome-wide mapping of 6mA in E. coli genome and mammalian mtDNA at single-nucleotide resolution.

2022-01-26 | GSE194212 | GEO

Quantitative and whole-transcriptome N6-Methyladenosine profiling at single-nucleotide resolution [polyA_RNA]

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The Rhino-Deadlock-Cutoff complex licenses non-canonical transcription of dual-strand piRNA clusters in Drosophila

Project description:Argonaute proteins of the PIWI clade are central to transposon silencing in animal gonads. Their target specificity is defined by 22-30nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. Here we show that the RDC complex composed of Rhino, Deadlock and Cutoff defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC complex is anchored to H3K9me3-marked chromatin in part via Rhino’s chromo-domain. Depletion of Piwi results in loss of the RDC and small RNAs at euchromatic piRNA source loci, demonstrating a feedback loop between Piwi and genomic piRNA sources. Intriguingly, profiles of RNA Polymerase II occupancy, nascent transcription and steady-state RNA levels reveal that the RDC licenses non-canonical transcription of dual-stranded piRNA clusters. Likely, this process involves 5’end protection of nascent RNAs and subsequent suppression of transcription termination. Together, our data provide a comprehensive model for the regulation and evolution of piRNA clusters.

2014-06-08 | GSE55824 | GEO

The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs

Project description:Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present in a single organism. Despite this complexity, specific Piwi proteins incorporate piRNAs with distinct nucleotide- and transposon strand-biases (antisense or sense) of unknown origin. Here we examined the contribution of structural domains in Piwi proteins towards defining these biases. We report the first crystal structure of the MID domain from a Piwi Argonaute and use docking experiments to show its ability to specify recognition of 5M-bM-^@M-2 uridine (1U-bias) of piRNAs. Mutational analyses reveal the importance of 5M-bM-^@M-^Y end-recognition within the MID domain for piRNA biogenesis in vivo. Finally, domain-swapping experiments uncover an unexpected role for the MID-PIWI module of a Piwi protein in dictating the strand-orientation of its bound piRNAs. Our work identifies structural features that allow distinguishing individual Piwi members during piRNA biogenesis Immunoprecipitated small RNA were purified from Bmn4 cells for preparation of high-throughput sequencing libraries.

2014-07-01 | E-GEOD-55451 | biostudies-arrayexpress

The Rhino-Deadlock-Cutoff complex licenses non-canonical transcription of dual-strand piRNA clusters in Drosophila

Project description:Argonaute proteins of the PIWI clade are central to transposon silencing in animal gonads. Their target specificity is defined by 22-30nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. Here we show that the RDC complex composed of Rhino, Deadlock and Cutoff defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC complex is anchored to H3K9me3-marked chromatin in part via RhinoM-bM-^@M-^Ys chromo-domain. Depletion of Piwi results in loss of the RDC and small RNAs at euchromatic piRNA source loci, demonstrating a feedback loop between Piwi and genomic piRNA sources. Intriguingly, profiles of RNA Polymerase II occupancy, nascent transcription and steady-state RNA levels reveal that the RDC licenses non-canonical transcription of dual-stranded piRNA clusters. Likely, this process involves 5M-bM-^@M-^Yend protection of nascent RNAs and subsequent suppression of transcription termination. Together, our data provide a comprehensive model for the regulation and evolution of piRNA clusters. This study aims at indentifying and characterizing genimc sources of piRNA percursour transcripts using genome-wide apporaches such as ChIP-seq, RNA-seq, smallRNA-seq and GRO-seq in adult Drosophila ovaries depleted for several factors implicated in piRNA cluster regulation

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