Direct RNA sequencing of the Escherichia coli epitranscriptome uncovers alterations under heat stress

Nucleic Acids Research, Journal Year: 2025, Volume and Issue: 53(6)

Published: March 20, 2025

Modifications of RNA, known as the epitranscriptome, affect gene expression, translation, and splicing in eukaryotes, with implications for developmental processes, cancer, viral infections. In prokaryotes, regulation at level epitranscriptome is still poorly understood. Here, we used nanopore direct RNA sequencing Escherichia coli to study modifications their changes under heat stress. With a single reaction, detected most modification types ribosomal (rRNA), transfer (tRNA), messenger (mRNA). was complemented by multifaceted approach that included mass spectrometry, deletion mutants, single-nucleotide polymerase chain vitro methylation. Known 5-methylcytidine (m5C) N6-methyladenosine (m6A) sites rRNA were confirmed, but these could not be localized mRNA. response stress, levels m5C, m6A, N6,N6-dimethyladenosine increased 16S rRNA. Sequencing spectrometry data demonstrated decrease tRNA abundance anticodon loop 45°C. general, mRNA 37°C enriched coding regions genes associated general metabolism processing, which shifted involved cell wall synthesis membrane transport This provides new insights into complexity post-transcriptional bacteria.

Language: Английский

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Application of Nanopore Long-Read Sequencing and Metabolomics in an in Vitro Dynamic Intestinal Digestion Model: A Genome-Centric Metatranscriptomic Approach to Investigating Microbial Tma and Scfa Metabolism

Published: Jan. 1, 2025

Language: Английский

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Nanopore Environmental Analysis

JACS Au, Journal Year: 2025, Volume and Issue: unknown

Published: April 1, 2025

As global pollution continues to escalate, timely and accurate monitoring is essential for guiding governance safeguarding public health. The increasing diversity of pollutants across environmental matrices poses a significant challenge instrumental analysis methods, which often require labor-intensive time-consuming sample pretreatment. Nanopore technology, an emerging single-molecule technique, presents promising solution by enabling the rapid identification multiple targets within complex mixtures with minimal preparation. A wide range have been characterized using natural biological nanopores or artificial solid-state nanopores, their distinct advantages include simple preparation, high sensitivity, onsite analysis. In particular, long-read nanopore sequencing has led dramatic improvements in analyses microbial communities, allows species-level taxonomic assignment amplicon sequencing, simplifies assembly metagenomes. this Perspective, we review latest advancements analyzing chemical through sensing techniques. We also explore challenges that remain rapidly evolving field provide outlook on potential transform monitoring, risk assessment, health protection.

Language: Английский

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Application of Nanopore Long-Read Sequencing and Metabolomics in an in vitro Dynamic Intestinal Digestion Model: A Genome-Centric Metatranscriptomic Approach to Investigating Microbial TMA and SCFA Metabolism

Journal of Pharmaceutical and Biomedical Analysis, Journal Year: 2025, Volume and Issue: unknown, P. 116896 - 116896

Published: April 1, 2025

Language: Английский

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Quantitative and Multiplexing Analysis of MicroRNAs by Direct Full-Length Sequencing in Nanopores

Journal of the American Chemical Society, Journal Year: 2025, Volume and Issue: unknown

Published: April 28, 2025

MicroRNAs (miRNAs) play important regulatory roles in biology. Direct sequencing of miRNAs full-length can reveal comprehensive information on their sequences, abundance, and modifications, which, however, has yet to be achieved due extremely short length (∼22 nt). Herein, we developed Direct-miR-seq, a nanopore-based direct RNA (DRS) method that elongates at both the 5' 3' ends by ligating with custom nucleic acid adaptors ensure high yield accuracy. Compared standard DRS, Direct-miR-seq enabled whole sequence miRNAs, 26-fold yield, exhibited reduced bias across miRNA species along low error rates. We applied native populations from cells human serum demonstrate its capability selectively capture known sequences complex environments for revealing quantitative abundance m6A modification single-molecule single-base resolution ∼100 single event. envision may translated toward variety biological medical applications other small RNAs.

Language: Английский

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Decoding theEscherichia coliepitranscriptome

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: July 8, 2024

ABSTRACT Modifications of RNA, known as the epitranscriptome, affect gene expression, translation, and splicing in eukaryotes, with implications for developmental processes, cancer, viral infections. In prokaryotes, regulation at level epitranscriptome is still poorly understood. Here, we used Nanopore direct RNA sequencing Escherichia coli to study modifications their changes under heat stress. With a single reaction, detected most modification types rRNA, tRNA, mRNA. was complemented by multifaceted approach that included mass spectrometry, deletion mutants, single-nucleotide PCR, vitro methylation. Known m 5 C 6 A sites rRNA were confirmed, but these could not be localized response stress, levels C, A, 6,6 increased 16S rRNA. Sequencing spectrometry data demonstrated decrease tRNA abundance anticodon loop 45 °C. general, mRNA 37 °C enriched coding regions genes associated general metabolism processing, which shifted involved cell wall synthesis membrane transport This provides new insights into complexity post-transcriptional bacteria. GRAPHICAL

Language: Английский

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