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Methodologies for bacterial ribonuclease characterization using RNA-seq.

ABSTRACT: Bacteria adjust gene expression at the post-transcriptional level through an intricate network of small regulatory RNAs and RNA-binding proteins, including ribonucleases (RNases). RNases play an essential role in RNA metabolism, regulating RNA stability, decay, and activation. These enzymes exhibit species-specific effects on gene expression, bacterial physiology, and different strategies of target recognition. Recent advances in high-throughput RNA sequencing (RNA-seq) approaches have provided a better understanding of the roles and modes of action of bacterial RNases. Global studies aiming to identify direct targets of RNases have highlighted the diversity of RNase activity and RNA-based mechanisms of gene expression regulation. Here, we review recent RNA-seq approaches used to study bacterial RNases, with a focus on the methods for identifying direct RNase targets.

SUBMITTER: Broglia L

PROVIDER: S-EPMC10503654 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Methodologies for bacterial ribonuclease characterization using RNA-seq.

Broglia Laura L Le Rhun Anaïs A Charpentier Emmanuelle E

FEMS microbiology reviews 20230901 5

Bacteria adjust gene expression at the post-transcriptional level through an intricate network of small regulatory RNAs and RNA-binding proteins, including ribonucleases (RNases). RNases play an essential role in RNA metabolism, regulating RNA stability, decay, and activation. These enzymes exhibit species-specific effects on gene expression, bacterial physiology, and different strategies of target recognition. Recent advances in high-throughput RNA sequencing (RNA-seq) approaches have provided ...[more]

PMID: 37656885

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Project description:Ribosome profiling (Ribo-Seq) is a powerful method to study translation in bacteria. However, Ribo-Seq signal can be observed across RNAs that one would not expect to be bound by ribosomes. For example, Escherichia coli Ribo-Seq libraries also capture reads from most noncoding RNAs (ncRNAs). While some of these ncRNAs may overlap coding regions, this alone does not explain the majority of observed signal across ncRNAs. These fragments of ncRNAs in Ribo-Seq data pass all size selection steps of the Ribo-Seq protocol and survive hours of micrococcal nuclease (MNase) treatment. In this work, we specifically focus on Ribo-Seq signal across ncRNAs and provide evidence to suggest that RNA structure, as opposed to ribosome binding, protects them from degradation and allows them to persist in the Ribo-Seq sequencing library preparation. By inspecting these "contaminant reads" in bacterial Ribo-Seq, we show that data previously disregarded in bacterial Ribo-Seq experiments may, in fact, be used to gain partial information regarding the in vivo secondary structure of ncRNAs.IMPORTANCE Structured ncRNAs are pivotal mediators of bioregulation in bacteria, and their functions are often reliant on their specific structures. Here, we first inspect Ribo-Seq reads across noncoding regions, identifying contaminant reads in these libraries. We observe that contaminant reads in bacterial Ribo-Seq experiments that are often disregarded, in fact, strongly overlap with structured regions of ncRNAs. We then perform several bioinformatic analyses to determine why these contaminant reads may persist in Ribo-Seq libraries. Finally, we highlight some structured RNA contaminants in Ribo-Seq and support the hypothesis that structures in the RNA protect them from MNase digestion. We conclude that researchers should be cautious when interpreting Ribo-Seq signal as coding without considering signal distribution. These findings also may enable us to partially resolve RNA structures, identify novel structured RNAs, and elucidate RNA structure-function relationships in bacteria at a large scale and in vivo through the reanalysis of existing Ribo-Seq data sets.

Dataset Information

Methodologies for bacterial ribonuclease characterization using RNA-seq.

Publications

Methodologies for bacterial ribonuclease characterization using RNA-seq.

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