Quantitative RNA pseudouridine landscape reveals dynamic modification patterns and evolutionary conservation across bacterial species
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eLife Assessment
This study illustrates a valuable application of BID-seq to bacterial RNA, allowing transcriptome-wide mapping of pseudouridine modifications across various bacterial species. The evidence presented includes a mix of solid and incomplete data and analyses, and would benefit from more rigorous approaches. The work will interest a specialized audience involved in RNA biology.
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Abstract
Abstract
Pseudouridine (Ψ) modifications are the most abundant RNA modifications; however, their distribution and functional significance in bacteria remain largely unexplored compared to eukaryotic systems. In this study, we present the first transcriptome-wide and quantitative mapping of Ψ modifications across five diverse bacterial species (Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Pseudomonas syringae) at single-base resolution, utilizing the optimized BID-seq method for bacterial RNA. Our analysis revealed growth phase-dependent dynamics of pseudouridylation in bacterial tRNA and mRNA, particularly in genes enriched in core metabolic pathways. Comparative analysis demonstrated evolutionarily conserved features of Ψ modifications, such as dominant motif contexts, Ψ clustering within operons, etc. Functional analysis indicated Ψ modifications influence bacterial mRNA stability, translation, and interactions with specific RNA-binding proteins (RBPs) in response to changing cellular demands during growth phase transitions. The integrated computational analysis on local RNA architecture was conducted to elucidate the structure-dependent Ψ modifications in bacterial RNA. Furthermore, we developed an integrated deep learning framework, combining Transformer-GNN-based neural networks (pseU_NN) to capture both RNA sequence and structural features for effective prediction of Ψ-modified sites. Overall, our study provides valuable insights into the landscapes of bacterial RNA Ψ modifications and establishes a foundation for future mechanistic investigations into the functions of Ψ in bacterial RNA regulation.
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eLife Assessment
This study illustrates a valuable application of BID-seq to bacterial RNA, allowing transcriptome-wide mapping of pseudouridine modifications across various bacterial species. The evidence presented includes a mix of solid and incomplete data and analyses, and would benefit from more rigorous approaches. The work will interest a specialized audience involved in RNA biology.
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Reviewer #1 (Public review):
Summary:
The manuscript by Xu et al. reported base-resolution mapping of RNA pseudouridylation in five bacterial species, utilizing recently developed BID-seq. They detected pseudouridine (Ψ) in bacterial rRNA, tRNA, and mRNA, and found growth phase-dependent Ψ changes in tRNA and mRNA. They then focused on mRNA and conducted a comparative analysis of Ψ profiles across different bacterial species. Finally, they developed a deep learning model to predict Ψ sites based on RNA sequence and structure.
Strengths:
This is the first comprehensive Ψ map across multiple bacterial species, and systematically reveals Ψ profiles in rRNA, tRNA, and mRNA under exponential and stationary growth conditions. It provides a valuable resource for future functional studies of Ψ in bacteria.
Weaknesses:
Ψ is highly abundant on …
Reviewer #1 (Public review):
Summary:
The manuscript by Xu et al. reported base-resolution mapping of RNA pseudouridylation in five bacterial species, utilizing recently developed BID-seq. They detected pseudouridine (Ψ) in bacterial rRNA, tRNA, and mRNA, and found growth phase-dependent Ψ changes in tRNA and mRNA. They then focused on mRNA and conducted a comparative analysis of Ψ profiles across different bacterial species. Finally, they developed a deep learning model to predict Ψ sites based on RNA sequence and structure.
Strengths:
This is the first comprehensive Ψ map across multiple bacterial species, and systematically reveals Ψ profiles in rRNA, tRNA, and mRNA under exponential and stationary growth conditions. It provides a valuable resource for future functional studies of Ψ in bacteria.
Weaknesses:
Ψ is highly abundant on non-coding RNA such as rRNA and tRNA, while its level on mRNA is very low. The manuscript focuses primarily on mRNA, which raises questions about the data quality and the rigor of the analysis. Many conclusions in the manuscript are speculative, based solely on the sequencing data but not supported by additional experiments.
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Reviewer #2 (Public review):
Summary:
In this study, Xu et al. present a transcriptome-wide, single-base resolution map of RNA pseudouridine modifications across evolutionarily diverse bacterial species using an adapted form of BID-Seq. By optimizing the method for bacterial RNA, the authors successfully mapped modifications in rRNA, tRNA, and, importantly, mRNA across both exponential and stationary growth phases. They uncover evolutionarily conserved Ψ motifs, dynamic Ψ regulation tied to bacterial growth state, and propose functional links between pseudouridylation and bacterial transcript stability, translation, and RNA-protein interactions. To extend these findings, they develop a deep learning model that predicts pseudouridine sites from local sequence and structural features.
Strengths:
The authors provide a valuable resource: a …
Reviewer #2 (Public review):
Summary:
In this study, Xu et al. present a transcriptome-wide, single-base resolution map of RNA pseudouridine modifications across evolutionarily diverse bacterial species using an adapted form of BID-Seq. By optimizing the method for bacterial RNA, the authors successfully mapped modifications in rRNA, tRNA, and, importantly, mRNA across both exponential and stationary growth phases. They uncover evolutionarily conserved Ψ motifs, dynamic Ψ regulation tied to bacterial growth state, and propose functional links between pseudouridylation and bacterial transcript stability, translation, and RNA-protein interactions. To extend these findings, they develop a deep learning model that predicts pseudouridine sites from local sequence and structural features.
Strengths:
The authors provide a valuable resource: a comprehensive Ψ atlas for bacterial systems, spanning hundreds of mRNAs and multiple species. The work addresses a gap in the field - our limited understanding of bacterial epitranscriptomics, by establishing both the method and datasets for exploring post-transcriptional modifications.
Weaknesses:
The main limitation of the study is that most functional claims (i.e., translation efficiency, mRNA stability, and RNA-binding protein interactions) are based on correlative evidence. While suggestive, these inferences would be significantly strengthened by targeted perturbation of specific Ψ synthases or direct biochemical validation of proposed RNA-protein interactions (e.g., with Hfq). Additionally, the GNN prediction model is a notable advance, but methodological details are insufficient to reproduce or assess its robustness.
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Reviewer #3 (Public review):
Summary:
This study aimed to investigate pseudouridylation across various RNA species in multiple bacterial strains using an optimized BID-seq approach. It examined both conserved and divergent modification patterns, the potential functional roles of pseudouridylation, and its dynamic regulation across different growth conditions.
Strengths:
The authors optimized the BID-seq method and applied this important technique to bacterial systems, identifying multiple pseudouridylation sites across different species. They investigated the distribution of these modifications, associated sequence motifs, their dynamics across growth phases, and potential functional roles. These data are of great interest to researchers focused on understanding the significance of RNA modifications, particularly mRNA modifications, in …
Reviewer #3 (Public review):
Summary:
This study aimed to investigate pseudouridylation across various RNA species in multiple bacterial strains using an optimized BID-seq approach. It examined both conserved and divergent modification patterns, the potential functional roles of pseudouridylation, and its dynamic regulation across different growth conditions.
Strengths:
The authors optimized the BID-seq method and applied this important technique to bacterial systems, identifying multiple pseudouridylation sites across different species. They investigated the distribution of these modifications, associated sequence motifs, their dynamics across growth phases, and potential functional roles. These data are of great interest to researchers focused on understanding the significance of RNA modifications, particularly mRNA modifications, in bacteria.
Weaknesses:
(1) The reliability of BID-seq data is questionable due to a lack of experimental validations.
(2) The manuscript is not well-written, and the presented work shows a major lack of scientific rigor, as several key pieces of information are missing.
(3) The manuscript's organization requires significant improvement, and numerous instances of missing or inconsistent information make it difficult to understand the key objectives and conclusions of the study.
(4) The rationale for selecting specific bacterial species is not clearly explained, and the manuscript lacks a systematic comparison of pseudouridylation among these species.
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