Quantitative mapping of pseudouridines in bacteria RNA

Area of Science:

• Molecular Biology
• Microbial Genomics
• RNA Biology

Background:

• RNA pseudouridylation is a common RNA modification, crucial in bacteria's transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs).
• The role and extent of pseudouridylation in bacterial messenger RNAs (mRNAs) are not well understood.
• Existing methods lack comprehensive quantification of pseudouridines across the bacterial transcriptome.

Purpose of the Study:

• To develop and apply a sequencing approach for comprehensive, quantitative mapping of RNA pseudouridines in bacteria.
• To investigate the prevalence and functional implications of pseudouridylation in bacterial mRNA.
• To demonstrate the utility of pseudouridine mapping for discovering novel transcripts and regulatory elements.

Main Methods:

• Utilized a bisulfite-based sequencing strategy for precise pseudouridine detection and quantification.
• Applied the method to *Escherichia coli* (*E. coli*) to establish proof of concept.
• Extended the application to human oral microbiome samples to assess broad applicability.

Main Results:

• Identified 1,954 high-confidence pseudouridine sites in 1,331 *E. coli* transcripts, covering approximately 30% of the transcriptome.
• Discovered differentially expressed genes related to stress response, missed by conventional RNA sequencing (RNA-seq).
• Identified a novel small RNA from the antisense strand of tRNA-Tyr, regulating distal gene expression.
• Successfully mapped pseudouridines in complex human oral microbiome samples.

Conclusions:

• Pseudouridine mapping provides a powerful tool for studying post-transcriptional regulation in bacteria.
• The approach reveals significant pseudouridylation in bacterial mRNA, impacting gene expression and stress response.
• This method facilitates the discovery of novel transcripts and regulatory mechanisms within microbial communities.