Precision run-on sequencing (PRO-seq) for microbiome transcriptomics.

Area of Science:

• Microbial genomics and transcriptomics
• Bacterial gene regulation
• Metagenomics and microbiome analysis

Background:

• Bacteria regulate gene expression via transcription initiation and elongation.
• Bulk RNA sequencing captures mature transcripts, missing actively transcribed regions.
• Nascent RNA capture methods were limited to culturable bacteria.

Purpose of the Study:

• To apply precision run-on sequencing (PRO-seq) for profiling nascent transcription in diverse bacteria, including uncultured species.
• To characterize transcription of small, structured, or modified RNAs missed by bulk RNA sequencing.
• To investigate transcriptional dynamics and RNA polymerase (RNAP) behavior in the human microbiome.

Main Methods:

• Precision run-on sequencing (PRO-seq) was employed to capture RNA polymerase (RNAP) complexed with nascent RNA.
• The method was applied to cultured E. coli and diverse uncultured bacteria.
• PRO-seq data from the human microbiome were analyzed for RNAP pause motifs and RNA processing.

Main Results:

• PRO-seq successfully profiled nascent transcription in both cultured and uncultured bacteria.
• The technique identified transcription of small, structured, and modified RNAs often absent in bulk RNA-seq.
• Analysis of the human microbiome revealed taxon-specific RNAP pause motifs and concurrent transcription-cleavage of CRISPR guide RNAs and tRNAs.

Conclusions:

• PRO-seq is a versatile tool for studying nascent transcription across diverse bacterial populations.
• The method provides insights into the transcription of small and modified RNAs.
• PRO-seq elucidates transcriptional dynamics, including RNAP pausing and RNA processing, within complex microbial communities like the human microbiome.