Tombusvirus p19 Captures RNase III-Cleaved Double-Stranded RNAs Formed by Overlapping Sense and Antisense Transcripts...

Area of Science:

• Bacterial molecular biology
• RNA biology
• Gene regulation

Background:

• Antisense transcription is common in bacteria, producing antisense RNAs (asRNA).
• AsRNAs form double-stranded RNAs (dsRNA) with sense RNAs, which are processed by RNase III.
• Identifying unstable asRNAs and their processing sites is challenging.

Purpose of the Study:

• To develop a method for characterizing unstable bacterial antisense RNAs (asRNA).
• To investigate the prevalence and regulation of asRNA by RNase III.
• To analyze sequence preferences of RNase III in dsRNA processing.

Main Methods:

• Ectopic expression of plant Tombusvirus p19 protein in Escherichia coli to stabilize dsRNA intermediates.
• Deep sequencing of p19-captured dsRNA to identify asRNA.
• Analysis of RNase III cleavage sites and sequence motifs in dsRNA.

Main Results:

• Widespread production of RNase III-generated small dsRNAs from bacterial genes and plasmids was detected.
• RNase III regulates asRNA and sense RNA decay at toxin-antitoxin loci and the rsd gene.
• RNase III exhibits sequence preferences for AU-rich regions near cleavage sites and GC-rich overhangs, with unexpected GC enrichment in dsRNA centers.

Conclusions:

• Ectopic expression of p19 is an effective method for identifying bacterial asRNA and RNase III cleavage sites.
• The study provides insights into the widespread nature of asRNA and its regulation by RNase III in bacteria.
• Potential RNase III-dependent regulation of CspD protein by asRNA was suggested.