RNA Sequencing Unveils Very Small RNAs With Potential Regulatory Functions in Bacteria.

Area of Science:

• Microbiology
• Molecular Biology
• Genomics

Background:

• Small non-coding RNAs are crucial regulators, but reads shorter than 16 nucleotides are typically discarded in RNA sequencing.
• A recent discovery of a 12 nt eukaryotic RNA modulating microRNA function prompted re-evaluation of short RNA exclusion criteria.

Purpose of the Study:

• To challenge the dogma of excluding very small RNAs (<16 nt) in RNA sequencing.
• To profile very small RNAs (vsRNAs) in various bacterial species and their derived outer membrane vesicles (OMVs).
• To investigate the abundance, biotypes, stability, and potential functions of bacterial vsRNAs.

Main Methods:

• Expanded RNA sequencing analysis to include reads as short as 8 nt.
• Conducted detailed profiling of vsRNAs in *Escherichia coli*, *E. coli*-derived OMVs, and five other bacterial strains.
• Analyzed vsRNA biotypes, abundance, thermodynamic stability, and predicted mRNA targets.

Main Results:

• Very small RNAs (8-15 nt) were found to be more abundant than larger RNAs (16-30 nt) across analyzed bacterial species.
• vsRNA biotypes were distinct, varied across species, and constituted one-third of reads in the 8-30 nt window.
• tRNA-derived fragments (tRFs), specifically Ile-tRF and Ala-tRF, were major vsRNA biotypes and were selectively loaded into OMVs.

Conclusions:

• Functional very small RNAs (<16 nt) exist in bacteria and are more abundant than previously recognized.
• tRF-derived vsRNAs are stable, potentially target host mRNAs, and may play roles in host-pathogen interactions.
• Bacterial vsRNAs and OMV-derived vsRNAs represent novel factors in modulating host-pathogen relationships.