Abstract
RNA sequencing (RNA-seq) is the gold standard for the discovery of small non-coding RNAs. Following a long-standing approach, reads shorter than 16 nucleotides (nt) are removed from the small RNA sequencing libraries or datasets. The serendipitous discovery of an eukaryotic 12 nt-long RNA species capable of modulating the microRNA from which they derive prompted us to challenge this dogma and, by expanding the window of RNA sizes down to 8 nt, to confirm the existence of functional very small RNAs (vsRNAs <16 nt). Here we report the detailed profiling of vsRNAs in Escherichia coli, E. coli-derived outer membrane vesicles (OMVs) and five other bacterial strains (Pseudomonas aeruginosa PA7, P. aeruginosa PAO1, Salmonella enterica serovar Typhimurium 14028S, Legionella pneumophila JR32 Philadelphia-1 and Staphylococcus aureus HG001). vsRNAs of 8-15 nt in length [RNAs (8-15 nt)] were found to be more abundant than RNAs of 16-30 nt in length [RNAs (16-30 nt)]. vsRNA biotypes were distinct and varied within and across bacterial species and accounted for one third of reads identified in the 8-30 nt window. The tRNA-derived fragments (tRFs) have appeared as a major biotype among the vsRNAs, notably Ile-tRF and Ala-tRF, and were selectively loaded in OMVs. tRF-derived vsRNAs appear to be thermodynamically stable with at least 2 G-C basepairs and stem-loop structure. The analyzed tRF-derived vsRNAs are predicted to target several human host mRNAs with diverse functions. Bacterial vsRNAs and OMV-derived vsRNAs could be novel players likely modulating the intricate relationship between pathogens and their hosts.