RNA sequencing unveils very small RNAs with potential regulatory functions in bacteria

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 a 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 <16nt). Here we report the profiling of vsRNAs in Escherichia coli (with different experimental conditions), E. coli-derived outer membrane vesicles (OMVs) and five other bacterial strains (Pseudomonas aeruginosa PA7, P. aeruginosa PAO1, Salmonella Typhimurium 14028S, Legionella pneumophila JR32 Philadelphia-1 and Staphylococcus aureus HG001). We analyzed by small RNA-seq the 8- to 30-nt window of RNA length of the gram-negative E. coli MG1655 model organism and 5 other bacterial strains (P. aeruginosa PA7, P. aeruginosa PAO1, S. Typhimurium 14028S, L. pneumophila JR32 Philadelphia-1 and S. aureus HG001).

RNA测序（RNA-seq）是发现小分子非编码RNA（small non-coding RNAs）的金标准。长期以来，研究人员普遍将长度短于16个核苷酸（nt）的读段从小RNA测序文库或数据集中移除。我们偶然发现了一种长度为12 nt的RNA物种，其能够调控其自身来源的微小RNA（microRNA），这一发现促使我们对这一学界既定惯例提出挑战；通过将RNA长度筛选窗口拓宽至8 nt，我们最终证实了功能性超小RNA（very small RNAs, vsRNAs <16 nt）的存在。本研究对不同实验条件下的大肠杆菌（Escherichia coli）、大肠杆菌来源的外膜囊泡（outer membrane vesicles, OMVs）以及另外5种细菌菌株——铜绿假单胞菌（Pseudomonas aeruginosa）PA7、铜绿假单胞菌PAO1、鼠伤寒沙门氏菌（Salmonella Typhimurium）14028S、嗜肺军团菌（Legionella pneumophila）JR32 Philadelphia-1以及金黄色葡萄球菌（Staphylococcus aureus）HG001——中的超小RNA进行了谱分析。我们通过小RNA-seq对革兰氏阴性模式生物大肠杆菌MG1655，以及上述5种细菌菌株的8~30 nt长度区间的RNA进行了分析。