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

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.

SUBMITTER: Diallo I

PROVIDER: S-EPMC9203972 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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

Diallo Idrissa I Ho Jeffrey J Lalaouna David D Massé Eric E Provost Patrick P

Frontiers in molecular biosciences 20220603

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 RN ...[more]

PMID: 35720117

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Project description:BackgroundNext-generation transcriptome sequencing (RNA-Seq) has become the standard practice for studying gene splicing, mutations and changes in gene expression to obtain valuable, accurate biological conclusions. However, obtaining good sequencing coverage and depth to study these is impeded by the difficulties of obtaining high quality total RNA with minimal genomic DNA contamination. With this in mind, we evaluated the performance of Phenol-free total RNA purification kit (Amresco) in comparison with TRI Reagent (MRC) and RNeasy Mini (Qiagen) for the extraction of total RNA of Pseudomonas aeruginosa which was grown in glucose-supplemented (control) and polyethylene-supplemented (growth-limiting condition) minimal medium. All three extraction methods were coupled with an in-house DNase I treatment before the yield, integrity and size distribution of the purified RNA were assessed. RNA samples extracted with the best extraction kit were then sequenced using the Illumina HiSeq 2000 platform.ResultsTRI Reagent gave the lowest yield enriched with small RNAs (sRNAs), while RNeasy gave moderate yield of good quality RNA with trace amounts of sRNAs. The Phenol-free kit, on the other hand, gave the highest yield and the best quality RNA (RIN value of 9.85 ± 0.3) with good amounts of sRNAs. Subsequent bioinformatic analysis of the sequencing data revealed that 5435 coding genes, 452 sRNAs and 7 potential novel intergenic sRNAs were detected, indicating excellent sequencing coverage across RNA size ranges. In addition, detection of low abundance transcripts and consistency of their expression profiles across replicates from the same conditions demonstrated the reproducibility of the RNA extraction technique.ConclusionsAmresco's Phenol-free Total RNA purification kit coupled with DNase I treatment yielded the highest quality RNAs containing good ratios of high and low molecular weight transcripts with minimal genomic DNA. These RNA extracts gave excellent non-biased sequencing coverage useful for comprehensive total transcriptome sequencing and analysis. Furthermore, our findings would be useful for those interested in studying both coding and non-coding RNAs from precious bacterial samples cultivated in growth-limiting condition, in a single sequencing run.

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

Publications

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

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