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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 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).

ORGANISM(S): synthetic construct Salmonella enterica subsp. enterica serovar Typhimurium Legionella pneumophila Staphylococcus aureus Escherichia coli str. K-12 substr. MG1655 Pseudomonas aeruginosa PAO1 Pseudomonas aeruginosa PA7

PROVIDER: GSE200758 | GEO | 2022/04/16

REPOSITORIES: GEO

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Project description:In response to infection, viral genomes are processed by Dicer-like (DCL) ribonuclease proteins into viral small RNAs (vsRNAs) of discrete sizes. vsRNAs are then used as guides for silencing the viral genome. The profile of vsRNAs produced during the infection process has been extensively studied for some group of viruses. However, nothing is known for members of the economically important family Luteoviridae, a group of phloem-restricted viruses. Here, the population of vsRNAs from cotton plants infected with Cotton leafroll dwarf virus (CLRDV), a member of the genus Polerovirus, family Luteoviridae, is characterized.Deep sequencing of small RNAs (sRNAs) from CLRDV-infected cotton leaves was performed. Results showed 21-nt to 24-nt long vsRNAs matching all the viral genome, with a higher frequency of matches in the 3M-CM-"M-BM-^@M-BM-^Y region. Equivalent amounts of sense and antisense vsRNAs were found, and the 22-nt long small RNA class was the most prominent one. Looking for cotton Dcl transcripts levels during infection, we could observe that Dcl4 seems to be up-regulated, while Dcl2 seems to be down-regulated.This is the first report on the profile of sRNAs coming from a plant infected with a member of the family Luteoviridae. Our sequence data strongly suggest that virus-derived double-stranded RNA functions as one of the main precursors of vsRNAsOur results indicate that secondary structures of the viral RNAs are not the main source of the viRNAs observed, as suggested for other viruses. Judging by the profiled size classes, all cotton DCLs might be working to silence the virus. The possible causes for the unexpected high accumulation of 22-nt viRNAvsRNAs are discussed. CLRDV is the causal agent of worldwide cotton pathology named Cotton blue disease. Our results are an important contribution for understanding the molecular mechanisms involved in this and related diseases. Total RNA obtained from leaves of the cotton plant 5 days post-infection with Cotton leafrol dawrf virus (CLRDV) compared to not-infected control.

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

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