| Yersinia pestis, the causative agent of bubonic and pneumonic plague, is primarily transmitted between fleas and mammals and is spread to humans through the bite of an infected flea or contact with affected animals. Small regulatory RNAs (sRNAs) are usually untranslated with lengths ranging from50to500nucleotides. sRNAs have been identified as regulators of gene expression, which mainly function at the post-transcriptional level through sRNAs-mRNA interactions. The RNA chaperone Hfq is required for trans-encoded sRNAs-mediated regulation. In recent years, sRNAs have been found to play key roles in cellular metabolism, host environmental adaptation, biofilm and pathogenesis in bacterial pathogens. RNA-seq (RNA sequencing) is a new research method which is based on high-throughput sequencing platforms which can be used to analyze the transcriptome of host and the pathogen simultaneously. RNA-seq can provide extensive information on transcription start sites (TSSs) and identify novel sRNAs.In this study, total RNAs of Yersinia pestis grown in vitro (in BHI and TMH broth separately) and in vivo (within lungs and spleens of infected mice) were extracted by Trizol reagent. The RNA fragments with the sizes of50～150and130～500nts were isolated, respectively. And then, RNA fragments were dephosphorylated and ligated with RNA adaptors to be reverse transcribed to single-stranded cDNAs. Finally, the nucleotide sequences were determined by Illumina/Solexa Genome Analyzer system.Totally, we found104sRNAs including26previously annotated sRNAs by searching against the Rfam database with78novel sRNAs candidates. Of78novel small RNAs,62sRNAs were located within the intergenic region and16on the antisense of annotated ORFs. Five novel sRNAs of Y. pestis were characterized that located on the Yersinia pestis specific plasmid pMTl and pPCPl.Transcription start sites (TSSs) of3novel sRNAs (sR034, sR035and sR084) were identified by primer extension assay that corresponded to the results observed by the deep sequencing. In addition, expression of14tested small RNAs was successfully validated and the transcript lengths detected by Northern Blot analysis corresponded to the lengths observed by the deep sequencing, suggesting data analysis of RNA-seq used in this study is feasible and it is accurate to find sRNAs expressed under two or more conditions. Moreover, Northern blot assay showed that9tested sRNAs were shown to be Hfq-dependent. Four sRNAs (CyaR, sR065, sR066and sR084) were identified as novel members of the transcription factor CRP regulon. |
