The Discovery And Validation Of Yersinia Pestis Non-coding RNA Under Different Pressure Conditions

BackgroundSmall non-coding RNAs (sRNAs) carry out a variety of biological functions that enable cells to adjust its physiology to environmental changes. Yersinia pestis is the etiological agent of the fatal disease, plague. Several studies have been done to investigate the regulatory proteins in Y. pestis. However, few are carried out to study sRNAs in Y. pestis, especially under different environmental modulations. Most of the earlier researches on discovering new kinds sRNAs are based on bioinformatics. Nervertheless, many sRNAs do not possess the typical features of putative promoters and terminators that could be employed to predict genes. It suggested that computational approaches for sRNA identification that rely on primary sequence conservation and on promoter and Rho-independent terminator detection are likely effective in identifying only a small subset of non-coding transcrips.MethodsOur research is intent to obtain novel and specific sRNAs in Y.pesitis. To study the effect of Y.pesitis under different enviormental stresses, the organism was grown under either suitable conditions or stress conditions. Then, we constructed cDNA library by combining RACE (rapid amplification of cDNA ends) technique and advanced RNA size selecting protocol. Thus, our strategy is inclined to gain authentic sRNA species with full length and rich abundance.ResultsBy sequencing the clones, 2540 reads are obtained and 81.73% of them are candidate sRNAs. tRNA and rRNA are only in a rate of 2.32% and 5.16%, respectively. By blasting against the genome sequence, 52 RNA species including 5 known sRNAs are obtained. Noticeably, a total of 21 antisense sRNAs were detected by cDNA sequencing, of which two sRNAs (0941 and 1555) were found to be complementary to a well-known sRNA, 6S RNA and tRNA-ser1, respectively. Twenty-four sRNAs were encoded on the intergenic region (IGR) or partially overlapping with the flanking ORF of the Y. pestis genome. Several RNA transcripts were detected by Northern Blotting using the corresponding RNA probes. The size of the RNA molecule detected was roughly in agreement with the results shown in the colony sequencing.We examined the expression patterns of sRNAs which are longer than 70 nucleotides in length under five stressful conditions by quantitative PCR. Almost all the tested sRNAs were transcribed under five different conditions. Twenty-one sRNAs were highly expressed during the stationary growth phase and the other four exhibited some degree of ion dependence.ConclusionThe strategy of cDNA library construction is feasible. This method is intent to gain full length sRNA with high abundant, and most of them are authentic. Our study is essential to the further research of sRNAs in Yersinia pestis.