Small RNA Discovery Using Conservation Analysis Enhancement

Small noncoding RNAs (sRNAs) have been described as posttranscriptional regulators of many cellular systems in bacteria, notably environmental stress responses. However, sRNAs are small and difficult to detect due to the lack of uniformity in which they are encoded in the genome. Since they may be found inn the 5' and 3' untranslated regions of genes and in intergenic regions, identification and characterization of sRNAs has been challenging. These elusive gene expression regulators may aid in explaining the interesting survival mechanisms of Deinococcus radiodurans under ionizing radation of > 12,000 Gy, while most bacteria find 1,000 Gy of radiation to be lethal. This study takes advantage of high-throughput experimental methods and computational prediction methods to develop a pipeline for more efficient sRNA discovery. Intergenic regions in 13 well-annotated bacterial species were analyzed for conservation levels to increase the likelihood that candidate intergenic regions would code for sRNAs. In total, more than 900 validated bacterial sRNAs and 23,000 intergenic regions were analyzed. The results indicated that sRNAs are found more abundantly in longer and highly conserved intergenic regions. This workflow was used to discover novel sRNA in D. radiodurans. 199 potential sRNA candidates were generated from whole-transcriptome deep sequencing analysis, and 41 were confirmed using northern blotting and reverse transcription PCR. Analysis of sRNA expression levels during recovery after actute radiation resulted in 8 confirmed sRNAs which demonstrated varying expression levels due to irradiation. Conservation comparison of novel D. radiodurans sRNAs led to the discovery and confirmation of 7 homologous sRNAs in the closely-related species Deinococcus geothermalis.