Biology II, University of Ulm, Ulm, Germany.
RNA Biol. 2012 Jul;9(7):1011-8. doi: 10.4161/rna.20826. Epub 2012 Jul 1.
To define the complete sRNA population of the halophilic archaeon Haloferax volcanii, we employed high throughput sequencing. cDNAs were generated from RNA ranging in size from 17 to 500 nucleotides isolated from cells grown at three different conditions to exponential and stationary phase, respectively. Altogether, 145 intergenic and 45 antisense sRNAs were identified. Comparison of the expression profile showed different numbers of reads at the six different conditions for the majority of sRNAs. A striking difference in the number of sRNA reads was observed between cells grown under standard vs. low salt conditions. Furthermore, the six highest numbers of reads were found for low salt conditions. In contrast, only slight differences between sRNA reads at different growth temperatures were detected. Attempts to delete four sRNA genes revealed that one sRNA gene is essential. The three viable sRNA gene deletion mutants possessed distinct phenotypes. According to microarray analyses, the removal of the sRNA gene resulted in a profound change of the transcriptome when compared with the wild type. High throughput sequencing also showed the presence of high concentrations of tRNA derived fragments in H. volcanii. These tRF molecules were shown to have different amounts of reads at the six conditions analyzed. Northern analysis was used to confirm the presence of the tRNA-derived fragments.
为了定义嗜盐古菌 Haloferax volcanii 的完整 sRNA 群体，我们采用了高通量测序。从分别在三种不同条件下生长至指数期和稳定期的细胞中分离出大小在 17 到 500 个核苷酸之间的 RNA，生成 cDNA。总共鉴定出 145 个基因间和 45 个反义 sRNA。对表达谱的比较显示，大多数 sRNA 在六种不同条件下的读取数不同。在标准盐度和低盐度条件下，sRNA 读取数的差异非常显著。此外，在低盐度条件下发现了六个最高的 sRNA 读取数。相比之下，在不同生长温度下，sRNA 读取数的差异很小。试图删除四个 sRNA 基因表明，一个 sRNA 基因是必需的。三个可存活的 sRNA 基因缺失突变体具有明显不同的表型。根据微阵列分析，与野生型相比，去除 sRNA 基因导致转录组发生了深刻的变化。高通量测序还显示了 H. volcanii 中存在高浓度的 tRNA 衍生片段。这些 tRF 分子在六种分析条件下的读取数不同。Northern 分析用于确认 tRNA 衍生片段的存在。
