Department of Biological Sciences, Ohio University, Athens, Ohio, USA.
Honors Tutorial College, Ohio University, Athens, Ohio, USA.
mSphere. 2020 Jul 29;5(4):e00439-20. doi: 10.1128/mSphere.00439-20.
Regulatory small RNAs (sRNAs) are known to play important roles in the Gram-positive bacterial pathogen ; however, their existence is often overlooked, primarily because sRNA genes are absent from genome annotation files. Consequently, transcriptome sequencing (RNA-Seq)-based experimental approaches, performed using standard genome annotation files as a reference, have likely overlooked data for sRNAs. Previously, we created an updated genome annotation file, which included annotations for 303 known sRNAs in USA300. Here, we utilized this updated reference file to reexamine publicly available RNA-Seq data sets in an attempt to recover lost information on sRNA expression, stability, and potential to encode peptides. First, we used transcriptomic data from 22 studies to identify how the expression of 303 sRNAs changed under 64 different experimental conditions. Next, we used RNA-Seq data from an RNA stability assay to identify highly stable/unstable sRNAs. We went on to reanalyze a ribosome profiling (Ribo-seq) data set to identify sRNAs that have the potential to encode peptides and to experimentally confirm the presence of three of these peptides in the USA300 background. Interestingly, one of these sRNAs/peptides, encoded at the locus, influences the ability of cells to autoaggregate. Finally, we reexamined two recently published RNA-Seq data sets, from the cystic fibrosis (CF) lung and a murine vaginal colonization study, and identified 29 sRNAs that may play a role Collectively, these results can help inform future studies of these important regulatory elements in and highlight the need for ongoing curating and updating of genome annotation files. Regulatory small RNAs (sRNAs) are a class of RNA molecules that are produced in bacterial cells but that typically do not encode proteins. Instead, they perform a variety of critical functions within the cell as RNA. Most bacterial genomes do not include annotations for sRNA genes, and any type of analysis that is performed using a bacterial genome as a reference will therefore overlook data for sRNAs. In this study, we reexamined hundreds of previously generated RNA-Seq data sets and reanalyzed them to generate data for sRNAs. To do so, we utilized an updated genome annotation file, previously generated by our group, which contains annotations for 303 sRNAs. The data generated (which were previously discarded) shed new light on sRNAs in , most of which are unstudied, and highlight certain sRNAs that are likely to play important roles in the cell.
调控性小 RNA（sRNA）在革兰氏阳性细菌病原体中发挥着重要作用；然而，它们的存在往往被忽视，主要是因为 sRNA 基因在基因组注释文件中缺失。因此，基于转录组测序（RNA-Seq）的实验方法，使用标准基因组注释文件作为参考，可能会忽略 sRNA 的数据。之前，我们创建了一个更新的基因组注释文件，其中包含了 USA300 中 303 个已知 sRNA 的注释。在这里，我们利用这个更新的参考文件重新检查了公开可用的 RNA-Seq 数据集，试图恢复关于 sRNA 表达、稳定性和编码肽潜力的丢失信息。首先，我们使用来自 22 项研究的转录组数据来确定 303 个 sRNA 的表达在 64 种不同实验条件下如何变化。接下来，我们使用 RNA 稳定性测定的 RNA-Seq 数据来识别高度稳定/不稳定的 sRNA。我们继续重新分析核糖体谱（Ribo-seq）数据集，以识别具有编码肽潜力的 sRNA，并通过实验确认这些肽在 USA300 背景下的存在。有趣的是，这些 sRNA/肽中的一个，编码在 基因座，影响细胞自动聚集的能力。最后，我们重新检查了两个最近发表的 USA300 RNA-Seq 数据集，来自囊性纤维化（CF）肺和小鼠阴道定植研究，并确定了 29 个可能在 USA300 中发挥作用的 sRNA。总的来说，这些结果可以帮助为未来在 USA300 中研究这些重要调控元件提供信息，并强调需要不断对基因组注释文件进行整理和更新。调控性小 RNA（sRNA）是一类在细菌细胞中产生的 RNA 分子，但通常不编码蛋白质。相反，它们作为 RNA 在细胞中发挥各种关键功能。大多数细菌基因组不包括 sRNA 基因的注释，因此任何使用细菌基因组作为参考的分析都将忽略 sRNA 的数据。在这项研究中，我们重新检查了数百个先前生成的 USA300 RNA-Seq 数据集，并对其进行重新分析以生成 sRNA 数据。为此，我们利用了我们小组之前生成的一个更新的 USA300 基因组注释文件，其中包含 303 个 sRNA 的注释。生成的数据（之前被丢弃）为 USA300 中的 sRNA 提供了新的认识，其中大多数 sRNA 尚未得到研究，并且突出了某些可能在细胞中发挥重要作用的 sRNA。
