Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA.
Department of Microbiology, Harvard Medical School, Boston, Massachusetts, USA
mBio. 2021 Feb 23;12(1):e03608-20. doi: 10.1128/mBio.03608-20.
A number of computational or experimental tools have been developed to identify targets of small RNA (sRNA) regulation. Here, we modified one of these methods, based on proximity ligation of sRNAs bound to their targets, referred to as rGRIL-seq, that can be used to capture sRNA regulators of a gene of interest. Intracellular expression of bacteriophage T4 RNA ligase leads to a covalent linking of sRNAs base-paired with mRNAs, and the chimeras are captured using oligonucleotides complementary to the mRNA, followed by sequencing. This allows the identification of known as well as novel sRNAs. We applied rGRIL-seq toward finding sRNA regulators of expression of the stress response sigma factor RpoS in , , and In , we confirmed the regulatory role of known sRNAs and discovered a new negative regulator, asYbiE. When applied to and , we identified two novel sRNAs (s03661 and s0223) in and two known sRNAs (TfoR and Vcr043) in as direct regulators of The use of rGRIL-seq for defining multiple posttranscriptional regulatory inputs into individual mRNAs represents a step toward a more comprehensive understanding of the workings of bacterial regulatory networks. With the recognition of the importance of posttranscriptional regulation mediated by bacterial small RNAs (sRNAs), their contribution to global gene expression regulatory networks needs to be addressed in a truly comprehensive manner. While a single sRNA targets multiple RNAs, an mRNA can be regulated by multiple sRNAs that can be either transcribed individually or derived by processing of mRNAs. In this paper, we developed a tool (referred to as rGRIL-seq) to identify sRNAs that regulate mRNAs regardless of their origin. We demonstrated the utility of this approach by identifying positive and negative sRNA regulators of the mRNA in three bacterial species. We not only described known sRNAs of or that control but also identified several new regulators in Therefore, rGRIL-seq can be used to identify species-specific sRNAs targeting a conserved mRNA, and they likely play an important role in bacterial adaptation to specific environmental niches.
已经开发出许多计算或实验工具来识别小 RNA（sRNA）调节的靶标。在这里，我们修改了其中一种方法，该方法基于与靶标结合的 sRNA 的接近连接，称为 rGRIL-seq，可用于捕获感兴趣基因的 sRNA 调节剂。噬菌体 T4 RNA 连接酶的细胞内表达导致 sRNA 与 mRNA 碱基配对的共价连接，并且使用与 mRNA 互补的寡核苷酸捕获嵌合体，然后进行测序。这允许鉴定已知和新的 sRNA。我们将 rGRIL-seq 应用于寻找应激反应σ因子 RpoS 在 、 和 中的表达的 sRNA 调节剂，在 中，我们证实了已知 sRNA 的调节作用，并发现了一个新的负调节因子 asYbiE。当应用于 和 时，我们在 中鉴定了两个新的 sRNA（s03661 和 s0223），在 中鉴定了两个已知的 sRNA（TfoR 和 Vcr043），它们是 的直接调节剂。使用 rGRIL-seq 定义单个 mRNA 的多个转录后调节输入代表了朝着更全面理解细菌调节网络的工作方式迈出的一步。随着对细菌小 RNA（sRNA）介导的转录后调节的重要性的认识，需要以真正全面的方式解决它们对全局基因表达调节网络的贡献。虽然单个 sRNA 靶向多个 RNA，但 mRNA 可以被多个 sRNA 调节，这些 sRNA 可以单独转录或由 mRNA 加工衍生。在本文中，我们开发了一种工具（称为 rGRIL-seq）来识别无论其来源如何调节 mRNA 的 sRNA。我们通过在三种细菌物种中鉴定 mRNA 的正和负 sRNA 调节剂证明了这种方法的实用性。我们不仅描述了控制 的 或 的已知 sRNA，而且还在 中鉴定了几个新的 调节剂。因此，rGRIL-seq 可用于鉴定靶向保守 mRNA 的物种特异性 sRNA，它们可能在细菌适应特定环境小生境中发挥重要作用。
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