Toward a Comprehensive Analysis of Posttranscriptional Regulatory Networks: a New Tool for the Identification of Small RNA Regulators of Specific mRNAs.
Toward a Comprehensive Analysis of Posttranscriptional Regulatory Networks: a New Tool for the Identification of Small RNA Regulators of Specific mRNAs.
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全面分析转录后调控网络：识别特定 mRNA 的小 RNA 调控因子的新工具。
DOI：
10.1128/mbio.03608-20
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发表时间：
2021-02-23
期刊：
影响因子：
6.4
通讯作者：
Lory S
中科院分区：
文献类型：
--
作者：
Han K;Lory S
已经开发了许多计算或实验工具来鉴定小RNA（sRNA）调节的靶点。在这里，我们修改了这些方法之一，基于sRNA结合到其靶点的体内邻近连接，称为rGRIL-seq，可用于捕获感兴趣基因的sRNA调节因子。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，然后测序。这允许鉴定已知的以及新的sRNA。我们应用rGRIL-seq寻找大肠杆菌、铜绿假单胞菌和霍乱弧菌中应激反应σ因子RpoS表达的sRNA调节因子。在大肠在大肠杆菌中，我们证实了已知的sRNA的调节作用，并发现了一个新的负调控因子asYbiE。当应用于铜绿假单胞菌和霍乱弧菌时，我们在铜绿假单胞菌中鉴定了两种新的sRNA（s 03661和s 0223），在霍乱弧菌中鉴定了两种已知的sRNA（TfoR和Vcr 043）作为rpoS的直接调节剂。使用rGRIL-seq将多个转录后调控输入定义为单个mRNA，这代表了更全面地理解细菌调控网络工作的一步。
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 in vivo 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 Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae. In E. coli, we confirmed the regulatory role of known sRNAs and discovered a new negative regulator, asYbiE. When applied to P. aeruginosa and V. cholerae, we identified two novel sRNAs (s03661 and s0223) in P. aeruginosa and two known sRNAs (TfoR and Vcr043) in V. cholerae as direct regulators of rpoS. 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.