Bacterial Regulatory RNA - Methods and Protocols
Bacterial Regulatory RNA - Methods and Protocols
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细菌调节 RNA - 方法和实验方案
DOI：
10.1007/978-1-0716-3565-0_17
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Esteban-Serna S
中科院分区：
文献类型：
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作者：
Esteban-Serna S
耐甲氧西林金黄色葡萄球菌(MRSA)是一种致病菌，在感染患者中死亡率很高。小RNA转录调控(SRNAs)已被证明可以调节促进抗生素耐药性和毒力INS的网络。金星。然而，大多数sRNA在MRSA宿主感染过程中的生物学作用仍不清楚。为了填补这一空白，我们与Jai Tree的实验室合作，在模拟宿主环境的条件下，使用Clash在MRSA中进行了全面的RNA-RNA相互作用组分析。在这里，我们介绍了我们最近开发的这种优化的CLASH(杂交的交联、连接和测序)协议的详细版本，该协议是为探索RNA相互作用组INS而量身定做的。金黄色葡萄球菌以及其他革兰氏阳性细菌。此外，我们还介绍了一组有用的Python函数，用于分析假定的RNA-RNA相互作用的折叠能量，并简化了碰撞数据中的sRNA和mRNA种子发现。在随附的计算演示中，我们的目标是建立一种标准化的策略，以评估观察到的嵌合体产生于真实的RNA-RNA相互作用的可能性。
Methicillin-resistantStaphylococcus aureus(MRSA) is a bacterial pathogen accounting for high mortality rates among infected patients. Transcriptomic regulation by small RNAs (sRNAs) has been shown to regulate networks promoting antibiotic resistance and virulence inS. aureus. Yet, the biological role of most sRNAs during MRSA host infection remains unknown. To fill this gap, in collaboration with the lab of Jai Tree, we performed comprehensive RNA-RNA interactome analyses in MRSA using CLASH under conditions that mimic the host environment. Here we present a detailed version of this optimized CLASH (cross-linking, ligation, and sequencing of hybrids) protocol we recently developed, which has been tailored to explore the RNA interactome inS. aureusas well as other Gram-positive bacteria. Alongside, we introduce a compilation of helpful Python functions for analyzing folding energies of putative RNA-RNA interactions and streamlining sRNA and mRNA seed discovery in CLASH data. In the accompanying computational demonstration, we aim to establish a standardized strategy to evaluate the likelihood that observed chimeras arise from true RNA-RNA interactions.