Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA–RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA–RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA–mRNA interactions are recovered and we find that an mRNA encoding a long 3′ untranslated region (UTR) (termed vigR 3′UTR) functions as a regulatory ‘hub’ within the RNA–RNA interaction network. We demonstrate that the vigR 3′UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA–mRNA base-pairing. Deletion of the vigR 3′UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3′UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.

耐甲氧西林金黄色葡萄球菌的治疗感染取决于万古霉素等一线抗生素的疗效。治疗失败通常与具有中间万古霉素耐药性（称为 VISA）的分离株有关。这些分离株积累了点突变，通常通过加厚细胞壁共同降低万古霉素敏感性。调节性小 RNA 表达的变化与 VISA 分离株中的抗生素应激相关，但大多数 RNA 调节剂的功能尚不清楚。在这里，我们使用 CLASH 捕获与 RNase III 相关的 RNA-RNA 相互作用。RNase III-CLASH 揭示了数百种新的体内 RNA-RNA 相互作用，首次允许对许多 sRNA 进行功能表征。令人惊讶的是，许多 mRNA-mRNA 相互作用被恢复，我们发现编码长 3' 非翻译区 (UTR)（称为vigR 3'UTR) 在 RNA-RNA 相互作用网络中起调节“中枢”的作用。我们证明vigR 3'UTR通过直接的 mRNA-mRNA 碱基配对促进folD和细胞壁裂解转糖基酶 isaA的表达。vigR 3'UTR的缺失使 VISA 对糖肽处理重新敏感，并且isaA和vigR 3'UTR 缺失都会影响细胞壁厚度。我们的结果证明了 RNase III-CLASH 的效用，并表明金黄色葡萄球菌使用 mRNA-mRNA 相互作用来协调基因表达，比以前认为的更广泛。