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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.

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Background Newborn screening (NBS) is an effective public health intervention that reduces death and disability from treatable genetic diseases, but many conditions are not screened due to a lack of a suitable assay. Whole genome and whole exome sequencing can potentially expand NBS but there remain many technical challenges preventing their use in population NBS. We investigated if targeted gene sequencing (TGS) is a feasible methodology for expanding NBS. Methods We constructed a TGS panel of 164 genes which screens for a broad range of inherited conditions. We designed a high-volume, low-turnaround laboratory and bioinformatics workflow that avoids the technical and data interpretation challenges associated with whole genome and whole exome sequencing. A methods-based analytical validation of the assay was completed and test performance in 2552 newborns examined. We calculated annual birth estimates for each condition to assess cost-effectiveness. Results Assay analytical sensitivity was >99% and specificity was 100%. Of the newborns screened, 1.3% tested positive for a condition. On average, each individual had 225 variants to interpret and 1.8% were variants of uncertain significance (VUS). The turnaround time was 7 to 10 days. Maximum batch size was 1536 samples. Conclusions We demonstrate that a TGS assay could be incorporated into an NBS program soon to increase the number of conditions screened. Additionally, we conclude that NBS using TGS may be cost-effective.