![]() Original images for the experiments shown in b, c are provided in the Source data. One percent of the total lysate was loaded for comparison. c Western blot results and Coomassie brilliant blue staining of the USA300 RNase III-HTF PTex experiments, performed with the indicated UV intensities (J/cm 2) in LPM medium. TSB tryptic soy broth, LPM low phosphate medium pH 7.5. Non-cross-linked cells were used as negative controls. The black asterisk indicates the Benzonase used to degrade the RNA. ‘Input’ indicates µg of total lysate loaded on the gel. RNase III-HTF (black triangle) was detected using anti-FLAG antibodies. The UV irradiation doses used (J/cm 2) are indicated. b Western blot and silver staining analysis of the USA300 RNase III-HTF 2C experiments. CL cross-linked, 2C complex capture, PTex phenol–toluol extraction, aq aqueous, inter interphase, org organic. See ‘Methods’ section for a detailed description of the approaches. aureus 2C and PTex RBPome capture workflows. Our RBPome protocols are widely applicable, and our work implies that the role of HTH domain proteins in regulating gene expression has been considerably underestimated.Ī S. We biochemically verified our findings in vitro as well as in vivo using ultraviolet (UV) cross-linking and cDNA analyses experiments (CRAC 14). aureus strains to obtain a global overview of S. Therefore, in this work, we applied silica- (complex capture (2C) 12) and organic extraction-based (phenol–toluol extraction (PTex) 13) approaches to two clinically relevant S. aureus also expresses many proteins that are not conserved in E. Thus far, RBPome studies uncovered RBPs for Gram-negative bacteria, many of which do not have conserved homologues in Gram-positive bacteria, such as S. As metabolic pathways are frequently targeted for drug development, the RNA-binding enzymes may be promising candidates. ![]() These may represent a “shortcut” that directly links the response to nutrient availability with post-transcriptional regulation of gene expression. This included many metabolic enzymes that moonlight as RBPs. ![]() Collectively, these studies revealed a surprisingly large number of proteins that previously did not have any known links to RNA metabolism, nor did they contain recognisable RNA-binding domains (RBDs). Recent in vivo high-throughput proteomic studies have identified hundreds of RBPs (RNA-binding proteome (RBPome)) in diverse organisms 6, 7, 8, 9, 10, 11. By directly binding to mRNAs, RBPs can shape gene expression profiles by modulating mRNA translation and/or degradation rates.Īlthough RBPs clearly play a key role in regulating adaptive responses, relatively few RBPs have been identified and characterised in S. Transcription factors were thought to be mainly responsible for directing this process by controlling gene expression at the DNA level however, it has become evident that a substantial amount of regulation occurs post-transcriptionally: RNA-binding proteins (RBPs), are now recognised as key players in controlling adaptive responses in pathogenic bacteria 4, 5. aureus remodels its transcriptome within minutes of stress imposition 3. The latter condition can promote resistance to antimicrobials and the host immune system, leading to chronic infections 2. Its capacity to very rapidly adapt to stress caused by environmental insults, such as alteration in host temperature because of an inflammatory response, enables it not only to survive in hostile conditions but also to persist within the host 1. aureus is such an effective pathogen because it expresses numerous virulence factors that facilitate its ability to colonise, evade immune defenses and transmit to other hosts 1. The rapid spread of highly virulent and methicillin-resistant Staphylococcus aureus strains (MRSA) is causing major health-care problems worldwide and is becoming increasingly difficult to treat 1.
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