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MS2-affinity purification coupled with RNA sequencing (MAPS) reveals S. aureus RsaI sRNA targetome

ABSTRACT: The virulon of Staphyloccocus aureus is controlled by intricate connections between transcriptional and post-transcriptional regulators including proteins and small non coding RNAs (sRNAs). Most of the sRNAs regulate gene expression through base-pairings with mRNAs. Here, we have applied and adapted the MS2-affinity purification approach coupled to RNA sequencing (MAPS) to determine the targetome of RsaI sRNA of S. aureus.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE122065 | GEO | 2018/11/02

REPOSITORIES: GEO

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MS2-affinity purification coupled with RNA sequencing (MAPS) reveals S. aureus RsaA sRNA targetome

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Project description:Despite the overwhelming information about sRNAs, one of the biggest challenges in the sRNA field is characterizing sRNA targetomes. Thus, we develop a novel method to identify RNAs that interact with a specific sRNA, regardless of the type of regulation (positive or negative) or targets (mRNA, tRNA, sRNA). This method is called MAPS: MS2 affinity purification coupled with RNA sequencing. As proof of principle, we identified RNAs bound to RybB, a well-characterized E. coli sRNA. Identification of RNAs co-purified with MS2-RybB in a rne131 ΔrybB strain. RybB (without MS2) was used as control

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Project description:The human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. Its length varies between different isolates due to the presence of repeated sequences at the 5’ end while its 3’ part is structurally independent and highly conserved. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, we identified sodA mRNA as a main target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. We demonstrated that in presence of RsaC, S. aureus cells were less resistant to oxidative stress, in relation with lower activity of SodA enzyme. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3’UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. By blocking the Mn-containing enzyme SodA synthesis, RsaC favors oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. Thus, RsaC may balance two interconnected defensive responses (i.e. against oxidative stress and manganese starvation) when S. aureus faces host immune cells.

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