Probing mRNA Structure and sRNA–mRNA Interactions in Bacteria Using Enzymes and Lead(II)

Chevalier, Clément; Geissmann, Thomas; Helfer, Anne‐Catherine; Romby, Pascale

doi:10.1007/978-1-59745-558-9_16

Cited by 24 publications

(19 citation statements)

References 43 publications

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“…Structure probing of the rnc mRNA was performed using the single-strand-specific RNases T2 and T1, and the double-strand-specific RNase V1 (Figure S2B and S2C). Enzymatic reactions were restricted to less than one cut per molecule, and cleavages were mapped by reverse transcription [35]. The structure probing supported the formation of three long hairpins in the CDS, as indicated by numerous RNase V1 cleavages located in the arms and strong RNase T2/T1 cuts occurring in the apical loops (I, II, and III) and the internal loop regions (Figure S2C).…”

Section: Resultsmentioning

confidence: 80%

Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression

Sharma²,

et al. 2012

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RNA turnover plays an important role in both virulence and adaptation to stress in the Gram-positive human pathogen Staphylococcus aureus. However, the molecular players and mechanisms involved in these processes are poorly understood. Here, we explored the functions of S. aureus endoribonuclease III (RNase III), a member of the ubiquitous family of double-strand-specific endoribonucleases. To define genomic transcripts that are bound and processed by RNase III, we performed deep sequencing on cDNA libraries generated from RNAs that were co-immunoprecipitated with wild-type RNase III or two different cleavage-defective mutant variants in vivo. Several newly identified RNase III targets were validated by independent experimental methods. We identified various classes of structured RNAs as RNase III substrates and demonstrated that this enzyme is involved in the maturation of rRNAs and tRNAs, regulates the turnover of mRNAs and non-coding RNAs, and autoregulates its synthesis by cleaving within the coding region of its own mRNA. Moreover, we identified a positive effect of RNase III on protein synthesis based on novel mechanisms. RNase III–mediated cleavage in the 5′ untranslated region (5′UTR) enhanced the stability and translation of cspA mRNA, which encodes the major cold-shock protein. Furthermore, RNase III cleaved overlapping 5′UTRs of divergently transcribed genes to generate leaderless mRNAs, which constitutes a novel way to co-regulate neighboring genes. In agreement with recent findings, low abundance antisense RNAs covering 44% of the annotated genes were captured by co-immunoprecipitation with RNase III mutant proteins. Thus, in addition to gene regulation, RNase III is associated with RNA quality control of pervasive transcription. Overall, this study illustrates the complexity of post-transcriptional regulation mediated by RNase III.

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Section: Resultsmentioning

confidence: 80%

Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression

Sharma²,

et al. 2012

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“…RNAIII carries hairpin loops with a C-rich motif that binds the G-rich sequence of the SD region of target mRNA and inhibits its translation [22], [30]. RNAIII forms an imperfect duplex with target mRNA, which is digested by RNase III, and decreases the stability of target mRNA [20], [31]. RNAIII is stable with a half-life of over 20 min [20], [30].…”

Section: Resultsmentioning

confidence: 99%

Mobile Genetic Element SCCmec-encoded psm-mec RNA Suppresses Translation of agrA and Attenuates MRSA Virulence

et al. 2013

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Community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) is a socially problematic pathogen that infects healthy individuals, causing severe disease. CA-MRSA is more virulent than hospital associated-MRSA (HA-MRSA). The underlying mechanism for the high virulence of CA-MRSA is not known. The transcription product of the psm-mec gene, located in the mobile genetic element SCCmec of HA-MRSA, but not CA-MRSA, suppresses the expression of phenol-soluble modulin α (PSMα), a cytolytic toxin of S. aureus. Here we report that psm-mec RNA inhibits translation of the agrA gene encoding a positive transcription factor for the PSMα gene via specific binding to agrA mRNA. Furthermore, 25% of 325 clinical MRSA isolates had a mutation in the psm-mec promoter that attenuated transcription, and 9% of the strains had no psm-mec. In most of these psm-mec-mutated or psm-mec-deleted HA-MRSAs, PSMα expression was increased compared with strains carrying intact psm-mec, and some mutated strains produced high amounts of PSMα comparable with that of CA-MRSA. Deletion of psm-mec from HA-MRSA strains carrying intact psm-mec increased the expression of AgrA protein and PSMα, and virulence in mice. Thus, psm-mec RNA suppresses MRSA virulence via inhibition of agrA translation and the absence of psm-mec function in CA-MRSA causes its high virulence property.

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“…As the efficiency of translation initiation correlates with the accessibility of the ribosome binding site (44,45), stabilizing and destabilizing point mutations should have an impact on RNA thermometer function. Selected sites for oligonucleotide-directed mutagenesis are outlined in Figure 1A.…”

Section: Resultsmentioning

confidence: 99%

Translation on demand by a simple RNA-based thermosensor

Kortmann

Sczodrok

Rinnenthal

et al. 2010

Nucleic Acids Research

115

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Structured RNA regions are important gene control elements in prokaryotes and eukaryotes. Here, we show that the mRNA of a cyanobacterial heat shock gene contains a built-in thermosensor critical for photosynthetic activity under stress conditions. The exceptionally short 5′-untranslated region is comprised of a single hairpin with an internal asymmetric loop. It inhibits translation of the Synechocystis hsp17 transcript at normal growth conditions, permits translation initiation under stress conditions and shuts down Hsp17 production in the recovery phase. Point mutations that stabilized or destabilized the RNA structure deregulated reporter gene expression in vivo and ribosome binding in vitro. Introduction of such point mutations into the Synechocystis genome produced severe phenotypic defects. Reversible formation of the open and closed structure was beneficial for viability, integrity of the photosystem and oxygen evolution. Continuous production of Hsp17 was detrimental when the stress declined indicating that shutting-off heat shock protein production is an important, previously unrecognized function of RNA thermometers. We discovered a simple biosensor that strictly adjusts the cellular level of a molecular chaperone to the physiological need.

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Probing mRNA Structure and sRNA–mRNA Interactions in Bacteria Using Enzymes and Lead(II)

Cited by 24 publications

References 43 publications

Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression

Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression

Mobile Genetic Element SCCmec-encoded psm-mec RNA Suppresses Translation of agrA and Attenuates MRSA Virulence

Translation on demand by a simple RNA-based thermosensor

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