Regulation of the small regulatory RNA MicA by ribonuclease III: a target-dependent pathway

Viegas, Sandra C.; Silva, Inês J.; Saramago, Margarida; Domingues, Susana; Arraiano, Cecília M.

doi:10.1093/nar/gkq1239

Cited by 76 publications

(64 citation statements)

References 60 publications

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“…The low levels of MicA found in hfq mutants strongly decrease the probability of base-pairing with target mRNAs. The down-regulation in sRNA-target mRNA duplexes probably explains the impairment in RNase III activity on MicA, in agreement with in vitro studies (Viegas et al 2011). Data suggest that the degradation of small RNAs that are not associated with Hfq mainly occurs in a targetindependent pathway, in which RNase III has a reduced impact.…”

Section: Discussionsupporting

confidence: 84%

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The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq

Andrade¹,

Pobre²,

Matos³

et al. 2012

RNA

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103

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The transient existence of small RNAs free of binding to the RNA chaperone Hfq is part of the normal dynamic lifecycle of a sRNA. Small RNAs are extremely labile when not associated with Hfq, but the mechanism by which Hfq stabilizes sRNAs has been elusive. In this work we have found that polynucleotide phosphorylase (PNPase) is the major factor involved in the rapid degradation of small RNAs, especially those that are free of binding to Hfq. The levels of MicA, GlmY, RyhB, and SgrS RNAs are drastically increased upon PNPase inactivation in Hfq À cells. In the absence of Hfq, all sRNAs are slightly shorter than their fulllength species as result of 39-end trimming. We show that the turnover of Hfq-free small RNAs is growth-phase regulated, and that PNPase activity is particularly important in stationary phase. Indeed, PNPase makes a greater contribution than RNase E, which is commonly believed to be the main enzyme in the decay of small RNAs. Lack of poly(A) polymerase I (PAP I) is also found to affect the rapid degradation of Hfq-free small RNAs, although to a lesser extent. Our data also suggest that when the sRNA is not associated with Hfq, the degradation occurs mainly in a target-independent pathway in which RNase III has a reduced impact. This work demonstrated that small RNAs free of Hfq binding are preferably degraded by PNPase. Overall, our data highlight the impact of 39-exonucleolytic RNA decay pathways and re-evaluates the degradation mechanisms of Hfq-free small RNAs.

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Section: Discussionsupporting

confidence: 84%

“…In vitro studies showed that Salmonella RNase III can cleave MicA when bound to its target ompA mRNA (Viegas et al 2011). To further analyze the role of RNase III in cells without Hfq, we constructed and analyzed double mutants lacking both Hfq and RNase III.…”

Section: Inactivation Of Pnpase Markedly Increases the Levels Of Smalmentioning

confidence: 99%

The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq

Andrade¹,

Pobre²,

Matos³

et al. 2012

RNA

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103

113

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“…For construction of the pWSK29hfq plasmid (pSVA-6) expressing hfq, a PCR fragment containing the entire hfq sequence was amplified from the SL1344 chromosome, digested with the enzymes HindIII and XbaI, and ligated into plasmid pWSK29 digested with the same enzymes. For the construction of plasmid pWSK29rnc (pSVA-7), the pSVDA-01 plasmid expressing His-tagged RNase III (20) was digested with BamHI and XbaI to obtain the rnc fragment, which was ligated with pWSK29 digested with the same enzymes. In the case of plasmid pSVA-8 expressing RNase E, a PCR fragment containing the entire rne sequence was amplified from SL1344 chromosome and was cloned into the XbaI and EcoRI sites of the pSE420 vector (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…Several genome-wide analyses reported that the absence of RNases E and III in E. coli (14) and RNase III in Bacillus subtilis (15) or Staphylococcus aureus (16,17) affects the abundance of a high number of mRNAs and sRNAs. Namely, in Salmonella and in other bacteria, RNases E and III have roles in the control of a number of sRNAs implicated in the regulation of outer membrane proteins (18)(19)(20) and important virulence factors (11,21,22).…”

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confidence: 99%

The Virulence of Salmonella enterica Serovar Typhimurium in the Insect Model Galleria mellonella Is Impaired by Mutations in RNase E and RNase III

Viegas

Mil‐Homens

Fialho

et al. 2013

Appl Environ Microbiol

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Salmonella enterica serovar Typhimurium is a Gram-negative bacterium able to invade and replicate inside eukaryotic cells. To cope with the host defense mechanisms, the bacterium has to rapidly remodel its transcriptional status. Regulatory RNAs and ribonucleases are the factors that ultimately control the fate of mRNAs and final protein levels in the cell. There is growing evidence of the direct involvement of these factors in bacterial pathogenicity. In this report, we validate the use of a Galleria mellonela model in S. Typhimurium pathogenicity studies through the parallel analysis of a mutant with a mutation in hfq, a wellestablished Salmonella virulence gene. The results obtained with this mutant are similar to the ones reported in a mouse model. Through the use of this insect model, we demonstrate a role for the main endoribonucleases RNase E and RNase III in Salmonella virulence. These ribonuclease mutants show an attenuated virulence phenotype, impairment in motility, and reduced proliferation inside the host. Interestingly, the two mutants trigger a distinct immune response in the host, and the two mutations seem to have an impact on distinct bacterial functions. Salmonella infections are a serious medical and veterinary problem worldwide. This pathogenic bacterium is able to invade and replicate within eukaryotic host cells. For infection, Salmonella relies upon a range of laterally acquired virulence regions, the so-called Salmonella pathogenicity islands (SPIs). Of these, SPI-1 and SPI-2 contain genes that encode type III secretion systems (TTSS), which deliver effector proteins into host cells to facilitate either cellular invasion or intracellular survival, respectively (for a review, see reference 1). Hundreds of genes are upregulated during infection and play important roles in adaptation, survival, and proliferation within mammalian cells (2). Transcriptome analysis of Salmonella enterica serovar Typhimurium within epithelial cells and macrophages revealed distinct patterns of expression linked to the different stages of infection (3, 4).Both the evolutionarily close relationship with Escherichia coli and the pathogen-specific aspects make Salmonella a very good model for studying the influence of RNA determinants in bacterial pathogenicity. In addition to transcriptional control, regulation of RNA decay has emerged as a major pathway in the fast adaptive process of bacteria to changes in the environment. RNAs may also act as regulatory molecules that can directly sense environmental clues and modulate the expression of target RNAs (for a review, see reference 5). The fate of RNA transcripts can be also controlled by proteins, including ribonucleases (RNases) and RNA chaperones.RNases are enzymes that govern the maturation and degradation of RNA molecules. RNA decay in Gram-negative bacteria usually begins with an endonucleolytic cleavage at one or more internal sites on the RNA molecule. This cleavage is normally performed by RNase E and/or RNase III (6, 7). The single-stranded specific endori...

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“…In addition to these diverse single-strand-specific RNases, most bacteria also encode the double-strand-specific RNase III (10). This enzyme is highly conserved in bacteria and eukaryotes and has a critical role in posttranscriptional regulation, where it cleaves double-stranded substrates, such as those resulting from the base pairing of an mRNA and noncoding RNA (11), or those associated with highly structured RNAs, such as ribosomal RNAs (rRNAs) (12,13). There can be considerable functional interplay between RNases, with a classic example being the generation of mature 23S, 16S, and 5S rRNAs from a single 30S transcript.…”

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confidence: 99%

Development, Antibiotic Production, and Ribosome Assembly in Streptomyces venezuelae Are Impacted by RNase J and RNase III Deletion

2014

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b RNA metabolism is a critical but frequently overlooked control element affecting virtually every cellular process in bacteria. RNA processing and degradation is mediated by a suite of ribonucleases having distinct cleavage and substrate specificity. Here, we probe the role of two ribonucleases (RNase III and RNase J) in the emerging model system Streptomyces venezuelae. We show that each enzyme makes a unique contribution to the growth and development of S. venezuelae and further affects the secondary metabolism and antibiotic production of this bacterium. We demonstrate a connection between the action of these ribonucleases and translation, with both enzymes being required for the formation of functional ribosomes. RNase III mutants in particular fail to properly process 23S rRNA, form fewer 70S ribosomes, and show reduced translational processivity. The loss of either RNase III or RNase J additionally led to the appearance of a new ribosomal species (the 100S ribosome dimer) during exponential growth and dramatically sensitized these mutants to a range of antibiotics. R ibonucleases (RNases) are enzymes that process and degrade RNA molecules; consequently, they are critical for RNA maturation, RNA stability, and posttranscriptional regulation (1). In bacterial cells, the finely tuned balance between RNA synthesis and RNA degradation allows for rapid adaptation to changing environments, proper processing of noncoding RNAs, and efficient recycling of ribonucleotides (2).Our understanding of RNA metabolism to date is based largely on studies of Escherichia coli and Bacillus subtilis, which employ distinct enzymes for RNA processing and degradation. In E. coli, RNase E is the central component of the RNA degradosome; as such, it is responsible for much of the RNA decay in this organism. B. subtilis lacks this enzyme, and acting in its place are three other nucleases: RNase J1/J2 and RNase Y (3, 4). Virtually all bacteria contain at least one of RNase E (or its paralog, RNase G), RNase J, or RNase Y (5). These RNases are unrelated in primary sequence and mechanism of catalysis but have similar substrate specificity: they all have single-strand-specific endonuclease activity and preferentially cleave AU-rich regions (4, 6, 7). Unlike RNase E and Y, however, RNase J has the capacity to act both as an endonuclease and as a 5= exonuclease (8, 9). The analysis of available genome sequences suggests that more than half of all bacteria, and over two-thirds of Archaea, possess an RNase J homologue (6,8). In addition to these diverse single-strand-specific RNases, most bacteria also encode the double-strand-specific RNase III (10). This enzyme is highly conserved in bacteria and eukaryotes and has a critical role in posttranscriptional regulation, where it cleaves double-stranded substrates, such as those resulting from the base pairing of an mRNA and noncoding RNA (11), or those associated with highly structured RNAs, such as ribosomal RNAs (rRNAs) (12, 13). There can be considerable functional interplay between RNases, ...

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Regulation of the small regulatory RNA MicA by ribonuclease III: a target-dependent pathway

Cited by 76 publications

References 60 publications

The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq

The crucial role of PNPase in the degradation of small RNAs that are not associated with Hfq

The Virulence of Salmonella enterica Serovar Typhimurium in the Insect Model Galleria mellonella Is Impaired by Mutations in RNase E and RNase III

Development, Antibiotic Production, and Ribosome Assembly in Streptomyces venezuelae Are Impacted by RNase J and RNase III Deletion

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