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

Andrade, José M.; Pobre, Vânia; Matos, Ana Marta de; Arraiano, Cecília M.

doi:10.1261/rna.029413.111

Cited by 100 publications

(120 citation statements)

References 71 publications

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“…The binding of sRNAs by Hfq protects them from degradation (Sledjeski et al 2001;Moller et al 2002;Andrade et al 2012;Fei et al 2015), but Hfq can also recruit the degradosome to sRNA-mRNA complexes, which leads to their accelerated decay (Ikeda et al 2011). Moreover, Hfq promotes the pairing of certain sRNAs to their mRNA targets (Moller et al 2002;Zhang et al 2002;Maki et al 2008;Soper and Woodson 2008), and facilitates the annealing of regulatory RNA-OUT to transposase-encoding RNA-IN (Ross et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

Wróblewska

Olejniczak

2016

RNA

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The bacterial protein Hfq participates in the regulation of translation by small noncoding RNAs (sRNAs). Several mechanisms have been proposed to explain the role of Hfq in the regulation by sRNAs binding to the 5 ′ -untranslated mRNA regions. However, it remains unknown how Hfq affects those sRNAs that target the coding sequence. Here, the contribution of Hfq to the annealing of three sRNAs, RybB, SdsR, and MicC, to the coding sequence of Salmonella ompD mRNA was investigated. Hfq bound to ompD mRNA with tight, subnanomolar affinity. Moreover, Hfq strongly accelerated the rates of annealing of RybB and MicC sRNAs to this mRNA, and it also had a small effect on the annealing of SdsR. The experiments using truncated RNAs revealed that the contributions of Hfq to the annealing of each sRNA were individually adjusted depending on the structures of interacting RNAs. In agreement with that, the mRNA structure probing revealed different structural contexts of each sRNA binding site. Additionally, the annealing of RybB and MicC sRNAs induced specific conformational changes in ompD mRNA consistent with local unfolding of mRNA secondary structure. Finally, the mutation analysis showed that the long AU-rich sequence in the 5 ′ -untranslated mRNA region served as an Hfq binding site essential for the annealing of sRNAs to the coding sequence. Overall, the data showed that the functional specificity of Hfq in the annealing of each sRNA to the ompD mRNA coding sequence was determined by the sequence and structure of the interacting RNAs.

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Section: Introductionmentioning

confidence: 99%

Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

Wróblewska

Olejniczak

2016

RNA

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“…Recent studies revealed that the deletion of pnp, encoding the 3= to 5= phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), paradoxically reduces the stability of several sRNAs, including CyaR and RyhB, during exponential growth (18,19). Deletion of pnp also resulted in the decreased regulation of mRNA targets by CyaR and other sRNAs (18,20).…”

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confidence: 99%

The Phosphorolytic Exoribonucleases Polynucleotide Phosphorylase and RNase PH Stabilize sRNAs and Facilitate Regulation of Their mRNA Targets

Cameron¹,

Lay

2016

J Bacteriol

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Gene regulation by base pairing between small noncoding RNAs (sRNAs) and their mRNA targets is an important mechanism that allows bacteria to maintain homeostasis and respond to dynamic environments. In Gram-negative bacteria, sRNA pairing and regulation are mediated by several RNA-binding proteins, including the sRNA chaperone Hfq and polynucleotide phosphorylase (PNPase). PNPase and its homolog RNase PH together represent the two 3= to 5= phosphorolytic exoribonucleases found in Escherichia coli; however, the role of RNase PH in sRNA regulation has not yet been explored and reported. Here, we have examined in detail how PNPase and RNase PH interact to support sRNA stability, activity, and base pairing in exponential and stationary growth conditions. Our results indicate that these proteins facilitate the stability and regulatory function of the sRNAs RyhB, CyaR, and MicA during exponential growth. PNPase further appears to contribute to pairing between RyhB and its mRNA targets. During stationary growth, each sRNA responded differently to the absence or presence of PNPase and RNase PH. Finally, our results suggest that PNPase and RNase PH stabilize only Hfq-bound sRNAs. Taken together, these results confirm and extend previous findings that PNPase participates in sRNA regulation and reveal that RNase PH serves a similar, albeit more limited, role as well. These proteins may, therefore, act to protect sRNAs from spurious degradation while also facilitating regulatory pairing with their targets. IMPORTANCEIn many bacteria, Hfq-dependent base-pairing sRNAs facilitate rapid changes in gene expression that are critical for maintaining homeostasis and responding to stress and environmental changes. While a role for Hfq in this process was identified more than 2 decades ago, the identity and function of the other proteins required for Hfq-dependent regulation by sRNAs have not been resolved. Here, we demonstrate that PNPase and RNase PH, the two phosphorolytic RNases in E. coli, stabilize sRNAs against premature degradation and, in the case of PNPase, also accelerate regulation by sRNA-mRNA pairings for certain sRNAs. These findings are the first to demonstrate that RNase PH influences and supports sRNA regulation and suggest shared and distinct roles for these phosphorolytic RNases in this process. Bacteria rapidly alter gene expression through small noncoding RNAs (sRNAs) that act by base pairing with target mRNAs. Although classically identified as important players in bacterial stress response, sRNAs also play general roles regulating the genes involved in cell growth, nutrient acquisition, social behavior, and virulence (1, 2). sRNAs often act through translational repression, in which a given sRNA base pairs with the leader sequence of a target mRNA, obscuring the site that would otherwise form contacts with the ribosomal 30S subunit (3). This pairing can also lead to subsequent RNase E-mediated decay of the mRNA (4). In other cases, pairing with an mRNA leader sequence promotes gene expression by revea...

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“…A pnp mutant accumulates numerous RNA fragments produced by endoribonucleic cleavages due to a major defect in poly (A)-dependent degradation (Braun et al 1996;Coburn and Mackie 1998). PNPase is involved in the turnover of sRNAs, the modulation of RNase II expression and in the processing of RliD-CRISPR in Listeria (Zilhao et al 1996;Andrade and Arraiano 2008;De Lay and Gottesman 2011;Andrade et al 2012;Sesto et al 2014). PNPase enzymatic activity is dependent on magnesium-chelated citrate, ATP and c-di-GMP, thereby creating a network between metabolism and RNA turnover (Del Favero et al 2008;Nurmohamed et al 2011;Tuckerman et al 2011).…”

Section: Discussionmentioning

confidence: 99%

“…Its association with the RhlB helicase, another component of the degradosome, allows PNPase to disrupt very stable secondary structures that cannot be degraded by the free enzyme in E. coli (Régnier and Hajnsdorf 2009). PNPase degrades certain small RNAs (sRNA) (Andrade and Arraiano 2008;Andrade et al 2012), but it can also interact with sRNAs in a nondestructive mode together with Hfq in E. coli or in complex with Rsr protein in Deinococcus radiodurans, where Y RNA act as adaptors between the two proteins (Chen et al 2013;Bandyra et al 2016).…”

Section: Introductionmentioning

confidence: 99%

The small RNA SraG participates in PNPase homeostasis

Fontaine

Gasiorowski

Gracia

et al. 2016

RNA

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The rpsO-pnp operon encodes ribosomal protein S15 and polynucleotide phosphorylase, a major 3 ′ -5 ′ exoribonuclease involved in mRNA decay in Escherichia coli. The gene for the SraG small RNA is located between the coding regions of the rpsO and pnp genes, and it is transcribed in the opposite direction relative to the two genes. No function has been assigned to SraG. Multiple levels of post-transcriptional regulation have been demonstrated for the rpsO-pnp operon. Here we show that SraG is a new factor affecting pnp expression. SraG overexpression results in a reduction of pnp expression and a destabilization of pnp mRNA; in contrast, inhibition of SraG transcription results in a higher level of the pnp transcript. Furthermore, in vitro experiments indicate that SraG inhibits translation initiation of pnp. Together, these observations demonstrate that SraG participates in the post-transcriptional control of pnp by a direct antisense interaction between SraG and PNPase RNAs. Our data reveal a new level of regulation in the expression of this major exoribonuclease.

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

Cited by 100 publications

References 71 publications

Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

Hfq assists small RNAs in binding to the coding sequence of ompD mRNA and in rearranging its structure

The Phosphorolytic Exoribonucleases Polynucleotide Phosphorylase and RNase PH Stabilize sRNAs and Facilitate Regulation of Their mRNA Targets

The small RNA SraG participates in PNPase homeostasis

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