Direct entry by RNase E is a major pathway for the degradation and processing of RNA in <i>Escherichia coli</i>

Clarke, Justin E.; Kime, Louise; Romero, A David; McDowall, Kenneth J.

doi:10.1093/nar/gku808

Cited by 95 publications

(144 citation statements)

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“…We refer to this approach, which builds upon work by Clarke and colleagues (Clarke et al., 2014) as transient inactivation of endoribonuclease followed by RNA-seq (TIER-Seq; see Figure 1A). At the permissive temperature (28°C), Salmonella wild-type (WT) rne and mutant rne TS strains both exhibit full RNase E activity, whereas upon shift to 44°C, only WT RNase E retains its activity to process RNA.…”

Section: Resultsmentioning

confidence: 99%

“…It can be inferred, from transcript accumulation upon its inactivation, that RNase E drives the decay of most mRNAs in E. coli (Bernstein et al., 2004, Clarke et al., 2014), and in Salmonella it processes the mRNA 3′ end-derived CpxQ and SroC sRNAs (Chao and Vogel, 2016, Miyakoshi et al., 2015a). RNase E also degrades several sRNAs in the absence of Hfq or upon base pairing with target mRNAs (Bandyra et al., 2012, Massé et al., 2003, Moll et al., 2003).…”

Section: Introductionmentioning

confidence: 99%

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In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways

et al. 2017

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SummaryUnderstanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3′ fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways

et al. 2017

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“…As in the case of 9S RNA, activity of RNase E was higher in the presence of Mn 2ϩ (see below). We investigated whether other di-or trivalent metal ions would support the activity of RNase E. We chose rpsT-332 RNA as the substrate since its cleavage is independent of the phosphorylation status of its 5= end (17), and it serves as a model for the "direct entry pathway" of RNA processing (21)(22)(23). The primary products of cleavage are 219 and 113 nucleotides (nt); the former can be cleaved further to 108 and 111 nt, which comigrate with the relatively stable 113-nt product (17) (Fig.…”

Section: Activity Of Rnase E With Different Metal Ionsmentioning

confidence: 99%

Altering the Divalent Metal Ion Preference of RNase E

Thompson¹,

Zong²,

Mackie³

2014

J. Bacteriol.

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RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg 2؉ RNase E is a 5=-end-dependent endoribonuclease that plays a central role in stable RNA processing and mRNA turnover in Escherichia coli (1). RNase E and its paralog, RNase G, are found in many bacteria but not all (1, 2). In common with many intracellular enzymes of nucleic acid metabolism, RNase E requires a divalent metal ion for activity; in addition, it also requires Zn 2ϩ to stabilize its quaternary structure (3-5). Pioneering work by Misra and Apirion on RNase E demonstrated that the partially purified enzyme requires divalent metal ions with a preference for Mn 2ϩ over Mg 2ϩ (5). With 9S pre-RNA as the substrate, these authors reported optimal concentrations of Mn 2ϩ and Mg 2ϩ as 1 and 5 mM, respectively. Later, Redko et al. used a 3=-fluorescein-labeled decaribonucleotide (BR10F) as the substrate for the purified catalytic domain of RNase E (residues 1 to 529) and reported the optimal Mg 2ϩ concentration as 25 mM (6). Subsequently, the crystal structure of the catalytic domain of RNase E (4) revealed details of how divalent ions contribute to the activity of RNase E. Two conserved residues in the catalytic core, D303 and D346, serve to chelate a Mg 2ϩ ion, while N305 donates an H-bond that helps to anchor D303 (4). The hydration shell surrounding the bound Mg 2ϩ likely serves as the source of a hydroxyl ion that attacks the scissile phosphate (7). In addition, the bound metal ion likely polarizes the phosphate to enhance its reactivity.The ability of RNase E to utilize alternative metal ions in vivo has not been explored. The intracellular concentration of Mg 2ϩ in E. coli can reach almost 200 mM (8, 9); however, most Mg 2ϩ ions are chelated (e.g., by ribosomes [10,11]) and the free concentration is only 1 to 5 mM (8)(9)(10)12). This implies that the activity of RNase E may be limited by the availability of divalent metal ions. However, since RNA binds Mg 2ϩ , the interaction of substrates with RNase E may increase the local concentration of metal ions (7). The concentration of free Mn 2ϩ inside E. coli has not been reported to our knowledge, but total Mn 2ϩ can range from 15 M in cells cultured in unsupplemented defined medium to 150 M in rich medium thanks to active transport mechanisms (13,14). Most intracellular Mn 2ϩ is likely to be chelated resulting in a free pool whose concentration lies in the low micromolar range. Moreover, although Mn 2ϩ is a requirement for pathogenesis in related organisms such as Salmonella enterica serovar Typhimurium (13), and at least 70 enzymes are reported to be able to utilize Mn 2ϩ in E. coli (www.ecocyc.org), relatively few enzymes in E. coli absolutely require Mn 2ϩ . Such enzymes include Mn-dependent superoxide dismutase (encoded by sodA), several glycolytic enzymes and guanosine 3=-diphosphate 5=-triphosphate 3=-diphosphatase encoded by spoT (13).We...

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“…Hundreds of mRNAs are the targets of the 59-end-dependent mRNA degradation pathway because the disruption of rppH results in the increase of their amounts or stabilities (Deana et al 2008). On the other hand, many other mRNAs appear to be degraded by RNase E using another mechanism, the direct cleavage, independently from RppH (Clarke et al 2014).Here, we strongly suggest that T4 Srd associates with the N-terminal half of RNase E and stimulates both the 59-end-dependent and -independent mRNA degradation activ ities of RNase E. Because T4 phage lacking srd exhibited reduced growth, the degradation of host mRNAs by RNase E activity that Srd stimulates after infection is required for efficient phage growth. …”

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confidence: 99%

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Rapid Degradation of Host mRNAs by Stimulation of RNase E Activity by Srd of Bacteriophage T4

Alawneh

Yonesaki

et al. 2015

Genetics

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Escherichia coli messenger RNAs (mRNAs) are rapidly degraded immediately after bacteriophage T4 infection, and the host RNase E contributes to this process. Here, we found that a previously uncharacterized factor of T4 phage, Srd (Similarity with rpoD), was involved in T4-induced host mRNA degradation. The rapid decay of ompA and lpp mRNAs was partially alleviated and a decay intermediate of lpp mRNA rapidly accumulated in cells infected with T4 phage lacking srd. Exogenous expression of Srd in uninfected cells significantly accelerated the decay of these mRNAs. In addition, lpp(T) RNA, with a sequence identical to the decay intermediate of lpp mRNA and a triphosphate at 59-end, was also destabilized by Srd. The destabilization of these RNAs by Srd was not observed in RNase E-defective cells. The initial cleavage of a primary transcript by RNase E can be either direct or dependent on the 59-end of transcript. In the latter case, host RppH is required to convert the triphosphate at 59-end to a monophosphate. lpp(T) RNA, but not lpp and ompA mRNAs, required RppH for Srd-stimulated degradation, indicating that Srd stimulates both 59-end-dependent and -independent cleavage activities of RNase E. Furthermore, pull-down and immunoprecipitation analyses strongly suggested that Srd physically associates with the N-terminal half of RNase E containing the catalytic moiety and the membrane target sequence. Finally, the growth of T4 phage was significantly decreased by the disruption of srd. These results strongly suggest that the stimulation of RNase E activity by T4 Srd is required for efficient phage growth. KEYWORDS Srd; RNase E; Escherichia coli; bacteriophage T4; mRNA decay B ACTERIOPHAGE T4 shuts off gene expression of the host, Escherichia coli, immediately after infection and quickly starts to express its own genes (Kutter et al. 1994). Multiple mechanisms, such as modifications of apparatuses for transcription and translation, are involved in this shift of gene expression from E. coli to T4 (Carlson et al. 1994). Our previous work revealed that the representative stable E. coli messenger RNAs (mRNAs), lpp and ompA, were rapidly degraded after T4 infection (T4-induced host mRNA degra dation) and that RNase E, which is an essential endoribonuclease in E. coli (Marcaida et al. 2006), primarily functions in T4-induced host mRNA degradation (Ueno and Yonesaki 2004). This rapid degradation of host mRNAs may contribute to the quick shift from E. coli to T4 metabolism because it leads to immediate cessation of host gene expression and consequently generation of ribonucleotides and free ribosomes, each of which would stimulate transcription and translation of T4 genes. In fact, deficiency of RNase E resulted in a slow start of T4 gene transcription, reducing the level of transcription (Otsuka and Yonesaki 2005) and retarding the growth of T4 (Mudd et al. 1990a). In eukaryotic cells, the degradation of host mRNAs after viral infection, such as in alphaherpesvirus, gammaherpesvirus, or betacoronavirus, also contribu...

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Direct entry by RNase E is a major pathway for the degradation and processing of RNA in Escherichia coli

Cited by 95 publications

References 73 publications

In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways

In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways

Altering the Divalent Metal Ion Preference of RNase E

Rapid Degradation of Host mRNAs by Stimulation of RNase E Activity by Srd of Bacteriophage T4

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