RNA Pyrophosphohydrolase RppH Research Articles

A distinct RNA recognition mechanism governs Np4 decapping by RppH

Dinucleoside tetraphosphates, often described as alarmones because their cellular concentration increases in response to stress, have recently been shown to function in bacteria as precursors to nucleoside tetraphosphate (Np4) RNA caps. Removal of this cap is critical for initiating 5' end-dependent degradation of those RNAs, potentially affecting bacterial adaptability to stress; however, the predominant Np4 decapping enzyme in proteobacteria, ApaH, is inactivated by the very conditions of disulfide stress that enable Np4-capped RNAs to accumulate to high levels. Here, we show that, in Escherichia coli cells experiencing such stress, the RNA pyrophosphohydrolase RppH assumes a leading role in decapping those transcripts, preferring them as substrates over their triphosphorylated and diphosphorylated counterparts. Unexpectedly, this enzyme recognizes Np4-capped 5' ends by a mechanism distinct from the one it uses to recognize other 5' termini, resulting in a one-nucleotide shift in substrate specificity. The unique manner in which capped substrates of this kind bind to the active site of RppH positions the δ-phosphate, rather than the β-phosphate, for hydrolytic attack, generating triphosphorylated RNA as the primary product of decapping. Consequently, a second RppH-catalyzed deprotection step is required to produce the monophosphorylated 5' terminus needed to stimulate rapid RNA decay. The unconventional manner in which RppH recognizes Np4-capped 5' ends and its differential impact on the rates at which such termini are deprotected as a prelude to RNA degradation could have major consequences for reprogramming gene expression during disulfide stress.

Multifaceted impact of a nucleoside monophosphate kinase on 5'-end-dependent mRNA degradation in bacteria.

A key pathway for mRNA degradation in bacterial cells begins with conversion of the initial 5′-terminal triphosphate to a monophosphate, a modification that renders transcripts more vulnerable to attack by ribonucleases whose affinity for monophosphorylated 5′ ends potentiates their catalytic efficacy. In Escherichia coli, the only proteins known to be important for controlling degradation via this pathway are the RNA pyrophosphohydrolase RppH, its heteromeric partner DapF, and the 5′-monophosphate-assisted endonucleases RNase E and RNase G. We have now identified the metabolic enzyme cytidylate kinase as another protein that affects rates of 5′-end-dependent mRNA degradation in E. coli. It does so by utilizing two distinct mechanisms to influence the 5′-terminal phosphorylation state of RNA, each dependent on the catalytic activity of cytidylate kinase and not its mere presence in cells. First, this enzyme acts in conjunction with DapF to stimulate the conversion of 5′ triphosphates to monophosphates by RppH. In addition, it suppresses the direct synthesis of monophosphorylated transcripts that begin with cytidine by reducing the cellular concentration of cytidine monophosphate, thereby disfavoring the 5′-terminal incorporation of this nucleotide by RNA polymerase during transcription initiation. Together, these findings suggest dual signaling pathways by which nucleotide metabolism can impact mRNA degradation in bacteria.

Defect of RNA pyrophosphohydrolase RppH enhances fermentative production of L-cysteine in Escherichia coli.

Fermentative production of L-cysteine has been established using Escherichia coli. In that procedure, thiosulfate is a beneficial sulfur source, whereas repressing sulfate utilization. We first found that thiosulfate decreased transcript levels of genes related to sulfur assimilation, particularly whose expression is controlled by the transcription factor CysB. Therefore, a novel approach, i.e. increment of expression of genes involved in sulfur-assimilation, was attempted for further improvement of L-cysteine overproduction. Disruption of the rppH gene significantly augmented transcript levels of the cysD, cysJ, cysM and yeeE genes (≥1.5-times) in medium containing sulfate as a sole sulfur source, probably because the rppH gene encodes mRNA pyrophosphohydrolase that triggers degradation of certain mRNAs. In addition, the ΔrppH strain appeared to preferentially uptake thiosulfate rather than sulfate, though thiosulfate dramatically reduced expression of the known sulfate/thiosulfate transporter complexes in both ΔrppH and wild-type cells. We also found that both YeeE and YeeD are required for the strain without the transporters to grow in the presence of thiosulfate as a sole sulfur source. Therefore, yeeE and yeeD are assigned as genes responsible for thiosulfate uptake (tsuA and tsuB, respectively). In final, we applied the ΔrppH strain to the fermentative production of L-cysteine. Disruption of the rppH gene enhanced L-cysteine biosynthesis, as a result, a strain producing approximately twice as much L-cysteine as the control strain was obtained.

Analysis of differentially upregulated proteins in ptsHIcrr- and rppH- mutants in Escherichia coli during an adaptive laboratory evolution experiment.

The previous deletion of the cytoplasmic components of the phosphotransferase system (PTS) in Escherichia coli JM101 resulted in the PTS- derivative strain PB11 with severely impaired growth capability in glucose as the sole carbon source. Previous adaptive laboratory evolution (ALE) experiment led to select a fast-growing strain named PB12 from PB11. Comparative genome analysis of PB12 showed a chromosomal deletion, which result in the loss of several genes including rppH which codes for the RNA pyrophosphohydrolase RppH, involved in the preparation of hundreds of mRNAs for further degradation by RNase E. Previous inactivation of rppH in PB11 (PB11rppH-) improved significantly its growing capabilities and increased several mRNAs respect its parental strain PB11. These previous results led to propose to the PB11rppH- mutant as an intermediate between PB11 and PB12 strains merged during the early ALE experiment. In this contribution, we report the metabolic response to the PTS- and rppH- mutations in the deep of a proteomic approach to understanding the relevance of rppH- phenotype during an ALE experiment. Differentially upregulated proteins between the wild-type JM101/PB11, PB11/PB11rppH-, and PB11/PB12 comparisons led to identifying 45 proteins between strain comparisons. Downregulated or upregulated proteins in PB11rppH- were found expressed at an intermediate level with respect to PB11 and PB12. Many of these proteins were found involved in non-previously metabolic traits reported in the study of the PTS- strains, including glucose, amino acids, ribose transport; amino acid biosynthesis; NAD biosynthesis/salvage pathway, biosynthesis of Ac-CoA precursors; detoxification and degradation pathways; stress response; protein synthesis; and possible mutator activities between comparisons. No changes were found in the expression of galactose permease GalP, previously proposed as the primary glucose transporter in the absence of PTS selected by the PTS- derivatives during the ALE experiment. This result suggests that the evolving PTS- population selected other transporters such as LamB, MglB, and ManX instead of GalP for glucose uptake during the early ALE experiment. Analysis of the biological relevance of the metabolic traits developed by the studied strains provided valuable information to understand the relevance of the rppH- mutation in the PTS- background during an ALE experiment as a strategy for the selection of valuable phenotypes for metabolic engineering purposes.

Importance of a diphosphorylated intermediate for RppH-dependent RNA degradation

ABSTRACTDeprotection of the 5′ end appears to be a universal mechanism for triggering the degradation of mRNA in bacteria and eukaryotes. In Escherichia coli, for example, converting the 5′ triphosphate of primary transcripts to a monophosphate accelerates cleavage at internal sites by the endonuclease RNase E. Previous studies have shown that the RNA pyrophosphohydrolase RppH catalyzes this transformation in vitro and generates monophosphorylated decay intermediates in vivo. Recently, we reported that purified E. coli RppH unexpectedly reacts faster with diphosphorylated than with triphosphorylated substrates. By using a novel assay, it was also determined that diphosphorylated mRNA decay intermediates are abundant in wild-type E. coli and that their fractional level increases to almost 100% for representative mRNAs in mutant cells lacking RppH. These findings indicate that the conversion of triphosphorylated to monophosphorylated RNA in E. coli is a stepwise process involving sequential phosphate removal and the transient formation of a diphosphorylated intermediate. The latter RNA phosphorylation state, which was previously unknown in bacteria, now appears to define the preferred biological substrates of E. coli RppH. The enzyme responsible for generating it remains to be identified.

The pyrophosphohydrolase RppH is involved in the control of RsmA/CsrA expression in Azotobacter vinelandii and Escherichia coli

In bacteria, the 5′-end-dependent RNA degradation is triggered by the RNA pyrophosphohydrolase RppH converting tri/diphosphate to monophosphate transcripts. This study shows that in the soil bacterium Azotobacter vinelandii, inactivation of rppH gene negatively affected the production of bioplastic poly-β-hydroxybutyrate (PHB) by reducing the expression at the translational level of PhbR, the specific transcriptional activator of the phbBAC biosynthetic operon. The effect of RppH on the translation of phbR seemed to be exerted through the translational repressor RsmA, as the inactivation of rsmA in the rppH mutant restored the phbR expression. Interestingly, in Escherichia coli inactivation of rppH also affected the expression of CsrA, the RsmA homolog. The level of the csrA transcript was higher and more stable in the E. coli rppH mutant than in the wild type strain. Additionally, and in contrast to the csrA mutants that are known to have a defective swimming phenotype, the E. coli rppH mutant showed a hyper-swimming phenotype that was suppressed by a csrA mutation, and the AvRppH restored to wild type level the swimming phenotype to the E. coli rppH mutant. We propose that in both A. vinelandii and E. coli, RppH activity plays a role in the expression of the translational regulator protein RsmA/CsrA.

Structural and kinetic insights into stimulation of RppH-dependent RNA degradation by the metabolic enzyme DapF.

Vitally important for controlling gene expression in eukaryotes and prokaryotes, the deprotection of mRNA 5′ termini is governed by enzymes whose activity is modulated by interactions with ancillary factors. In Escherichia coli, 5′-end-dependent mRNA degradation begins with the generation of monophosphorylated 5′ termini by the RNA pyrophosphohydrolase RppH, which can be stimulated by DapF, a diaminopimelate epimerase involved in amino acid and cell wall biosynthesis. We have determined crystal structures of RppH–DapF complexes and measured rates of RNA deprotection. These studies show that DapF potentiates RppH activity in two ways, depending on the nature of the substrate. Its stimulatory effect on the reactivity of diphosphorylated RNAs, the predominant natural substrates of RppH, requires a substrate long enough to reach DapF in the complex, while the enhanced reactivity of triphosphorylated RNAs appears to involve DapF-induced changes in RppH itself and likewise increases with substrate length. This study provides a basis for understanding the intricate relationship between cellular metabolism and mRNA decay and reveals striking parallels with the stimulation of decapping activity in eukaryotes.

Effect of the RNA pyrophosphohydrolase RppH on envelope integrity in Escherichia coli

The bacterial enzyme RppH initiates mRNA decay by removing pyrophosphate from 5΄-triphosphorylated mRNA. Escherichia coli RppH has promiscuous substrate specificity, but relatively few transcripts are affected by loss of RppH. The phenotypic analysis of the rppH mutant is required for understanding the physiological role of RppH, but the phenotype of the rppH mutant has not yet been determined. In this study, we provide several phenotypes of the rppH mutant associated with envelope integrity. Through phenotype analysis and drug susceptibility testing, we found that the rppH mutant is sensitive to a variety of chemicals including antibiotics, and is also significantly sensitive to envelope stresses, such as osmotic stress, ethanol and sodium dodecyl sulfate. All phenotypes of the rppH mutant were caused by loss of its enzymatic activity. The rppH mutant exhibited increased envelope permeability, compared to wild-type cells. In contrast, an increase of RppH activity significantly inhibited the growth of wild-type cells under low-temperature conditions. In conclusion, various phenotypes of the rppH mutant propose that RppH is associated with regulation of envelope integrity.

A Novel RNA Phosphorylation State Enables 5′ End-Dependent Degradation in Escherichia coli

RNA modifications that once escaped detection are now thought to be pivotal for governing RNA lifetimes in both prokaryotes and eukaryotes. For example, converting the 5'-terminal triphosphate of bacterial transcripts to a monophosphate triggers 5' end-dependent degradation by RNase E. However, the existence of diphosphorylated RNA in bacteria has never been reported, and no biological role for such a modification has ever been proposed. By using a novel assay, we show here for representative Escherichia coli mRNAs that ~35%-50% of each transcript is diphosphorylated. The remainder is primarily monophosphorylated, with surprisingly little triphosphorylated RNA evident. Furthermore, diphosphorylated RNA is the preferred substrate of the RNA pyrophosphohydrolase RppH, whose biological function was previously assumed to be pyrophosphate removal from triphosphorylated transcripts. We conclude that triphosphate-to-monophosphate conversion to induce 5' end-dependent RNA degradation is a two-step process in E. coli involving γ-phosphate removal by an unidentified enzyme to enable subsequent β-phosphate removal by RppH.

Identification of the RNA Pyrophosphohydrolase RppH of Helicobacter pylori and Global Analysis of Its RNA Targets

RNA degradation is crucial for regulating gene expression in all organisms. Like the decapping of eukaryotic mRNAs, the conversion of the 5'-terminal triphosphate of bacterial transcripts to a monophosphate can trigger RNA decay by exposing the transcript to attack by 5'-monophosphate-dependent ribonucleases. In both biological realms, this deprotection step is catalyzed by members of the Nudix hydrolase family. The genome of the gastric pathogen Helicobacter pylori, a Gram-negative epsilonproteobacterium, encodes two proteins resembling Nudix enzymes. Here we present evidence that one of them, HP1228 (renamed HpRppH), is an RNA pyrophosphohydrolase that triggers RNA degradation in H. pylori, whereas the other, HP0507, lacks such activity. In vitro, HpRppH converts RNA 5'-triphosphates and diphosphates to monophosphates. It requires at least two unpaired nucleotides at the 5' end of its substrates and prefers three or more but has only modest sequence preferences. The influence of HpRppH on RNA degradation in vivo was examined by using RNA-seq to search the H. pylori transcriptome for RNAs whose 5'-phosphorylation state and cellular concentration are governed by this enzyme. Analysis of cDNA libraries specific for transcripts bearing a 5'-triphosphate and/or monophosphate revealed at least 63 potential HpRppH targets. These included mRNAs and sRNAs, several of which were validated individually by half-life measurements and quantification of their 5'-terminal phosphorylation state in wild-type and mutant cells. These findings demonstrate an important role for RppH in post-transcriptional gene regulation in pathogenic Epsilonproteobacteria and suggest a possible basis for the phenotypes of H. pylori mutants lacking this enzyme.

Structures of RNA Complexes with the Escherichia coli RNA Pyrophosphohydrolase RppH Unveil the Basis for Specific 5′-End-dependent mRNA Decay

5'-End-dependent RNA degradation impacts virulence, stress responses, and DNA repair in bacteria by controlling the decay of hundreds of mRNAs. The RNA pyrophosphohydrolase RppH, a member of the Nudix hydrolase superfamily, triggers this degradation pathway by removing pyrophosphate from the triphosphorylated RNA 5' terminus. Here, we report the x-ray structures of Escherichia coli RppH (EcRppH) in apo- and RNA-bound forms. These structures show distinct conformations of EcRppH·RNA complexes on the catalytic pathway and suggest a common catalytic mechanism for Nudix hydrolases. EcRppH interacts with RNA by a bipartite mechanism involving specific recognition of the 5'-terminal triphosphate and the second nucleotide, thus enabling discrimination against mononucleotides as substrates. The structures also reveal the molecular basis for the preference of the enzyme for RNA substrates bearing guanine in the second position by identifying a protein cleft in which guanine interacts with EcRppH side chains via cation-π contacts and hydrogen bonds. These interactions explain the modest specificity of EcRppH at the 5' terminus and distinguish the enzyme from the highly selective RppH present in Bacillus subtilis. The divergent means by which RNA is recognized by these two functionally and structurally analogous enzymes have important implications for mRNA decay and the regulation of protein biosynthesis in bacteria.

Specificity and Evolutionary Conservation of the Escherichia coli RNA Pyrophosphohydrolase RppH

Bacterial RNA degradation often begins with conversion of the 5'-terminal triphosphate to a monophosphate by the RNA pyrophosphohydrolase RppH, an event that triggers rapid ribonucleolytic attack. Besides its role as the master regulator of 5'-end-dependent mRNA decay, RppH is important for the ability of pathogenic bacteria to invade host cells, yet little is known about how it chooses its targets. Here, we show that Escherichia coli RppH (EcRppH) requires at least two unpaired nucleotides at the RNA 5' end and prefers three or more such nucleotides. It can tolerate any nucleotide at the first three positions but has a modest preference for A at the 5' terminus and either a G or A at the second position. Mutational analysis has identified EcRppH residues crucial for substrate recognition or catalysis. The promiscuity of EcRppH differentiates it from its Bacillus subtilis counterpart, which has a strict RNA sequence requirement. EcRppH orthologs likely to share its relaxed sequence specificity are widespread in all classes of Proteobacteria, except Deltaproteobacteria, and in flowering plants. By contrast, the phylogenetic range of recognizable B. subtilis RppH orthologs appears to be restricted to the order Bacillales. These findings help to explain the selective influence of RppH on bacterial mRNA decay and show that RppH-dependent degradation has diversified significantly during the course of evolution.

Way to go, RNA.

Way to go, RNA.

NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs.

A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. In eukaryotes, 5',5'-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5'-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5'-NAD modification is shown in vitro to stabilize RNA against 5'-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, ∼13% of the abundant sRNA RNAI is NAD-capped in the presence, and ∼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.

Specificity of RppH-dependent RNA degradation in Bacillus subtilis

Bacterial RNA degradation often begins with conversion of the 5'-terminal triphosphate to a monophosphate, creating a better substrate for subsequent ribonuclease digestion. For example, in Bacillus subtilis and related organisms, removal of the gamma and beta phosphates of primary transcripts by the RNA pyrophosphohydrolase RppH triggers rapid 5'-exonucleolytic degradation by RNase J. However, the basis for the selective targeting of a subset of cellular RNAs by this pathway has remained largely unknown. Here we report that purified B. subtilis RppH requires at least two unpaired nucleotides at the 5' end of its RNA substrates and prefers three or more. The second of these 5'-terminal nucleotides must be G, whereas a less strict preference for a purine is evident at the third position, and A is slightly favored over G at the first position. The same sequence requirements are observed for RppH-dependent mRNA degradation in B. subtilis cells. By contrast, a parallel pathway for 5'-end-dependent RNA degradation in that species appears to involve an alternative phosphate-removing enzyme that is relatively insensitive to sequence variation at the first three positions.

1H, 13C and 15N resonance assignments of RNA pyrophosphohydrolase RppH from Escherichia coli

The mRNA degradation is an important regulatory mechanism which controls gene expression by limiting the number of translation times. Previous studies demonstrated that this process is essential for organisms. Escherichia coli RNA pyrophosphohydrolase (RppH) is an enzyme that triggers mRNA degradation by removing the 5' pyrophosphate, which is a rate-determining step. In order to understand the molecular mechanism of the biological function, the structural information of RppH is required. Herein, we report the resonance assignments of (1)H, (15)N, (13)C atoms of the E. coli RppH.