RppH-dependent pyrophosphohydrolysis of mRNAs is regulated by direct interaction with DapF in Escherichia coli

Abstract

Similar to decapping of eukaryotic mRNAs, the RppH-catalyzed conversion of 5′-terminal triphosphate to monophosphate has recently been identified as the rate-limiting step for the degradation of a subset of mRNAs in Escherichia coli. However, the regulation of RppH pyrophosphohydrolase activity is not well understood. Because the overexpression of RppH alone does not affect the decay rate of most target mRNAs, the existence of a mechanism regulating its activity has been suggested. In this study, we identified DapF, a diaminopimelate (DAP) epimerase catalyzing the stereoinversion of L,L-DAP to meso-DAP, as a regulator of RppH. DapF showed a high affinity interaction with RppH and increased its RNA pyrophosphohydrolase activity. The simultaneous overexpression of both DapF and RppH increased the decay rates of RppH target RNAs by about a factor of two. Together, our data suggest that the cellular level of DapF is a critical factor regulating the RppH-catalyzed pyrophosphate removal and the subsequent degradation of target mRNAs.

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

“…Furthermore, contrary to the phenotype expected for a triphosphatase-deficient mutant, deleting the rppH gene does not diminish the abundance of diphosphorylated 5′ ends in E. coli (Figures 3C and 4B). For similar reasons (Lee et al, 2014 and PACO analysis of RNA from ΔdapF cells [data not shown]), it is also unlikely that DapF, which forms a complex with RppH, functions as an RNA triphosphatase in E. coli . Nor does E. coli contain a homolog of the eukaryotic RNA triphosphatases that catalyze the first step in RNA capping.…”

“…By contrast, overproducing RppH has little effect on the phosphorylation state or half-life of the mRNAs it targets, suggesting that the rate of formation of the full-length monophosphorylated intermediate may be limited by the cellular concentration of an ancillary factor or by a hypothetical step that precedes attack by RppH (Luciano et al, 2012). This observation led to the discovery that the specific activity of RppH is boosted 2–3 fold in E. coli by binding to the diaminopimelate epimerase DapF (Lee et al, 2014). However, the possibility of a prior step that triggers the action of RppH has not previously been explored.…”

Glucose induces delocalization of a flagellar biosynthesis protein from the flagellated pole

“…Purified EIIA Glc was immobilized on the carboxymethylated dextran surface of a CM5 sensor chip by an NHS/EDC reaction, and real‐time interaction of EIIA Glc with FapA was monitored by Surface Plasmon Resonance (SPR) detection using a BIAcore 3000 (GE Healthcare Life Sciences) as previously described (Lee et al ., ; Park et al ., ; Lee et al ., ). The indicated concentrations of FapA in standard running buffer (10 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM KCl and 1 mM MgCl 2 ) were flowed over the EIIA Glc surface at a flow rate of 5 μl min −1 .…”

Noncanonical features and modifications on the 5′‐end of bacterial sRNAs and mRNAs

“…Biochemical and structural studies discovered a 2:2 stoichiometry of the complex, consistent with dimerization of an RppH‐DapF heterodimer through DapF‐DapF interactions (Figure d) (Gao et al, ; C. R. Lee et al, ; Q. Wang et al, ). The structures showed that the RppH‐DapF interface is located away from both the catalytic site and RNA‐binding cleft of RppH, therefore, DapF cannot directly participate in 5′‐end binding and catalysis (Gao et al, ; Q. Wang et al, ).…”

“…However, the structures of the individual enzymes and RppH‐DapF complex in the apo and RNA‐binding states did not reveal pronounced allosteric changes in RppH that would explain its higher reactivity upon DapF binding (Gao et al, ). Allosteric modulation was expected at least for short triphosphorylated substrates of RppH, which cannot reach DapF in the complex but which are processed by DapF‐bound RppH at greater rate than by RppH alone (Gao et al, ; C. R. Lee et al, ). Remarkably, RppH reactivity on short diphosphorylated RNAs was not affected while a rate acceleration was observed on substrates ≥8 nucleotides long, a length sufficient to reach DapF in the complex (Gao et al, ).…”