Stresses that Raise Np4A Levels Induce Protective Nucleoside Tetraphosphate Capping of Bacterial RNA.

Present in all realms of life, dinucleoside tetraphosphates (Np4Ns) are generally considered signaling molecules. However, only a single pathway for Np4N signaling has been delineated in eukaryotes, and no receptor that mediates the influence of Np4Ns has ever been identified in bacteria. Here we show that, under disulfide stress conditions that elevate cellular Np4N concentrations, diverse Escherichia coli mRNAs and sRNAs acquire a cognate Np4 cap. Purified E. coli RNA polymerase and lysyl-tRNA synthetase are both capable of adding such 5' caps. Cap removal by either of two pyrophosphatases, ApaH or RppH, triggers rapid RNA degradation in E. coli. ApaH, the predominant decapping enzyme, functions as both a sensor and an effector of disulfide stress, which inactivates it. These findings suggest that the physiological changes attributed to elevated Np4N concentrations in bacteria may result from widespread Np4 capping, leading to altered RNA stability and consequent changes in gene expression.

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 ß-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.

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.

Direct inhibition of P/Q-type voltage-gated Ca2+ channels by Gem does not require a direct Gem/Cavbeta interaction.

The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca(2+) channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca(2+) channel beta subunits (Ca(v)beta), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca(2+) channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca(v)beta but not the Gem/Ca(v)beta interaction, and that Gem coimmunoprecipitates with the P/Q channel alpha(1) subunit (Ca(v)alpha(1)) in a Ca(v)beta-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca(v)alpha(1) critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca(v)alpha(1) abolishes Gem inhibition of P/Q channels and confers Ca(v)beta-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca(v)alpha(1) that binds Ca(v)beta). Our results challenge the prevailing view regarding the role of Ca(v)beta in RGK inhibition of high voltage-activated Ca(2+) channels and prompt a paradigm in which Gem directly binds and inhibits Ca(v)beta-primed Ca(v)alpha(1) on the plasma membrane.

Inactivation influences the extent of inhibition of voltage-gated Ca+2 channels by Gem-implications for channelopathies.

Voltage-gated Ca2+ channels (VGCC) directly control muscle contraction and neurotransmitter release, and slower processes such as cell differentiation, migration, and death. They are potently inhibited by RGK GTP-ases (Rem, Rem2, Rad, and Gem/Kir), which decrease Ca2+ channel membrane expression, as well as directly inhibit membrane-resident channels. The mechanisms of membrane-resident channel inhibition are difficult to study because RGK-overexpression causes complete or near complete channel inhibition. Using titrated levels of Gem expression in Xenopus oocytes to inhibit WT P/Q-type calcium channels by ∼50%, we show that inhibition is dependent on channel inactivation. Interestingly, fast-inactivating channels, including Familial Hemiplegic Migraine mutants, are more potently inhibited than WT channels, while slow-inactivating channels, such as those expressed with the Cavß2a auxiliary subunit, are spared. We found similar results in L-type channels, and, remarkably, Timothy Syndrome mutant channels were insensitive to Gem inhibition. Further results suggest that RGKs slow channel recovery from inactivation and further implicate RGKs as likely modulating factors in channelopathies.