The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis.

Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp.

The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria, where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.

Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance.

Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a posttranscriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction.

Single-Cell Microscopy Reveals That Levels of Cyclic di-GMP Vary among Bacillus subtilis Subpopulations.

The synthesis of signaling molecules is one strategy bacteria employ to sense alterations in their environment and rapidly adjust to those changes. In Gram-negative bacteria, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile state. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. To that end, we constructed a fluorescent yfp reporter based on a c-di-GMP-responsive riboswitch to visualize the relative abundance of c-di-GMP for single cells of the Gram-positive model organism Bacillus subtilis Coupled with cell-type-specific fluorescent reporters, this riboswitch reporter revealed that c-di-GMP levels are markedly different among B. subtilis cellular subpopulations. For example, cells that have made the decision to become matrix producers maintain higher intracellular c-di-GMP concentrations than motile cells. Similarly, we find that c-di-GMP levels differ between sporulating and competent cell types. These results suggest that biochemical measurements of c-di-GMP abundance are likely to be inaccurate for a bulk ensemble of B. subtilis cells, as such measurements will average c-di-GMP levels across the population. Moreover, the significant variation in c-di-GMP levels between cell types hints that c-di-GMP might play an important role during B. subtilis biofilm formation. This study therefore emphasizes the importance of using single-cell approaches for analyzing metabolic trends within ensemble bacterial populations.IMPORTANCE Many bacteria have been shown to differentiate into genetically identical yet morphologically distinct cell types. Such population heterogeneity is especially prevalent among biofilms, where multicellular communities are primed for unexpected environmental conditions and can efficiently distribute metabolic responsibilities. Bacillus subtilis is a model system for studying population heterogeneity; however, a role for c-di-GMP in these processes has not been thoroughly investigated. Herein, we introduce a fluorescent reporter, based on a c-di-GMP-responsive riboswitch, to visualize the relative abundance of c-di-GMP for single B. subtilis cells. Our analysis shows that c-di-GMP levels are conspicuously different among B. subtilis cellular subtypes, suggesting a role for c-di-GMP during biofilm formation. These data highlight the utility of riboswitches as tools for imaging metabolic changes within individual bacterial cells. Analyses such as these offer new insight into c-di-GMP-regulated phenotypes, especially given that other biofilms also consist of multicellular communities.

A Subset of Exoribonucleases Serve as Degradative Enzymes for pGpG in c-di-GMP Signaling.

Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that regulates processes, such as biofilm formation and virulence. During degradation, c-di-GMP is first linearized to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG) and subsequently hydrolyzed to two GMPs by a previously unknown enzyme, which was recently identified in Pseudomonas aeruginosa as the 3'-to-5' exoribonuclease oligoribonuclease (Orn). Mutants of orn accumulated pGpG, which inhibited the linearization of c-di-GMP. This product inhibition led to elevated c-di-GMP levels, resulting in increased aggregate and biofilm formation. Thus, the hydrolysis of pGpG is crucial to the maintenance of c-di-GMP homeostasis. How species that utilize c-di-GMP signaling but lack an orn ortholog hydrolyze pGpG remains unknown. Because Orn is an exoribonuclease, we asked whether pGpG hydrolysis can be carried out by genes that encode protein domains found in exoribonucleases. From a screen of these genes from Vibrio cholerae and Bacillus anthracis, we found that only enzymes known to cleave oligoribonucleotides (orn and nrnA) rescued the P. aeruginosa Δorn mutant phenotypes to the wild type. Thus, we tested additional RNases with demonstrated activity against short oligoribonucleotides. These experiments show that only exoribonucleases previously reported to degrade short RNAs (nrnA, nrnB, nrnC, and orn) can also hydrolyze pGpG. A B. subtilisnrnA nrnB mutant had elevated c-di-GMP, suggesting that these two genes serve as the primary enzymes to degrade pGpG. These results indicate that the requirement for pGpG hydrolysis to complete c-di-GMP signaling is conserved across species. The final steps of RNA turnover and c-di-GMP turnover appear to converge at a subset of RNases specific for short oligoribonucleotides.IMPORTANCE The bacterial bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling molecule regulates complex processes, such as biofilm formation. c-di-GMP is degraded in two-steps, linearization into pGpG and subsequent cleavage to two GMPs. The 3'-to-5' exonuclease oligoribonuclease (Orn) serves as the enzyme that degrades pGpG in Pseudomonas aeruginosa Many phyla contain species that utilize c-di-GMP signaling but lack an Orn homolog, and the protein that functions to degrade pGpG remains uncharacterized. Here, systematic screening of genes encoding proteins containing domains found in exoribonucleases revealed a subset of genes encoded within the genomes of Bacillus anthracis and Vibrio cholerae that degrade pGpG to GMP and are functionally analogous to Orn. Feedback inhibition by pGpG is a conserved process, as strains lacking these genes accumulate c-di-GMP.

Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli.

During amino acid starvation, bacterial cells rapidly synthesize the nucleotides (p)ppGpp, causing a massive re-programming of the transcriptional profile known as the stringent response. The (p)ppGpp synthase RelA is activated by ribosomes harboring an uncharged tRNA at the A site. It is unclear whether synthesis occurs while RelA is bound to the ribosome or free in the cytoplasm. We present a study of three Escherichia coli strains, each expressing a different RelA-fluorescent protein (RelA-FP) construct: RelA-YFP, RelA-mEos2 and RelA-Dendra2. Single-molecule localization and tracking studies were carried out under normal growth conditions and during amino acid starvation. Study of three labeling schemes enabled us to assess potential problems with FP labeling of RelA. The diffusive trajectories and axial spatial distributions indicate that amino acid starvation induces net binding of all three RelA-FP constructs to 70S ribosomes. The data are most consistent with a model in which RelA synthesizes (p)ppGpp while bound to the 70S ribosome. We suggest a 'short hopping time' model of RelA activity during starvation. Our results contradict an earlier study of RelA-Dendra2 diffusion that inferred off-ribosome synthesis of (p)ppGpp. The reasons for the discrepancy remain unclear.

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis.

UNLABELLED: The bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp. IMPORTANCE: Accumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

Escherichia coli O157:H7 of genotype lineage-specific polymorphism assay 211111 and clade 8 are common clinical isolates within Pennsylvania.

Molecular subtyping methods have previously shown that there is a nonrandom distribution of Escherichia coli O157:H7 strains among clinical and nonclinical isolates. Two examples include the lineage-specific polymorphism assay (LSPA) and clade typing assay. The clade typing method was previously used to identify a phylogenetic group of E. coli O157:H7 designated clade 8, which is believed to be more virulent than non-clade 8 isolates, and the LSPA previously indicated that clade 8 isolates are LSPA genotype 211111. Published screens have suggested that LSPA 211111 comprise anywhere from 3.9% to greater than 46% of clinical isolates. To determine the prevalence of such isolates within Pennsylvania, we applied LSPA and screened 52 clinical isolates. We found that 31% of isolates were LSPA 211111 and that 13/16 of these could be classified as clade 8. A rapid polymerase chain reaction screen for clade 8 isolates was developed and shown to have a specificity and sensitivity of 0.92 and 1.0, respectively. Polymerase chain reaction screens indicated that all isolates carried hlyA and eaeA and that all but one of the isolates carried katP. The most common LSPA genotype seen within our collection was 111111, and 29 of 30 of these carried both stx1 and stx2. Clade 8 isolates were more diverse, with four different Shiga toxin profiles observed. We conclude that E. coli O157:H7 of LSPA 211111 and clade 8 are common to clinical isolates in Pennsylvania and suggest that further studies are needed to determine whether their prevalence is increasing as observed elsewhere.

Autophagy sustains glutamate and aspartate synthesis in Saccharomyces cerevisiae during nitrogen starvation.

Autophagy catabolizes cellular constituents to promote survival during nutrient deprivation. Yet, a metabolic comprehension of this recycling operation, despite its crucial importance, remains incomplete. Here, we uncover a specific metabolic function of autophagy that exquisitely adjusts cellular metabolism according to nitrogen availability in the budding yeast Saccharomyces cerevisiae. Autophagy enables metabolic plasticity to promote glutamate and aspartate synthesis, which empowers nitrogen-starved cells to replenish their nitrogen currency and sustain macromolecule synthesis. Our findings provide critical insights into the metabolic basis by which autophagy recycles cellular components and may also have important implications in understanding the role of autophagy in diseases such as cancer.

Regulation of translation by methylation multiplicity of 18S rRNA.

N6-methyladenosine (m6A) is a conserved ribonucleoside modification that regulates many facets of RNA metabolism. Using quantitative mass spectrometry, we find that the universally conserved tandem adenosines at the 3' end of 18S rRNA, thought to be constitutively di-methylated (m62A), are also mono-methylated (m6A). Although present at substoichiometric amounts, m6A at these positions increases significantly in response to sulfur starvation in yeast cells and mammalian cell lines. Combining yeast genetics and ribosome profiling, we provide evidence to suggest that m6A-bearing ribosomes carry out translation distinctly from m62A-bearing ribosomes, featuring a striking specificity for sulfur metabolism genes. Our work thus reveals methylation multiplicity as a mechanism to regulate translation.

SAM homeostasis is regulated by CFIm-mediated splicing of MAT2A.

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism.

Evolution of (p)ppGpp-HPRT regulation through diversification of an allosteric oligomeric interaction.

The alarmone (p)ppGpp regulates diverse targets, yet its target specificity and evolution remain poorly understood. Here, we elucidate the mechanism by which basal (p)ppGpp inhibits the purine salvage enzyme HPRT by sharing a conserved motif with its substrate PRPP. Intriguingly, HPRT regulation by (p)ppGpp varies across organisms and correlates with HPRT oligomeric forms. (p)ppGpp-sensitive HPRT exists as a PRPP-bound dimer or an apo- and (p)ppGpp-bound tetramer, where a dimer-dimer interface triggers allosteric structural rearrangements to enhance (p)ppGpp inhibition. Loss of this oligomeric interface results in weakened (p)ppGpp regulation. Our results reveal an evolutionary principle whereby protein oligomerization allows evolutionary change to accumulate away from a conserved binding pocket to allosterically alter specificity of ligand interaction. This principle also explains how another (p)ppGpp target GMK is variably regulated across species. Since most ligands bind near protein interfaces, we propose that this principle extends to many other protein-ligand interactions.

Diversity in (p)ppGpp metabolism and effectors.

Bacteria produce guanosine tetraphosphate and pentaphosphate, collectively named (p)ppGpp, in response to a variety of environmental stimuli. These two remarkable molecules regulate many cellular processes, including the central dogma processes and metabolism, to ensure survival and adaptation. Work in Escherichia coli laid the foundation for understanding the molecular details of (p)ppGpp and its cellular functions. As recent studies expand to other species, it is apparent that there exists considerable variation, with respect to not only (p)ppGpp metabolism, but also to its mechanism of action. From an evolutionary standpoint, this diversification is an elegant example of how different species adapt a particular regulatory network to their diverse lifestyles.

Basal levels of (p)ppGpp in Enterococcus faecalis: the magic beyond the stringent response.

UNLABELLED: The stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. In Enterococcus faecalis, production of (p)ppGpp is controlled by the bifunctional protein RSH (for "Rel SpoT homologue"; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization of E. faecalis strains lacking rsh, relQ, or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays of E. faecalis strains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the rsh relQ [(p)ppGpp(0)] strain, suggesting that a lack of basal (p)ppGpp places the cell in a "transcriptionally relaxed" state. Alterations in the fermentation profile and increased production of H2O2 in the (p)ppGpp(0) strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen in Bacillus subtilis, (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions. IMPORTANCE: Drug-resistant bacterial infections continue to pose a significant public health threat by limiting therapeutic options available to care providers. The stringent response (SR), mediated by the accumulation of two modified guanine nucleotides collectively known as (p)ppGpp, is a highly conserved stress response that broadly remodels bacterial physiology to a survival state. Given the strong correlation of the SR with the ability of bacteria to survive antibiotic treatment and the direct association of (p)ppGpp production with bacterial infectivity, understanding how bacteria produce and utilize (p)ppGpp may reveal potential targets for the development of new antimicrobial therapies. Using the multidrug-resistant pathogen Enterococcus faecalis as a model, we show that small alterations to (p)ppGpp levels, well below concentrations needed to trigger the SR, severely affected bacterial metabolism and antibiotic survival. Our findings highlight the often-underappreciated contribution of basal (p)ppGpp levels to metabolic balance and stress tolerance in bacteria.