Identification,Characterization, and Structure Analysis of the Cyclic di-AMP-binding PII-like Signal Transduction Protein DarA

The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to P_II signal transducer proteins. In contrast to P_II proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3′3′-cGAMP) but not c-di-GMP or 2′3′-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA.     

Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP)

Although it was only recently identified as a second messenger, c-di-AMP was found to have fundamental importance in numerous bacterial functions such as ion transport. The potassium transporter protein, KtrA, was identified as a c-di-AMP receptor. However, the co-crystallization of c-di-AMP with the protein has not been studied. Here, we determined the crystal structure of the KtrA RCK_C domain in complex with c-di-AMP. The c-di-AMP nucleotide, which adopts a U-shaped conformation, is bound at the dimer interface of RCK_C close to helices α3 and α4. c-di-AMP interacts with KtrA RCK_C mainly by forming hydrogen bonds and hydrophobic interactions. c-di-AMP binding induces the contraction of the dimer, bringing the two monomers of KtrA RCK_C into close proximity. The KtrA RCK_C was able to interact with only c-di-AMP, but not with c-di-GMP, 3′,3-cGAMP, ATP, and ADP. The structure of the KtrA RCK_C domain and c-di-AMP complex would expand our understanding about the mechanism of inactivation in Ktr transporters governed by c-di-AMP.     

Identification and characterization of phosphodiesterases that specifically degrade 3′3′-cyclic GMP-AMP

Cyclic dinucleotides act as intracellular second messengers, modulating a variety of cellular activities including innate immune activation. Although phosphodiesterases (PDEs) hydrolyzing c-di-GMP and c-di-AMP have been identified, no PDEs for cGAMPs have been reported. Here we identified the first three cGAMP-specific PDEs in V. cholerae (herein designated as V-cGAP1/2/3). V-cGAPs are HD-GYP domain-containing proteins and specifically break 3′3′-cGAMP, but not other forms of cGAMP. 3′3′-cGAMP is first linearized by all three V-cGAPs to produce 5′-pApG, which is further hydrolyzed into 5′-ApG by V-cGAP1. In this two-step reaction, V-cGAP1 functions as both a PDE and a 5′-nucleotidase. In vivo experiments demonstrated that V-cGAPs play non-redundant roles in cGAMP degradation. The high specificity of V-cGAPs on 3′3′-cGAMP suggests the existence of specific PDEs for other cGAMPs, including 2′3′-cGAMP in mammalian cells. The absolute requirement of the GYP motif for 3′3′-cGAMP degradation suggests that HD domain-containing PDEs in eukaryotes are probably unable to hydrolyze cGAMPs. The fact that all V-cGAPs attack 3′3′-cGAMP on one specific phosphodiester bond suggests that PDEs for other cGAMPs would utilize a similar strategy. These results will provide valuable information for identification and characterization of mammalian 2′3′-cGAMP-specific PDEs in future studies.     

V-cGAPs: attenuators of 3′3′-cGAMP signaling

Cyclic GMP-AMPs (cGAMPs) are new members of the cyclic dinucleotide family of second messenger signaling molecules identified in both bacteria and mammalian cells. A recent study by Gao et al. published in Cell Research has identified and characterized three 3′3′-cGAMP-specific phosphodiesterases (termed as V-cGAP1/2/3) in V. cholerae, thereby providing mechanistic insights into the function of these enzymes that degrade cGAMPs.Despite their indispensable roles in the composition of DNA and RNA, as well as serving as energy sources, nucleotides are also well known as crucial signaling molecules in all domains of life. Cyclic dinucleotides (CDNs) represent an important and growing family of second messengers, which have been previously recognized as key modulators governing a variety of cellular activities in bacteria, and more recently, in mammalian cells. c-di-GMP and c-di-AMP, the first two members of the CDN family, have been implicated in central bacterial processes, and likely act as universal bacterial secondary messengers^1,2. The latest addition to the bacterial CDN family is 3′3′-cGAMP, a hybrid molecule that is synthesized from ATP and GTP by DncV (a cyclase from V. cholerae) and shown to promote intestinal colonization of V. cholerae by downregulating chemotaxis³. Predicted homologs of DncV are present in many other bacterial species³, indicating that 3′3′-cGAMP may also regulate a wide range of cellular functions, similar to c-di-GMP and c-di-AMP. The research on CDNs as second messengers reached new heights following the recent identification of 2′3′-cGAMP, a noncanonical CDN in mammalian cells containing mixed 2′,5′ (at GpA step) and 3′,5′ (at ApG step) linkages, which is synthesized by cGAMP synthase (cGAS) in response to the presence of DNA in the cytosol^4,5,6. A remarkable set of new discoveries have revealed that all the CDNs described above are able to bind and activate STING, the central adaptor in the cytosolic DNA sensing pathway, thereby promoting the innate immune response in mammalian cells by inducing the expression of Type I interferon (IFN)^7,8,9.Given their critical roles in a variety of important cellular processes, the cellular levels of CDNs have to be tightly controlled by the coordinated action of counteracting cyclases and degradation enzymes. To date, several phosphodiesterases (PDEs) have been found to hydrolyze c-di-GMP (EAL or HD-GYP domain-containing enzymes)¹ and c-di-AMP (DHH-DHHA or HD domain-containing enzymes)^2,10 (Figure 1). In addition, recent research reported that ENPP1 (ecto-nucleotide pyrophosphatase/phosphodiesterase) is the dominant 2′3′-cGAMP hydrolyzing enzyme in mammalian cells¹¹ (Figure 1). A new study by Gao et al.¹² has now identified the first three 3′3′-cGAMP-specific PDEs in V. cholerae and provided detailed insights into their enzymatic mechanisms.Open in a separate windowFigure 1Schematic representation of degradation enzymes identified for different cyclic dinucleotides and the related hydrolysis products. The various protein domains are highlighted by different shapes and colors. Note that the newly identified V-cGAPs belong to the HD-GYP domain-containing PDEs.There are a total of 36 potential PDE genes (containing EAL, HD-GYP or DHH domains) in the V. cholerae genome. To search for 3′3′-cGAMP-specific PDE(s), Gao et al.¹² established an efficient and sensitive eukaryotic screening system by taking advantage of the ability of 3′3′-cGAMP to activate STING and induce type I IFN expression in mammalian cells. By overexpressing the 3′3′-cGAMP synthetase DncV together with the 36 potential PDEs in 293 cells, the authors could monitor IFN-β promoter activation to identify the PDE(s) that could degrade 3′3′-cGAMP. To exclude false-positives, Gao et al. further purified the PDEs that potentially target 3′3′-cGAMP based on the initial screening, and incubated these enzymes with chemically synthesized 3′3′-cGAMP. The treated 3′3′-cGAMP molecules were further assayed by either adding to PFO-permeabilized THP-1 cells to examine IRF3 phosphorylation levels or through loading on HPLC to monitor the generation of new products. As a result of the screening and validation, the authors successfully identified three HD-GYP domain-containing proteins that could degrade 3′3′-cGAMP, named VCA0681, VCA0210 and VCA0931 (designated as V-cGAP1, 2 and 3, respectively).To determine the substrate specificity of V-cGAPs, different cGAMP linkage isomers (3′3′-, 3′2′-, 2′3′-, and 2′2′-cGAMPs) were incubated with the purified V-cGAPs. The results of both IRF3 phosphorylation in THP-1 cells and HPLC assays clearly indicated that V-cGAPs only degrade 3′3′-cGAMP, but not other cGAMP linkage isomers. The 3′3′-cGAMP PDE activity of V-cGAPs was further confirmed by dosage- and time-dependent enzymatic assays. By using mutant proteins, the authors also confirmed that both the HD and GYP motifs within V-cGAPs are critical for PDE activity.Combining detailed HPLC analysis, mass spectrometry and enzymatic treatment, Gao et al. definitively established that 3′3′-cGAMP is first hydrolyzed by all three V-cGAPs to generate linear 5′-pApG, which is further hydrolyzed into 5′-ApG only by V-cGAP1. These results show that V-cGAP2 and V-cGAP3 have only PDE activity, while V-cGAP1 has both PDE and 5′-nucleotidase activities. The authors also found that V-cGAP1 has a much higher activity for linearization of 3′3′-cGAMP to 5′-pApG than V-cGAP2 and 3, with the later two V-cGAPs exhibiting similar kinetics of degradation.The cellular level of 3′3′-cGAMP has to be tightly regulated by a combination of counteracting synthesis and degradation enzymes. Since the expression level of DncV was found to be inducible by outside signals to enhance intestinal colonization and infectivity, it is very likely that the expression level of V-cGAPs will also be regulated by 3′3′-cGAMP production. Indeed, the authors proved that V-cGAP expression is greatly and readily enhanced after arabinose-induced DncV expression in a ΔdncV mutant V. cholerae strain, at both mRNA (by qRT-PCR) and protein (by immunoblot analysis) levels. To confirm the in vivo function of V-cGAPs, the authors performed both “chemotactic” and “infant mouse colonization competition” assays by using V-cGAP1/2/3 single-, double-, or triple-deletion V. cholerae strains. All the in vivo data clearly established that V-cGAPs counteract DncV function and exert a crucial role in regulating bacterial infectivity.The large amount of insightful data presented by Gao et al. has elucidated detailed information regarding the identification and characterization of 3′3′-cGAMP-specific phosphodiesterases, thereby providing valuable insights into our understanding of the regulatory mechanisms of cGAMP signaling in bacteria. Clearly, further structural work will be necessary to understand the intermolecular interactions between 3′3′-cGAMP and V-cGAPs, and provide insights into the mechanism by which V-cGAPs preferentially attack the phosphodiester bond at the GpA step.     

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

The intracellular infections of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, are regulated by many cyclic dinucleotide signaling. Rv2837c from M. tuberculosis is a soluble, stand-alone DHH-DHHA1 domain phosphodiesterase that down-regulates c-di-AMP through catalytic degradation and plays an important role in M. tuberculosis infections. Here, we report the crystal structure of Rv2837c (2.0 Å), and its complex with hydrolysis intermediate 5′-pApA (2.35 Å). Our structures indicate that both DHH and DHHA1 domains are essential for c-di-AMP degradation. Further structural analysis shows that Rv2837c does not distinguish adenine from guanine, which explains why Rv2837c hydrolyzes all linear dinucleotides with almost the same efficiency. We observed that Rv2837c degraded other c-di-NMPs at a lower rate than it did on c-di-AMP. Nevertheless, our data also showed that Rv2837c significantly decreases concentrations of both c-di-AMP and c-di-GMP in vivo. Our results suggest that beside its major role in c-di-AMP degradation Rv2837c could also regulate c-di-GMP signaling pathways in bacterial cell.     

The Smac mimetic BV6 cooperates with STING to induce necroptosis in apoptosis-resistant pancreatic carcinoma cells

Pancreatic cancer (PC) still remains a major cause of cancer-related death worldwide and alternative treatments are urgently required. A common problem of PC is the development of resistance against apoptosis that limits therapeutic success. Here we demonstrate that the prototypical Smac mimetic BV6 cooperates with the stimulator of interferon (IFN) genes (STING) ligand 2′,3′-cyclic guanosine monophosphate–adenosine monophosphate (2′3′-cGAMP) to trigger necroptosis in apoptosis-deficient PC cells. Pharmacological inhibition of key components of necroptosis signaling, such as receptor-interacting protein 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), significantly rescues PC cells from 2′3′-cGAMP/BV6/zVAD.fmk-mediated cell death, suggesting the induction of necroptosis. Consistently, 2′3′-cGAMP/BV6 co-treatment promotes phosphorylation of MLKL. Furthermore, we show that 2′3′-cGAMP stimulates the production of type I IFNs, which cooperate with BV6 to trigger necroptosis in apoptosis-deficient settings. STING silencing via siRNA or CRISPR/Cas9-mediated gene knockout protects PC cells from 2′3′-cGAMP/BV6/zVAD.fmk-mediated cell death. Interestingly, we demonstrate that nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNFα), and IFN-regulatory factor 1 (IRF1) signaling are involved in triggering 2′3′-cGAMP/BV6/zVAD.fmk-induced necroptosis. In conclusion, we show that activated STING and BV6 act together to exert antitumor effects on PC cells with important implications for the design of new PC treatment concepts.Subject terms: Cancer, Cancer     

Structure–activity relationships in human RNA 3′-phosphate cyclase

RNA 3′-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2′,3′ cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP_i; transfer of AMP from Rtc1 to an RNA 3′-phosphate to form an RNA(3′)pp(5′)A intermediate; and attack by the terminal nucleoside O2′ on the 3′-phosphate to form an RNA 2′,3′ cyclic phosphate product and release AMP. Here we used the crystal structure of Escherichia coli RtcA to guide a mutational analysis of the human RNA cyclase Rtc1. An alanine scan defined seven conserved residues as essential for the Rtc1 RNA cyclization and autoadenylylation reactions. Structure–activity relationships were clarified by conservative substitutions. Our results are consistent with a mechanism of adenylate transfer in which attack of the Rtc1 His320 nucleophile on the ATP α phosphorus is facilitated by proper orientation of the PP_i leaving group via contacts to Arg21, Arg40, and Arg43. We invoke roles for Tyr294 in binding the adenine base and Glu14 in binding the divalent cation cofactor. We find that Rtc1 forms a stable binary complex with a 3′-phosphate terminated RNA, but not with an otherwise identical 3′-OH terminated RNA. Mutation of His320 had little impact on RNA 3′-phosphate binding, signifying that covalent adenylylation of Rtc1 is not a prerequisite for end recognition.     

The Mucosal Adjuvant Cyclic di-AMP Exerts Immune Stimulatory Effects on Dendritic Cells and Macrophages

The cyclic di-nucleotide bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP) is a candidate mucosal adjuvant with proven efficacy in preclinical models. It was shown to promote specific humoral and cellular immune responses following mucosal administration. To date, there is only fragmentary knowledge on the cellular and molecular mode of action of c-di-AMP. Here, we report on the identification of dendritic cells and macrophages as target cells of c-di-AMP. We show that c-di-AMP induces the cell surface up-regulation of T cell co-stimulatory molecules as well as the production of interferon-β. Those responses were characterized by in vitro experiments with murine and human immune cells and in vivo studies in mice. Analyses of dendritic cell subsets revealed conventional dendritic cells as principal responders to stimulation by c-di-AMP. We discuss the impact of the reported antigen presenting cell activation on the previously observed adjuvant effects of c-di-AMP in mouse immunization studies.     

Co-occupancy of two Pumilio molecules on a single hunchback NRE

Pumilio controls a number of processes in eukaryotes, including the translational repression of hunchback (hb) mRNA in early Drosophila embryos. The Pumilio Puf domain binds to a pair of 32 nucleotide (nt) Nanos response elements (NRE1 and NRE2) within the 3′ untranslated region of hb mRNA. Despite the elucidation of structures of human Pumilio Puf domain in complex with hb RNA elements, the nature of hb mRNA recognition remains unclear. In particular, the site that mediates regulation in vivo is significantly larger than the 8–10-nt RNA elements bound to single Puf molecules in crystal structures. Here we present biophysical and biochemical data that partially resolve the paradox. We show that each NRE is composed of two binding sites (Box A and Box B) and that two Puf domains can co-occupy a single NRE. The Puf domains have a higher affinity for the 3′ Box B site than the 5′ Box A site; binding to the intact NRE appears to be cooperative (at least in some experiments). We suggest that the 2 Pumilio:1 NRE complex is the functional regulatory unit in vivo.     

Multiple hPOT1–TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3′ toward 5′, a feature due to a 3′-end binding preference and to structuring of telomeric DNA

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K⁺ and in Li⁺, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.     

Two-Step Synthesis and Hydrolysis of Cyclic di-AMP in Mycobacterium tuberculosis

Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5′-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5′-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5′-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE.     

Specific recognition of a multiply phosphorylated motif in the DNA repair scaffold XRCC1 by the FHA domain of human PNK

Short-patch repair of DNA single-strand breaks and gaps (SSB) is coordinated by XRCC1, a scaffold protein that recruits the DNA polymerase and DNA ligase required for filling and sealing the damaged strand. XRCC1 can also recruit end-processing enzymes, such as PNK (polynucleotide kinase 3′-phosphatase), Aprataxin and APLF (aprataxin/PNK-like factor), which ensure the availability of a free 3′-hydroxyl on one side of the gap, and a 5′-phosphate group on the other, for the polymerase and ligase reactions respectively. PNK binds to a phosphorylated segment of XRCC1 (between its two C-terminal BRCT domains) via its Forkhead-associated (FHA) domain. We show here, contrary to previous studies, that the FHA domain of PNK binds specifically, and with high affinity to a multiply phosphorylated motif in XRCC1 containing a pSer-pThr dipeptide, and forms a 2:1 PNK:XRCC1 complex. The high-resolution crystal structure of a PNK–FHA–XRCC1 phosphopeptide complex reveals the basis for this unusual bis-phosphopeptide recognition, which is probably a common feature of the known XRCC1-associating end-processing enzymes.     

DNA3′pp5′G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition

DNA_3′pp_5′G caps synthesized by the 3′-PO₄/5′-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3′-OH ends. Aprataxin, an enzyme that repairs A_5′pp_5′DNA ends formed during abortive ligation by classic 3′-OH/5′-PO₄ ligases, is also a DNA 3′ de-capping enzyme, converting DNAppG to DNA_3′p and GMP. By taking advantage of RtcB''s ability to utilize certain GTP analogs to synthesize DNAppN caps, we show that aprataxin hydrolyzes inosine and 6-O-methylguanosine caps, but is not adept at removing a deoxyguanosine cap. We report a 1.5 Å crystal structure of aprataxin in a complex with GMP, which reveals that: (i) GMP binds at the same position and in the same anti nucleoside conformation as AMP; and (ii) aprataxin makes more extensive nucleobase contacts with guanine than with adenine, via a hydrogen bonding network to the guanine O6, N1, N2 base edge. Alanine mutations of catalytic residues His147 and His149 abolish DNAppG de-capping activity, suggesting that the 3′ de-guanylylation and 5′ de-adenylylation reactions follow the same pathway of nucleotidyl transfer through a covalent aprataxin-(His147)–NMP intermediate. Alanine mutation of Asp63, which coordinates the guanosine ribose hydroxyls, impairs DNAppG de-capping.     

Analysis of the DNA joining repertoire of Chlorella virus DNA ligase and a new crystal structure of the ligase-adenylate intermediate

Chlorella virus DNA ligase is the smallest eukaryotic ATP-dependent DNA ligase known; it suffices for yeast cell growth in lieu of the essential yeast DNA ligase Cdc9. The Chlorella virus ligase–adenylate intermediate has an intrinsic nick sensing function and its DNA footprint extends 8–9 nt on the 3′-hydroxyl (3′-OH) side of the nick and 11–12 nt on the 5′-phosphate (5′-PO₄) side. Here we establish the minimal length requirements for ligatable 3′-OH and 5′-PO₄ strands at the nick (6 nt) and describe a new crystal structure of the ligase–adenylate in a state construed to reflect the configuration of the active site prior to nick recognition. Comparison with a previous structure of the ligase–adenylate bound to sulfate (a mimetic of the nick 5′-PO₄) suggests how the positions and contacts of the active site components and the bound adenylate are remodeled by DNA binding. We find that the minimal Chlorella virus ligase is capable of catalyzing non-homologous end-joining reactions in vivo in yeast, a process normally executed by the structurally more complex cellular Lig4 enzyme. Our results suggest a model of ligase evolution in which: (i) a small ‘pluripotent’ ligase is the progenitor of the much larger ligases found presently in eukaryotic cells and (ii) gene duplications, variations within the core ligase structure and the fusion of new domains to the core structure (affording new protein–protein interactions) led to the compartmentalization of eukaryotic ligase function, i.e. by enhancing some components of the functional repertoire of the ancestral ligase while disabling others.     

Structure of RNA 3′-phosphate cyclase bound to substrate RNA

RNA 3′-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3′-phosphate to form a 2′,3′-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA–AMP and RNA(3′)pp(5′)A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3′-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3′-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3′-phosphate is poised for in-line attack on the P–N bond that links the phosphorous atom of AMP to N^ε of His307. Thus, we provide the first insights into RNA 3′-phosphate termini recognition and the mechanism of 3′-phosphate activation by an Rtc enzyme.     

The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms

The Lsm1-7-Pat1 complex binds to the 3′ end of cellular mRNAs and promotes 3′ end protection and 5′–3′ decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.     

High-resolution crystal structure of the intramolecular d(TpA) thymine-adenine photoadduct and its mechanistic implications

A high-resolution crystal structure is reported for d(TpA)*, the intramolecular thymine–adenine photoadduct that is produced by direct ultraviolet excitation of the dinucleoside monophosphate d(TpA). It confirms the presence of a central 1,3-diazacyclooctatriene ring linking the remnants of the T and A bases, as previously deduced from heteronuclear NMR measurements by Zhao et al. (The structure of d(TpA)*, the major photoproduct of thymidylyl-(3′-5′)-deoxyadenosine. Nucleic Acids Res., 1996, 24, 1554–1560). Within the crystal, the d(TpA)* molecules exist as zwitterions with a protonated amidine fragment of the eight-membered ring neutralizing the charge of the internucleotide phosphate monoanion. The absolute configuration at the original thymine C5 and C6 atoms is determined as 5S,6R. This is consistent with d(TpA)* arising by valence isomerization of a precursor cyclobutane photoproduct with cis–syn stereochemistry that is generated by [2 + 2] photoaddition of the thymine 5,6-double bond across the C6 and C5 positions of adenine. This mode of photoaddition should be favoured by the stacked conformation of adjacent T and A bases in B-form DNA. It is probable that the primary photoreaction is mechanistically analogous to pyrimidine dimerization despite having a much lower quantum yield.     

The C-terminal extension of Lsm4 interacts directly with the 3′ end of the histone mRNP and is required for efficient histone mRNA degradation

Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem–loop sequence, which is bound to the stem–loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3′ end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3′hExo, forms a ternary complex with SLBP and the stem–loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3′hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3′hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.     

The telomeric 5′ end nucleotide is regulated in the budding yeast Naumovozyma castellii

The junction between the double-stranded and single-stranded telomeric DNA (ds–ss junction) is fundamental in the maintenance of the telomeric chromatin, as it directs the assembly of the telomere binding proteins. In budding yeast, multiple Rap1 proteins bind the telomeric dsDNA, while ssDNA repeats are bound by the Cdc13 protein. Here, we aimed to determine, for the first time, the telomeric 5′ end nucleotide in a budding yeast. To this end, we developed a permutation-specific PCR-based method directed towards the regular 8-mer telomeric repeats in Naumovozyma castellii. We find that, in logarithmically growing cells, the 320 ± 30 bp long telomeres mainly terminate in either of two specific 5′ end permutations of the repeat, both corresponding to a terminal adenine nucleotide. Strikingly, two permutations are completely absent at the 5′ end, indicating that not all ds‐ss junction structures would allow the establishment of the protective telomere chromatin cap structure. Using in vitro DNA end protection assays, we determined that binding of Rap1 and Cdc13 around the most abundant ds–ss junction ensures the protection of both 5′ ends and 3′ overhangs from exonucleolytic degradation. Our results provide mechanistic insights into telomere protection, and reveal that Rap1 and Cdc13 have complementary roles.     

Cyclic Dinucleotide-Controlled Regulatory Pathways in Streptomyces Species

The cyclic dinucleotides cyclic 3′,5′-diguanylate (c-di-GMP) and cyclic 3′,5′-diadenylate (c-di-AMP) have emerged as key components of bacterial signal transduction networks. These closely related second messengers follow the classical general principles of nucleotide signaling by integrating diverse signals into regulatory pathways that control cellular responses to changing environments. They impact distinct cellular processes, with c-di-GMP having an established role in promoting bacterial adhesion and inhibiting motility and c-di-AMP being involved in cell wall metabolism, potassium homeostasis, and DNA repair. The involvement of c-dinucleotides in the physiology of the filamentous, nonmotile streptomycetes remained obscure until recent discoveries showed that c-di-GMP controls the activity of the developmental master regulator BldD and that c-di-AMP determines the level of the resuscitation-promoting factor A(RpfA) cell wall-remodelling enzyme. Here, I summarize our current knowledge of c-dinucleotide signaling in Streptomyces species and highlight the important roles of c-di-GMP and c-di-AMP in the biology of these antibiotic-producing, multicellular bacteria.