ABSTRACT
The DNA sensor cGAS and its adaptor STING constitute an ancient pathogen detection mechanism, but it is unclear to what extent its function is conserved across the animal kingdom. In this issue of Cell, Kranzusch and colleagues identify thousands of cGAS-like receptors and discover networks of second messengers that activate innate immune responses in animals.
Subject(s)
Membrane Proteins , Signal Transduction , Animals , Signal Transduction/physiology , Nucleotidyltransferases/metabolism , Immunity, Innate , Protein Serine-Threonine Kinases/metabolismABSTRACT
DNA damage can be sensed as a danger-associated molecular pattern by the innate immune system. Here we find that keratinocytes and other human cells mount an innate immune response within hours of etoposide-induced DNA damage, which involves the DNA sensing adaptor STING but is independent of the cytosolic DNA receptor cGAS. This non-canonical activation of STING is mediated by the DNA binding protein IFI16, together with the DNA damage response factors ATM and PARP-1, resulting in the assembly of an alternative STING signaling complex that includes the tumor suppressor p53 and the E3 ubiquitin ligase TRAF6. TRAF6 catalyzes the formation of K63-linked ubiquitin chains on STING, leading to the activation of the transcription factor NF-κB and the induction of an alternative STING-dependent gene expression program. We propose that STING acts as a signaling hub that coordinates a transcriptional response depending on its mode of activation.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , Cell Nucleus/genetics , DNA Damage/genetics , Membrane Proteins/genetics , NF-kappa B/genetics , Nuclear Proteins/genetics , Phosphoproteins/genetics , Signal Transduction/genetics , Cell Line , Cytosol/metabolism , DNA/genetics , HEK293 Cells , Humans , Immunity, Innate/genetics , Keratinocytes/physiology , Poly (ADP-Ribose) Polymerase-1/genetics , Tumor Suppressor Protein p53/genetics , Ubiquitin/genetics , Ubiquitin-Protein Ligases/geneticsABSTRACT
The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at â¼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway.
Subject(s)
DNA/chemistry , Nuclear Proteins/chemistry , Phosphoproteins/chemistry , Crystallography, X-Ray , DNA/metabolism , Models, Molecular , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , Protein Domains , Protein MultimerizationABSTRACT
The detection of intracellular microbial DNA is critical to appropriate innate immune responses; however, knowledge of how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-ß (IFN-ß). IFI16 directly associated with IFN-ß-inducing viral DNA motifs. STING, a critical mediator of IFN-ß responses to DNA, was recruited to IFI16 after DNA stimulation. Lowering the expression of IFI16 or its mouse ortholog p204 by RNA-mediated interference inhibited gene induction and activation of the transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 (p204) is the first PYHIN protein to our knowledge shown to be involved in IFN-ß induction. Thus, the PYHIN proteins IFI16 and AIM2 form a new family of innate DNA sensors we call 'AIM2-like receptors' (ALRs).
Subject(s)
DNA, Viral/immunology , Immunity, Innate , Intracellular Space/immunology , Nuclear Proteins/immunology , Phosphoproteins/immunology , Animals , Cell Line , DNA-Binding Proteins , Herpesvirus 1, Human/immunology , Humans , Interferon-beta/immunology , Interferon-beta/metabolism , Membrane Proteins/immunology , Mice , Monocytes/immunology , Signal TransductionABSTRACT
Recognition of DNA by the innate immune system is central to antiviral and antibacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.
Subject(s)
DNA, B-Form/metabolism , DNA-Binding Proteins/chemistry , Inflammasomes/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Amino Acid Sequence , Cell Line , Crystallography, X-Ray , DNA, B-Form/chemistry , DNA, B-Form/immunology , Humans , Immunity, Innate , Inflammasomes/genetics , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/chemistry , Protein Binding , Protein Folding , Protein Structure, Tertiary , Signal TransductionABSTRACT
Intracellular DNA and RNA sensors play a vital part in the innate immune response to viruses and other intracellular pathogens, causing the secretion of type I interferons, cytokines and chemokines from infected cells. Pathogen RNA can be detected by retinoic-acid inducible gene I-like receptors in the cytosol, whereas cytosolic DNA is recognized by DNA sensors such as cyclic GMP-AMP synthase (cGAS). The resulting local immune response, which is initiated within hours of infection, is able to eliminate many pathogens before they are able to establish an infection in the host. For this reason, all viruses, and some intracellular bacteria and protozoa, need to evade detection by nucleic acid sensors. Immune evasion strategies include the sequestration and modification of nucleic acids, and the inhibition or degradation of host factors involved in innate immune signalling. Large DNA viruses, such as herpesviruses, often use multiple viral proteins to inhibit signalling cascades at several different points; for instance herpes simplex virus 1 targets both DNA sensors cGAS and interferon-γ-inducible protein 16, as well as the adaptor protein STING (stimulator of interferon genes) and other signalling factors in the pathway. Viruses with a small genome encode only a few immunomodulatory proteins, but these are often multifunctional, such as the NS1 protein from influenza A virus, which inhibits RNA sensing in multiple ways. Intracellular bacteria and protozoa can also be detected by nucleic acid sensors. However, as the type I interferon response is not always beneficial for the host under these circumstances, some bacteria subvert, rather than evade, these signalling cascades for their own gain.
Subject(s)
Immune Evasion/immunology , Nucleic Acids/immunology , Animals , Bacteria/immunology , Cytoplasm/immunology , Humans , Immunity, Innate/immunology , Interferon Type I/immunology , Signal Transduction/immunology , Viruses/immunologySubject(s)
Nucleotides, Cyclic , Nucleotidyltransferases , DNA , Dinucleoside Phosphates , Immunity, InnateABSTRACT
The transcription factor NF-κB is essential for immune responses against pathogens and its activation requires the phosphorylation, ubiquitination and proteasomal degradation of IκBα. Here we describe an inhibitor of NF-κB from vaccinia virus that has a closely related counterpart in variola virus, the cause of smallpox, and mechanistic similarity with the HIV protein Vpu. Protein A49 blocks NF-κB activation by molecular mimicry and contains a motif conserved in IκBα which, in IκBα, is phosphorylated by IKKß causing ubiquitination and degradation. Like IκBα, A49 binds the E3 ligase ß-TrCP, thereby preventing ubiquitination and degradation of IκBα. Consequently, A49 stabilised phosphorylated IκBα (p-IκBα) and its interaction with p65, so preventing p65 nuclear translocation. Serine-to-alanine mutagenesis within the IκBα-like motif of A49 abolished ß-TrCP binding, stabilisation of p-IκBα and inhibition of NF-κB activation. Remarkably, despite encoding nine other inhibitors of NF-κB, a VACV lacking A49 showed reduced virulence in vivo.
Subject(s)
Molecular Mimicry , NF-kappa B/antagonists & inhibitors , Ubiquitin-Protein Ligases/metabolism , Vaccinia virus/pathogenicity , Variola virus/pathogenicity , beta-Transducin Repeat-Containing Proteins/metabolism , Animals , Cell Line , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Immune Evasion , Mice , Mice, Inbred BALB C , Mutagenesis, Site-Directed , NF-kappa B/genetics , NF-kappa B/metabolism , Phosphorylation , Protein Binding , Ubiquitin-Protein Ligases/genetics , Vaccinia virus/genetics , Vaccinia virus/immunology , Variola virus/genetics , Variola virus/immunology , Virulence , beta-Transducin Repeat-Containing Proteins/geneticsABSTRACT
The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9 and that nonimmune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, such as macrophages and conventional dendritic cells, detect infections with herpesviruses. In this study, we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFN-γ-inducible 16 is important for induction of IFN-ß in human macrophages postinfection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as did IFN-γ-inducible 16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors.
Subject(s)
Capsid/metabolism , Cytomegalovirus/metabolism , DNA, Viral/genetics , Herpesvirus 1, Human/metabolism , Macrophages/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Chlorocebus aethiops , Cytomegalovirus/genetics , Cytosol/metabolism , DNA, Viral/immunology , Dendritic Cells/metabolism , Dendritic Cells/virology , Gene Silencing , Herpesvirus 1, Human/genetics , Humans , Interferon-beta/biosynthesis , Interferon-beta/immunology , Macrophages/virology , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/immunology , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/immunology , RNA, Small Interfering/genetics , Ubiquitination , Vero CellsABSTRACT
Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-ß (IFN-ß) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-ß expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-ß promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.
Subject(s)
Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factor-7/metabolism , Protein Serine-Threonine Kinases/metabolism , Vaccinia virus/genetics , Viral Proteins/genetics , Gene Expression Regulation, Viral , Genes, Regulator , HEK293 Cells , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Immune Evasion , Immunity, Innate , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-7/genetics , Interferon-beta/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Open Reading Frames , Phosphorylation , Plasmids , Protein Binding/genetics , Protein Serine-Threonine Kinases/genetics , Receptors, Pattern Recognition/metabolism , Signal Transduction , Transcription, Genetic , Vaccinia virus/metabolism , Vaccinia virus/physiology , Viral Proteins/metabolism , Virulence Factors/genetics , Virulence Factors/metabolism , Virus ReplicationABSTRACT
The DNA sensor cGAS (cyclic GMP-AMP synthase) and its adaptor protein STING (Stimulator of Interferon Genes) detect the presence of cytosolic DNA as a sign of infection or damage. In cancer cells, this pathway can be activated through persistent DNA damage and chromosomal instability, which results in the formation of micronuclei and the exposure of DNA fragments to the cytosol. DNA damage from radio- or chemotherapy can further activate DNA sensing responses, which may occur in the cancer cells themselves or in stromal and immune cells in the tumour microenvironment (TME). cGAS-STING signalling results in the production of type I interferons, which have been linked to immune cell infiltration in 'hot' tumours that are susceptible to immunosurveillance and immunotherapy approaches. However, recent research has highlighted the complex nature of STING signalling, with tumours having developed mechanisms to evade and hijack this signalling pathway for their own benefit. In this mini-review we will explore how cGAS-STING signalling in different cells in the TME can promote both anti-tumour and pro-tumour responses. This includes the role of type I interferons and the second messenger cGAMP in the TME, and the influence of STING signalling on local immune cell populations. We examine how alternative signalling cascades downstream of STING can promote chronic interferon signalling, the activation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and the production of inflammatory cytokines, which can have pro-tumour functions. An in-depth understanding of DNA sensing in different cell contexts will be required to harness the anti-tumour functions of STING signalling.
Subject(s)
Interferon Type I , Neoplasms , Humans , Immunity, Innate/genetics , DNA/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Interferon Type I/genetics , Interferon Type I/metabolism , Neoplasms/therapy , Tumor MicroenvironmentABSTRACT
Type I interferons (IFNs) are critical for anti-viral responses, and also drive autoimmunity when dysregulated. Upon viral sensing, monocytes elicit a sequential cascade of IFNß and IFNα production involving feedback amplification, but how exactly this cascade is regulated in human cells is incompletely understood. Here we show that the PYHIN protein myeloid cell nuclear differentiation antigen (MNDA) is required for IFNα induction in monocytes. Unlike other PYHINs, this is not due to a pathogen sensing role, but rather MNDA regulated expression of IRF7, a transcription factor essential for IFNα induction. Mechanistically, MNDA is required for recruitment of STAT2 and RNA polymerase II to the IRF7 gene promoter, and in fact MNDA is itself recruited to the IRF7 promoter after type I IFN stimulation. These data implicate MNDA as a critical regulator of the type I IFN cascade in human myeloid cells and reveal a new role for human PYHINs in innate immune gene induction.
Subject(s)
Antigens, Differentiation, Myelomonocytic/metabolism , Immunity, Innate , Interferon Type I/metabolism , Myeloid Cells/metabolism , Transcription Factors/metabolism , Cell Line , Gene Expression Regulation , Humans , Immunity, Innate/genetics , Immunity, Innate/physiology , Monocytes/metabolismABSTRACT
Rabies represents a typical model for spillover of zoonotic viral diseases among multiple hosts. Understanding the success of rabies virus (RV) in switching hosts requires the analysis of viral evolution and host interactions. In this study, we have investigated the structural and sequence analysis of host receptors among different RV susceptible host species. Our extensive bioinformatic analysis revealed the absence of the integrin plexin domain in the integrin ß1 (ITGB1) receptor of the black fruit bats in the current annotation of the genome. Interestingly, the nicotinic acetyl choline receptor (nAChR) interaction site with the glycoprotein (G) of RV was conserved among different species. To study the interaction dynamics between RV-G protein and the RV receptors, we constructed and analyzed structures of RV receptors and G proteins using homology modeling. The molecular docking of protein-protein interaction between RV-G protein and different host receptors highlighted the variability of interacting residues between RV receptors of different species. These in silico structural analysis and interaction mapping of viral protein and host receptors establish the foundation to understand complex entry mechanisms of RV entry, which may facilitate the understanding of receptor mediated spillover events in RV infections and guide the development of novel vaccines to contain the infection.
Subject(s)
Rabies virus , Rabies , Glycoproteins/genetics , Humans , Molecular Docking Simulation , Receptors, VirusABSTRACT
Self-DNA has previously been thought to be protected from immune detection by compartmentalisation in the nucleus or mitochondria. Here, we describe the discovery of a signalling cascade that links the detection of DNA damage in the nucleus to the activation of the innate immune adaptor STING (STimulator of INterfern Genes).
ABSTRACT
The immediate response to viral infection relies on pattern-recognition receptors (PRRs), most prominently the Toll-like receptors (TLRs) and the RNA helicases RIG-I and MDA5, as well as double stranded RNA-dependent protein kinase (PKR) and the DNA receptor, DAI. These PRRs recognize pathogen-associated molecular patterns (PAMPs) such as viral proteins and nucleic acids. The engagement of these receptors then initiates intracellular signaling cascades which ultimately cause the activation of transcription factors and the expression of type I interferons and pro-inflammatory cytokines. This innate response establishes an anti-viral state in the infected cell and its neighbours and alerts immune cells to the danger. In order to establish a productive infection, viruses need to overcome this initial anti-viral response. Evasion of innate immune defences is achieved by means of viral proteins that inhibit the signaling cascades emanating from the PRRs. The same innate signal transduction pathways have been implicated in conditions of sterile inflammation, such as rheumatoid arthritis and multiple sclerosis, and in autoimmunity. Because viral proteins target crucial host proteins involved in these pathways, they can point the way to key drug targets. Further, the viral proteins themselves or derivatives of them may be of use therapeutically to curtail inflammation and autoimmunity.
Subject(s)
Immunity, Innate , Signal Transduction/physiology , Virus Diseases/immunology , Animals , DEAD Box Protein 58 , DEAD-box RNA Helicases/physiology , Humans , Interferon Regulatory Factor-3/antagonists & inhibitors , Interferon Regulatory Factor-7/antagonists & inhibitors , Interferon-Induced Helicase, IFIH1 , NF-kappa B/physiology , Receptors, Immunologic , Toll-Like Receptors/physiology , Virus Diseases/drug therapy , eIF-2 Kinase/physiologyABSTRACT
Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.
Subject(s)
DNA/metabolism , Keratinocytes/metabolism , Membrane Proteins/metabolism , Nuclear Proteins/metabolism , Nucleotidyltransferases/metabolism , Phosphoproteins/metabolism , Cell Line , DNA Viruses/metabolism , Gene Expression , Humans , Immunity, Innate , Interferon-beta/genetics , Interferon-beta/metabolism , Keratinocytes/immunology , Mutation , Nuclear Proteins/genetics , Nucleotides, Cyclic/metabolism , Phosphoproteins/genetics , Phosphorylation , Protein TransportABSTRACT
The detection of intracellular DNA has emerged to be a key event in the innate immune response to viruses and intracellular bacteria, and during conditions of sterile inflammation and autoimmunity. One of the consequences of the detection of DNA as a 'stranger' and a 'danger' signal is the production of type I interferons and pro-inflammatory cytokines. Much work has been dedicated to the elucidation of the signalling cascades that activate this DNA-induced gene expression programme. However, while many proteins have been proposed to act as sensors for intracellular DNA in recent years, none has been met with universal acceptance, and a theory linking all the recent observations is, as yet, lacking. This review presents the evidence for the various interferon-inducing DNA receptors proposed to date, and examines the hypotheses that might explain why so many different receptors appear to be involved in the innate immune recognition of intracellular DNA.
Subject(s)
DNA/immunology , Interferon Type I/immunology , Receptors, Cell Surface/immunology , Animals , Autoimmunity/immunology , Gene Expression/immunology , Humans , Immunity, Innate/genetics , Immunity, Innate/immunology , Inflammation/immunology , Virus Diseases/immunologyABSTRACT
It has been assumed that cells distinguish viral from cellular DNA due to the former's presence in the cytosol. However, in this issue, Kerur et al. (2011) propose that the DNA genome of Kaposi's sarcoma-associated herpesvirus (KSHV) is recognized inside the nucleus by the DNA sensor IFI16, leading inflammasome activation.
ABSTRACT
The expression of pattern-recognition receptors (PRRs) by immune and tissue cells provides the host with the ability to detect and respond to infection by viruses and other microorganisms. Significant progress has been made from studying this area, including the identification of PRRs, such as Toll-like receptors and RIG-I-like receptors, and the description of the molecular basis of their signalling pathways, which lead to the production of interferons and other cytokines. In parallel, common mechanisms used by viruses to evade PRR-mediated responses or to actively subvert these pathways for their own benefit are emerging. Accumulating evidence on how viral infection and PRR signalling pathways intersect is providing further insights into the function of the pathways involved, their constituent proteins and ways in which they could be manipulated therapeutically.
Subject(s)
Receptors, Pattern Recognition/immunology , Virus Diseases/immunology , Viruses/immunology , Animals , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate/immunology , Interferons/immunology , Signal Transduction , Viral Proteins/immunology , Virus Diseases/virologyABSTRACT
In metazoa, regulation of the phosphorylation state of UPF1 is crucial for nonsense-mediated mRNA decay (NMD), a process by which aberrant mRNAs containing nonsense mutations are degraded. UPF1 is targeted for dephosphorylation by three related proteins, SMG5, SMG6, and SMG7. We report here the crystal structure of the N-terminal domain of SMG7. The structure reveals that SMG7 contains a 14-3-3-like domain. Residues that bind phosphoserine-containing peptides in 14-3-3 are conserved at the equivalent positions in SMG7. Mutation of these residues impairs UPF1 binding to SMG7 in vitro and UPF1 recruitment to cytoplasmic mRNA decay foci in vivo, suggesting that SMG7 acts as an adaptor in targeting mRNAs associated with phosphorylated UPF1 for degradation. The 14-3-3 site of SMG7 is conserved in SMG5 and SMG6. These data also imply that the homologous human Est1 might have a 14-3-3 function at telomeres, and that phosphorylation events may be important for telomerase regulation.