ABSTRACT
The Zα domain of ADARp150 is critical for proper Z-RNA substrate binding and is a key factor in the type-I interferon response pathway. Two point-mutations in this domain (N173S and P193A), which cause neurodegenerative disorders, are linked to decreased A-to-I editing in disease models. To understand this phenomenon at the molecular level, we biophysically and structurally characterized these two mutated domains, revealing that they bind Z-RNA with a decreased affinity. Less efficient binding to Z-RNA can be explained by structural changes in beta-wing, part of the Z-RNA-protein interface, and alteration of conformational dynamics of the proteins.
Subject(s)
Adenosine Deaminase , Autoimmune Diseases of the Nervous System , Nervous System Malformations , Humans , Adenosine Deaminase/genetics , Adenosine Deaminase/chemistry , Adenosine Deaminase/metabolism , Autoimmune Diseases of the Nervous System/enzymology , Autoimmune Diseases of the Nervous System/genetics , Binding Sites , Nervous System Malformations/enzymology , Nervous System Malformations/genetics , RNA/chemistry , Protein Domains/genetics , Point Mutation , Nucleic Acid ConformationABSTRACT
The positive regulatory (PR) domain containing 13 (PRDM13) putative chromatin modifier and transcriptional regulator functions downstream of the transcription factor PTF1A, which controls GABAergic fate in the spinal cord and neurogenesis in the hypothalamus. Here, we report a recessive syndrome associated with PRDM13 mutation. Patients exhibited intellectual disability, ataxia with cerebellar hypoplasia, scoliosis, and delayed puberty with congenital hypogonadotropic hypogonadism (CHH). Expression studies revealed Prdm13/PRDM13 transcripts in the developing hypothalamus and cerebellum in mouse and human. An analysis of hypothalamus and cerebellum development in mice homozygous for a Prdm13 mutant allele revealed a significant reduction in the number of Kisspeptin (Kiss1) neurons in the hypothalamus and PAX2+ progenitors emerging from the cerebellar ventricular zone. The latter was accompanied by ectopic expression of the glutamatergic lineage marker TLX3. Prdm13-deficient mice displayed cerebellar hypoplasia and normal gonadal structure, but delayed pubertal onset. Together, these findings identify PRDM13 as a critical regulator of GABAergic cell fate in the cerebellum and of hypothalamic kisspeptin neuron development, providing a mechanistic explanation for the cooccurrence of CHH and cerebellar hypoplasia in this syndrome. To our knowledge, this is the first evidence linking disrupted PRDM13-mediated regulation of Kiss1 neurons to CHH in humans.
Subject(s)
Cerebellum/abnormalities , Histone-Lysine N-Methyltransferase , Hypogonadism , Hypothalamus/enzymology , Mutation , Nervous System Malformations , Transcription Factors , Animals , Cerebellum/enzymology , Developmental Disabilities/enzymology , Developmental Disabilities/genetics , Disease Models, Animal , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Hypogonadism/enzymology , Hypogonadism/genetics , Mice , Mice, Mutant Strains , Nervous System Malformations/enzymology , Nervous System Malformations/genetics , Neurons/enzymology , Transcription Factors/genetics , Transcription Factors/metabolismSubject(s)
Autoimmune Diseases of the Nervous System/genetics , Autoimmune Diseases of the Nervous System/immunology , DNA/metabolism , Hydroxychloroquine/pharmacology , Mutation/genetics , Nervous System Malformations/genetics , Nervous System Malformations/immunology , RNA/metabolism , Ribonuclease H/genetics , Antibodies/pharmacology , Autoimmune Diseases of the Nervous System/enzymology , Autophagy/drug effects , Child, Preschool , Humans , Lysosomes/drug effects , Lysosomes/metabolism , Nervous System Malformations/enzymology , Nucleotidyltransferases/metabolismABSTRACT
Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for the immune response to cancer and pathogen infection. Here, we discover that cGAS-DNA phase separation is required to resist negative regulation and allow efficient sensing of immunostimulatory DNA. We map the molecular determinants of cGAS condensate formation and demonstrate that phase separation functions to limit activity of the cytosolic exonuclease TREX1. Mechanistically, phase separation forms a selective environment that suppresses TREX1 catalytic function and restricts DNA degradation to an outer shell at the droplet periphery. We identify a TREX1 mutation associated with the severe autoimmune disease Aicardi-Goutières syndrome that increases penetration of TREX1 into the repressive droplet interior and specifically impairs degradation of phase-separated DNA. Our results define a critical function of cGAS-DNA phase separation and reveal a molecular mechanism that balances cytosolic DNA degradation and innate immune activation.
Subject(s)
Autoimmune Diseases of the Nervous System/enzymology , Cytosol/metabolism , DNA/metabolism , Exodeoxyribonucleases/metabolism , Mutation , Nervous System Malformations/enzymology , Nucleotidyltransferases/metabolism , Phosphoproteins/metabolism , Autoimmune Diseases of the Nervous System/genetics , Catalysis , Cell Line, Tumor , DNA/genetics , Exodeoxyribonucleases/genetics , HEK293 Cells , Humans , Nervous System Malformations/genetics , Nucleotidyltransferases/genetics , Phosphoproteins/geneticsABSTRACT
The oligoadenylate synthetase (OAS)-RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.
Subject(s)
Adenosine Deaminase/deficiency , Autoimmune Diseases of the Nervous System/enzymology , Endoribonucleases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Nervous System Malformations/enzymology , Phenol/pharmacology , 2',5'-Oligoadenylate Synthetase/genetics , 2',5'-Oligoadenylate Synthetase/metabolism , Adenine Nucleotides/metabolism , Adenosine Deaminase/genetics , Autoimmune Diseases of the Nervous System/genetics , Autoimmune Diseases of the Nervous System/physiopathology , Cell Death/drug effects , Endoribonucleases/genetics , Endoribonucleases/metabolism , Enzyme Inhibitors/chemistry , Humans , Nervous System Malformations/genetics , Nervous System Malformations/physiopathology , Oligoribonucleotides/metabolism , Phenol/chemistry , RNA-Binding Proteins/geneticsABSTRACT
Hypomorphic mutations in the DNA repair enzyme RNase H2 cause the neuroinflammatory autoimmune disorder Aicardi-Goutières syndrome (AGS). Endogenous nucleic acids are believed to accumulate in patient cells and instigate pathogenic type I interferon expression. However, the underlying nucleic acid species amassing in the absence of RNase H2 has not been established yet. Here, we report that murine RNase H2 knockout cells accumulated cytosolic DNA aggregates virtually indistinguishable from micronuclei. RNase H2-dependent micronuclei were surrounded by nuclear lamina and most of them contained damaged DNA. Importantly, they induced expression of interferon-stimulated genes (ISGs) and co-localized with the nucleic acid sensor cGAS. Moreover, micronuclei associated with RNase H2 deficiency were cleared by autophagy. Consequently, induction of autophagy by pharmacological mTOR inhibition resulted in a significant reduction of cytosolic DNA and the accompanied interferon signature. Autophagy induction might therefore represent a viable therapeutic option for RNase H2-dependent disease. Endogenous retroelements have previously been proposed as a source of self-nucleic acids triggering inappropriate activation of the immune system in AGS. We used human RNase H2-knockout cells generated by CRISPR/Cas9 to investigate the impact of RNase H2 on retroelement propagation. Surprisingly, replication of LINE-1 and Alu elements was blunted in cells lacking RNase H2, establishing RNase H2 as essential host factor for the mobilisation of endogenous retrotransposons.
Subject(s)
Autoimmune Diseases of the Nervous System/enzymology , Micronucleus, Germline/enzymology , Nervous System Malformations/enzymology , Ribonuclease H/deficiency , Animals , Autoimmune Diseases of the Nervous System/genetics , Autoimmune Diseases of the Nervous System/metabolism , Autoimmune Diseases of the Nervous System/pathology , Autophagy/genetics , DNA/genetics , DNA Damage , DNA Replication , Mice , Mice, Knockout , Micronucleus, Germline/genetics , Micronucleus, Germline/immunology , Mutation , Nervous System Malformations/genetics , Nervous System Malformations/metabolism , Nervous System Malformations/pathology , Ribonuclease H/genetics , Ribonuclease H/metabolismABSTRACT
SAMHD1 plays diverse roles in innate immunity, autoimmune diseases and HIV restriction, but the mechanisms involved are still unclear. SAMHD1 has been reported to have both dNTPase and RNase activities. However, whether SAMHD1 possesses RNase activity remains highly controversial. Here, we found that, unlike conventional hydrolytic exoribonucleases, SAMHD1 requires inorganic phosphate to degrade RNA substrates and produces nucleotide diphosphates rather than nucleoside monophosphates, which indicated that SAMHD1 is a phosphorolytic but not hydrolytic 3'-5' exoribonuclease. Furthermore, SAMHD1 preferentially cleaved single-stranded RNAs comprising A20 or U20, whereas neither C20 nor G20 was susceptible to SAMHD1-mediated degradation. Our findings will facilitate more advanced studies into the role of the SAMHD1 RNase function in the cellular pathogenesis implicated in nucleic acid-triggered inflammatory responses and the anti-retroviral function of SAMHD1.
Subject(s)
Autoimmune Diseases of the Nervous System/enzymology , Dinucleoside Phosphates/chemistry , Monomeric GTP-Binding Proteins/chemistry , Nervous System Malformations/enzymology , RNA/chemistry , Retroviridae Proteins/chemistry , Ribonucleases/chemistry , Binding Sites , Enzyme Activation , Humans , Hydrolysis , Phosphorylation , Protein Binding , SAM Domain and HD Domain-Containing Protein 1ABSTRACT
INTRODUCTION: Aicardi-Goutieres syndrome is a rare immune disorder due to mutations in seven different genes that encode proteins called TREX1, ribonuclease H2 complex, SAMHD1, ADAR and IDIH1 (MDA5), which are involved in acid nucleic metabolism. Two cases are described in detail below caused by RNASEH2B gene mutation, one of which displays a mutation no described to date. CASE REPORTS: Case 1: male consulting because from 5-month-old shows loss of maturity items acquired until then, coming with several fever episodes. Case 2: a 4-month-old boy showing since 2-month-old great irritability and oral-feeding trouble with severe psychomotor impairment. In both cases it was found an increase of pterines in the cerebrospinal fluid, mainly neopterine, with calcifications in the basal ganglia. The diagnosis was proved by sequencing RNASEH2B gene, founding in case 2 a new mutation not described previously. CONCLUSIONS: The reported cases belong to the description already done by Aicardi-Goutieres, it should be noticed this syndrome in a patient with a subacute encephalopathy of debut in the first year of life, dystonia/spasticity in variable degree and important affectation/regression of psychomotor development, particularly in those with increase of pterines (neopterine) in the cerebrospinal fluid and calcifications in the basal ganglia.
TITLE: Variaciones fenotipicas en el sindrome de Aicardi-Goutieres causado por mutaciones en el gen RNASEH2B: presentacion de dos nuevos casos.Introduccion. El sindrome de Aicardi-Goutieres es un trastorno inmunitario raro debido a mutaciones en siete genes que codifican proteinas llamadas TREX1, el complejo ribonucleasa H2, SAMHD1, ADAR e IFIH1 (MAD5), las cuales estan implicadas en el metabolismo de los acidos nucleicos. A continuacion se presentan dos nuevos casos por mutacion en el gen RNASEH2B, uno de los cuales presenta una mutacion no descrita hasta la fecha. Casos clinicos. Caso 1: varon que consulto porque desde los 5 meses, coincidiendo con cuadros febriles de repeticion, presentaba perdida de los items madurativos adquiridos hasta la fecha. Caso 2: niño de 4 meses que desde los 2 meses mostraba gran irritabilidad con dificultades en la alimentacion, asociado a un grave retraso psicomotor. En ambos casos se constato un aumento de las pterinas en el liquido cefalorraquideo, principalmente de la neopterina, con calcificaciones en los ganglios basales. El diagnostico se confirmo mediante secuenciacion del gen RNASEH2B; el caso 2 presentaba una mutacion no descrita en la literatura medica. Conclusiones. Los casos corresponden a la descripcion clasica realizada por Aicardi-Goutieres. Debe tenerse en cuenta este sindrome ante un paciente con un cuadro de encefalopatia subaguda de comienzo en el primer año de vida, distonia/espasticidad en grado variable e importante afectacion/regresion del desarrollo psicomotor, especialmente si asocia aumento de las pterinas (neopterina) en el liquido cefalorraquideo y calcificaciones en los ganglios basales.
Subject(s)
Autoimmune Diseases of the Nervous System/genetics , Mutation, Missense , Nervous System Malformations/genetics , Ribonuclease H/genetics , Autoimmune Diseases of the Nervous System/diagnostic imaging , Autoimmune Diseases of the Nervous System/enzymology , Basal Ganglia/diagnostic imaging , Basal Ganglia/pathology , Biopterins/cerebrospinal fluid , Calcinosis/etiology , Calcinosis/pathology , Feeding and Eating Disorders of Childhood/genetics , Genetic Association Studies , Heterozygote , Humans , Infant , Intellectual Disability/genetics , Magnetic Resonance Imaging , Male , Muscle Spasticity/genetics , Neopterin/cerebrospinal fluid , Nervous System Malformations/diagnostic imaging , Nervous System Malformations/enzymology , Neuroimaging , Phenotype , Ribonuclease H/deficiency , Sequence Analysis, DNAABSTRACT
In human cells, ribonuclease (RNase) H2 complex is the predominant source of RNase H activities with possible roles in nucleic acid metabolism to preserve genome stability and to prevent immune activation. Dysfunction mutations in any of the three subunits of human RNase H2 complex can result in embryonic/perinatal lethality or cause Aicardi-Goutières syndrome (AGS). Most recently, increasing findings have shown that human RNase H2 proteins play roles beyond the RNase H2 enzymatic activities in health and disease. Firstly, the biochemical and structural properties of human RNase H2 proteins allow their interactions with various partner proteins that may support functions other than RNase H2 enzymatic activities. Secondly, the disparities of clinical presentations of AGS with different AGS-mutations and the biochemical and structural analysis of AGS-mutations, especially the results from both AGS-knockin and RNase H2-null mouse models, suggest that human RNase H2 complex has certain cellular functions beyond the RNase H2 enzymatic activities to prevent the innate-immune-mediated inflammation. Thirdly, the subunit proteins RNASEH2A and RNASEH2B respectively, not related to the RNase H2 enzymatic activities, have been shown to play a certain role in the pathophysiological processes of different cancer types. In this minireview, we aims to provide a brief overview of the most recent investigations into the biological functions of human RNase H2 proteins and the underlying mechanisms of their actions, emphasizing on the new insights into the roles of human RNase H2 proteins playing beyond the RNase H2 enzymatic activities in health and disease.
Subject(s)
Ribonuclease H/metabolism , Animals , Autoimmune Diseases of the Nervous System/enzymology , Embryonic Development , Genomic Instability , Humans , Neoplasms/enzymology , Nervous System Malformations/enzymologyABSTRACT
Polymicrogyria (PMG) is a structural brain abnormality involving the cerebral cortex that results from impaired neuronal migration and although several genes have been implicated, many cases remain unsolved. In this study, exome sequencing in a family where three fetuses had all been diagnosed with PMG and cerebellar hypoplasia allowed us to identify regions of the genome for which both chromosomes were shared identical-by-descent, reducing the search space for causative variants to 8.6% of the genome. In these regions, the only plausibly pathogenic mutations were compound heterozygous variants in PI4KA, which Sanger sequencing confirmed segregated consistent with autosomal recessive inheritance. The paternally transmitted variant predicted a premature stop mutation (c.2386C>T; p.R796X), whereas the maternally transmitted variant predicted a missense substitution (c.5560G>A; p.D1854N) at a conserved residue within the catalytic domain. Functional studies using expressed wild-type or mutant PI4KA enzyme confirmed the importance of p.D1854 for kinase activity. Our results emphasize the importance of phosphoinositide signalling in early brain development.
Subject(s)
Arthrogryposis/enzymology , Cerebellum/abnormalities , Fetal Diseases/enzymology , Germ-Line Mutation , Nervous System Malformations/enzymology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Polymicrogyria/enzymology , Amino Acid Sequence , Arthrogryposis/embryology , Arthrogryposis/genetics , Base Sequence , Brain/embryology , Brain/enzymology , Cerebellum/embryology , Cerebellum/enzymology , Developmental Disabilities/enzymology , Developmental Disabilities/genetics , Exome , Female , Fetal Diseases/genetics , Humans , Infant , Male , Minor Histocompatibility Antigens , Molecular Sequence Data , Mutation, Missense , Nervous System Malformations/embryology , Nervous System Malformations/genetics , Pedigree , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Polymicrogyria/embryology , Polymicrogyria/genetics , Polymorphism, Single Nucleotide , Sequence AlignmentABSTRACT
BACKGROUND: Heterozygous loss-of-function mutations in the X-linked CASK gene cause progressive microcephaly with pontine and cerebellar hypoplasia (MICPCH) and severe intellectual disability (ID) in females. Different CASK mutations have also been reported in males. The associated phenotypes range from nonsyndromic ID to Ohtahara syndrome with cerebellar hypoplasia. However, the phenotypic spectrum in males has not been systematically evaluated to date. METHODS: We identified a CASK alteration in 8 novel unrelated male patients by targeted Sanger sequencing, copy number analysis (MLPA and/or FISH) and array CGH. CASK transcripts were investigated by RT-PCR followed by sequencing. Immunoblotting was used to detect CASK protein in patient-derived cells. The clinical phenotype and natural history of the 8 patients and 28 CASK-mutation positive males reported previously were reviewed and correlated with available molecular data. RESULTS: CASK alterations include one nonsense mutation, one 5-bp deletion, one mutation of the start codon, and five partial gene deletions and duplications; seven were de novo, including three somatic mosaicisms, and one was familial. In three subjects, specific mRNA junction fragments indicated in tandem duplication of CASK exons disrupting the integrity of the gene. The 5-bp deletion resulted in multiple aberrant CASK mRNAs. In fibroblasts from patients with a CASK loss-of-function mutation, no CASK protein could be detected. Individuals who are mosaic for a severe CASK mutation or carry a hypomorphic mutation still showed detectable amount of protein. CONCLUSIONS: Based on eight novel patients and all CASK-mutation positive males reported previously three phenotypic groups can be distinguished that represent a clinical continuum: (i) MICPCH with severe epileptic encephalopathy caused by hemizygous loss-of-function mutations, (ii) MICPCH associated with inactivating alterations in the mosaic state or a partly penetrant mutation, and (iii) syndromic/nonsyndromic mild to severe ID with or without nystagmus caused by CASK missense and splice mutations that leave the CASK protein intact but likely alter its function or reduce the amount of normal protein. Our findings facilitate focused testing of the CASK gene and interpreting sequence variants identified by next-generation sequencing in cases with a phenotype resembling either of the three groups.
Subject(s)
Guanylate Kinases/genetics , Microcephaly/enzymology , Adolescent , Adult , Cerebellum/abnormalities , Cerebellum/enzymology , Child , Child, Preschool , Developmental Disabilities/enzymology , Developmental Disabilities/etiology , Developmental Disabilities/genetics , Humans , Infant , Intellectual Disability/enzymology , Intellectual Disability/etiology , Intellectual Disability/genetics , Male , Microcephaly/complications , Microcephaly/genetics , Middle Aged , Mutation , Nervous System Malformations/enzymology , Nervous System Malformations/etiology , Nervous System Malformations/genetics , Phenotype , Young AdultABSTRACT
Lissencephaly comprises a heterogeneous group of developmental brain disorders of varying severity, involving abnormal cortical gyration. We studied a highly consanguineous Israeli Moslem family with a lethal form of autosomal recessive lissencephaly with cerebellar hypoplasia (LCH). Using microarray-based homozygosity mapping in the reported family, combined with whole exome sequencing in one affected infant, we identified a homozygous splice site mutation g.IVS8+1G>A in cyclin-dependent kinase 5 (CDK5), causing complete skipping of exon 8, and leading to a frame shift and premature stop codon (p.V162SfsX19). The mutation co-segregated with the disease phenotype in all 29 study participants (4 patients and 25 healthy relatives), and was not identified in 200 ethnically matched control chromosomes. The p.V162SfsX19 mutation causes lack of endogenous CDK5 expression in affected dermal fibroblasts and brain tissue at the mRNA and protein levels, consistent with nonsense-mediated mRNA decay. Functional analysis of the p.V162SfsX19 mutation, using a yeast complementation assay, showed loss-of-function of the mutant CDK5 gene product, thereby implicating its role in the pathogenesis of autosomal recessive LCH in the studied family.
Subject(s)
Cerebellum/abnormalities , Cyclin-Dependent Kinase 5/genetics , Lissencephaly/genetics , Nervous System Malformations/genetics , Base Sequence , Cells, Cultured , Cerebellum/enzymology , Consanguinity , DNA Mutational Analysis , Developmental Disabilities/enzymology , Developmental Disabilities/genetics , Female , Genes, Recessive , Genetic Association Studies , Genetic Complementation Test , Homozygote , Humans , Infant , Infant, Newborn , Lissencephaly/enzymology , Male , Mutation, Missense , Nervous System Malformations/enzymology , PedigreeABSTRACT
Innate immune sensing of nucleic acids provides resistance against viral infection and is important in the aetiology of autoimmune diseases. AGS (Aicardi-Goutières syndrome) is a monogenic autoinflammatory disorder mimicking in utero viral infection of the brain. Phenotypically and immunologically, it also exhibits similarities to SLE (systemic lupus erythaematosus). Three of the six genes identified to date encode components of the ribonuclease H2 complex. As all six encode enzymes involved in nucleic acid metabolism, it is thought that pathogenesis involves the accumulation of nucleic acids to stimulate an inappropriate innate immune response. Given that AGS is a monogenic disorder with a defined molecular basis, we use it as a model for common autoimmune disease to investigate cellular processes and molecular pathways responsible for nucleic-acid-mediated autoimmunity. These investigations have also provided fundamental insights into the biological roles of the RNase H2 endonuclease enzyme. In the present article, we describe how human RNase H2 and its role in AGS were first identified, and give an overview of subsequent structural, biochemical, cellular and developmental studies of this enzyme. These investigations have culminated in establishing this enzyme as a key genome-surveillance enzyme required for mammalian genome stability.
Subject(s)
Ribonuclease H/metabolism , Autoimmune Diseases of the Nervous System/enzymology , Autoimmune Diseases of the Nervous System/genetics , Autoimmune Diseases of the Nervous System/metabolism , Genomic Instability/genetics , Genomic Instability/physiology , Humans , Inflammation/enzymology , Inflammation/genetics , Inflammation/metabolism , Nervous System Malformations/enzymology , Nervous System Malformations/genetics , Nervous System Malformations/metabolism , Ribonuclease H/geneticsABSTRACT
Ribonuclease H2 plays an essential role for genome stability as it removes ribonucleotides misincorporated into genomic DNA by replicative polymerases and resolves RNA/DNA hybrids. Biallelic mutations in the genes encoding the three RNase H2 subunits cause Aicardi-Goutières syndrome (AGS), an early-onset inflammatory encephalopathy that phenotypically overlaps with the autoimmune disorder systemic lupus erythematosus. Here we studied the intracellular dynamics of RNase H2 in living cells during DNA replication and in response to DNA damage using confocal time-lapse imaging and fluorescence cross-correlation spectroscopy. We demonstrate that the RNase H2 complex is assembled in the cytosol and imported into the nucleus in an RNase H2B-dependent manner. RNase H2 is not only recruited to DNA replication foci, but also to sites of PCNA-dependent DNA repair. By fluorescence recovery after photobleaching, we demonstrate a high mobility and fast exchange of RNase H2 at sites of DNA repair and replication. We provide evidence that recruitment of RNase H2 is not only PCNA-dependent, mediated by an interaction of the B subunit with PCNA, but also PCNA-independent mediated via the catalytic domain of the A subunit. We found that AGS-associated mutations alter complex formation, recruitment efficiency and exchange kinetics at sites of DNA replication and repair suggesting that impaired ribonucleotide removal contributes to AGS pathogenesis.
Subject(s)
Autoimmune Diseases of the Nervous System/enzymology , DNA Damage , DNA Replication , Nervous System Malformations/enzymology , Ribonuclease H/metabolism , Autoimmune Diseases of the Nervous System/genetics , Cell Nucleus/enzymology , Cell Nucleus/genetics , Cytosol/enzymology , Humans , Nervous System Malformations/genetics , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Protein Multimerization , Protein Transport , Ribonuclease H/chemistry , Ribonuclease H/geneticsABSTRACT
TREX1 is a 3'-deoxyribonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA) to prevent inappropriate nucleic acid-mediated immune activation. More than 40 different disease-causing TREX1 mutations have been identified exhibiting dominant and recessive genetic phenotypes in a spectrum of autoimmune disorders. Mutations in TREX1 at positions Asp-18 and Asp-200 to His and Asn exhibit dominant autoimmune phenotypes associated with the clinical disorders familial chilblain lupus and Aicardi-Goutières syndrome. Our previous biochemical studies showed that the TREX1 dominant autoimmune disease phenotype depends upon an intact DNA-binding process coupled with dysfunctional active site chemistry. Studies here show that the TREX1 Arg-62 residues extend across the dimer interface into the active site of the opposing protomer to coordinate substrate DNA and to affect catalysis in the opposing protomer. The TREX1(R62A/R62A) homodimer exhibits â¼50-fold reduced ssDNA and dsDNA degradation activities relative to TREX1(WT). The TREX1 D18H, D18N, D200H, and D200N dominant mutant enzymes were prepared as compound heterodimers with the TREX1 R62A substitution in the opposing protomer. The TREX1(D18H/R62A), TREX1(D18N/R62A), TREX1(D200H/R62A), and TREX1(D200N/R62A) compound heterodimers exhibit higher levels of ss- and dsDNA degradation activities than the homodimers demonstrating the requirement for TREX1 Arg-62 residues to provide necessary structural elements for full catalytic activity in the opposing TREX1 protomer. This concept is further supported by the loss of dominant negative effects in the TREX1 D18H, D18N, D200H, and D200N compound heterodimers. These data provide compelling evidence for the required TREX1 dimeric structure for full catalytic function.
Subject(s)
DNA, Single-Stranded/chemistry , Exodeoxyribonucleases/chemistry , Phosphoproteins/chemistry , Protein Multimerization/physiology , Protein Subunits/chemistry , Amino Acid Substitution , Arginine/chemistry , Arginine/genetics , Arginine/metabolism , Autoimmune Diseases of the Nervous System/enzymology , Autoimmune Diseases of the Nervous System/genetics , Catalysis , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Humans , Mutation, Missense , Nervous System Malformations/enzymology , Nervous System Malformations/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Structure, Quaternary , Protein Subunits/genetics , Protein Subunits/metabolismABSTRACT
Ribonuclease H2 (RNase H2) belongs to the family of RNase H enzymes, which process RNA/DNA hybrids. Apart from cleaving the RNA moiety of a plain RNA/DNA hybrid, RNase H2 participates in the removal of single ribonucleotides embedded in a DNA duplex. Mutations in RNase H2 lead to the chronic inflammatory disorder Aicardi-Goutières syndrome (AGS), which has significant phenotypic overlaps with the autoimmune disease systemic lupus erythematosus. RNase H2 knock-out mice are embryonic lethal. Mouse embryos lacking RNase H2 accumulate DNA damage and exhibit a p53-mediated growth arrest commencing at gastrulation. On a molecular level, the knock-out mice reveal that RNase H2 represents an essential DNA repair enzyme, whose main cellular function is the removal of accidentally misincorporated ribonucleotides from genomic DNA. Ribonucleotides strongly accumulate within the genomic DNA of RNase H2-deficient cells, in turn resulting in a massive build-up of DNA damage in these cells. The DNA lesions that arise from misincorporated ribonucleotides constitute the by far most frequent type of naturally occurring DNA damage. AGS-causing mutations have also been found in the genes of the 3'-exonuclease TREX1, the dNTP triphosphatase SAMHD1, as well as the RNA-editing enzyme ADAR1, defining defects in nucleic acid metabolism pathways as a common hallmark of AGS pathology. However, recent evidence gathered from RNase H2 knock-out mice might provide additional insight into the molecular mechanisms underlying AGS development and a potential role of DNA damage as a trigger of autoimmunity is discussed.
Subject(s)
Autoimmune Diseases of the Nervous System/enzymology , Autoimmune Diseases of the Nervous System/genetics , Nervous System Malformations/enzymology , Nervous System Malformations/genetics , Ribonuclease H/genetics , Animals , Autoimmune Diseases of the Nervous System/pathology , DNA Damage , Disease Models, Animal , Humans , Mice , Mice, Knockout , Nervous System Malformations/pathology , Ribonuclease H/analysis , Ribonuclease H/metabolismABSTRACT
Aicardi-Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families.
Subject(s)
Autoimmune Diseases of the Nervous System/genetics , Nervous System Malformations/genetics , Point Mutation , RNA Splice Sites , Ribonuclease H/genetics , Autoimmune Diseases of the Nervous System/diagnosis , Autoimmune Diseases of the Nervous System/enzymology , Female , Genetic Variation , Humans , Infant , Infant, Newborn , Male , Mutation, Missense , Nervous System Malformations/diagnosis , Nervous System Malformations/enzymology , Ribonuclease H/metabolismABSTRACT
The human HD domain protein SAMHD1 is implicated in the Aicardi-Goutières autoimmune syndrome and in the restriction of HIV-1 replication in myeloid cells. Recently, this protein has been shown to possess dNTP triphosphatase activity, which is proposed to inhibit HIV-1 replication and the autoimmune response by hydrolyzing cellular dNTPs. Here, we show that the purified full-length human SAMHD1 protein also possesses metal-dependent 3'â5' exonuclease activity against single-stranded DNAs and RNAs in vitro. In double-stranded substrates, this protein preferentially cleaved 3'-overhangs and RNA in blunt-ended DNA/RNA duplexes. Full-length SAMHD1 also exhibited strong DNA and RNA binding to substrates with complex secondary structures. Both nuclease and dNTP triphosphatase activities of SAMHD1 are associated with its HD domain, but the SAM domain is required for maximal activity and nucleic acid binding. The nuclease activity of SAMHD1 could represent an additional mechanism contributing to HIV-1 restriction and suppression of the autoimmune response through direct cleavage of viral and endogenous nucleic acids. In addition, we demonstrated the presence of dGTP triphosphohydrolase and nuclease activities in several microbial HD domain proteins, suggesting that these proteins might contribute to antiviral defense in prokaryotes.
Subject(s)
Autoimmune Diseases of the Nervous System/enzymology , Exonucleases/physiology , HIV-1/physiology , Monomeric GTP-Binding Proteins/chemistry , Nervous System Malformations/enzymology , Amino Acid Substitution , Catalytic Domain , DNA Cleavage , DNA, Single-Stranded/chemistry , Humans , Hydrolysis , Magnesium/chemistry , Molecular Sequence Annotation , Monomeric GTP-Binding Proteins/genetics , Mutagenesis, Site-Directed , Protein Binding , RNA/chemistry , RNA Cleavage , RNA, Viral/chemistry , SAM Domain and HD Domain-Containing Protein 1 , Virus Replication , gag Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
SP600125 (anthrapyrazolone) is a synthetic polyaromatic chemical that inhibits c-Jun N-terminal kinase (JNK) signaling by interfering with phosphorylation of c-Jun. To determine the pharmacological impact of SP600125 on zebrafish development, we incubated embryos in various concentrations of SP600125 from 18 h postfertilization (hpf) to 48 hpf. Embryos treated with 1.25 µm appeared with occasional pericardium edema. Treatment with 12.5 µm resulted in complete mortality by 120 hpf, preventing an assessment of physiological defects. Embryos treated with 5 µm exhibited slowed overall growth, a delay in hatching and numerous morphological defects such as pericardium edema, yolk sac edema, swim bladder deflation, bent vertebrae and eye and jaw malformations. Whole-mount immunohistochemical studies using an anti-acetylated ß-tubulin antibody confirmed developmental defects in the nervous system. Within the retina, fish treated with 1.25 µm showed a mild reduction of immunoreactivity. Immunoreactivity in the retina was further reduced in fish treated with 5 µm of SP600125. In these fish, eyes and olfactory organs were half the size compared with other groups. Multiple lenses were observed in 67% of these fish. A second experiment with a shorter exposure period of SP600125 (6 h) presented significantly fewer morphological defects. The treatment led to a delay in hatching, and increased incidences of swim bladder deflation and pericardium edema with increasing concentrations. In summary, SP600125 caused developmental abnormalities during zebrafish organogenesis starting at 1.25 µm and the defects were exacerbated with increasing concentrations. Our study suggests that SP600125 at 1.25 µm and beyond has devastating consequences for zebrafish development.
Subject(s)
Abnormalities, Drug-Induced/etiology , Anthracenes/toxicity , Embryo, Nonmammalian/drug effects , Enzyme Inhibitors/toxicity , Nervous System Malformations/chemically induced , Abnormalities, Drug-Induced/enzymology , Abnormalities, Drug-Induced/pathology , Air Sacs/abnormalities , Air Sacs/drug effects , Air Sacs/enzymology , Animals , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/enzymology , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Longevity/drug effects , Nervous System Malformations/enzymology , Nervous System Malformations/pathology , Olfactory Bulb/abnormalities , Olfactory Bulb/drug effects , Olfactory Bulb/enzymology , Pericardial Effusion/chemically induced , Pericardial Effusion/enzymology , Pericardial Effusion/pathology , Retina/abnormalities , Retina/drug effects , Retina/enzymology , Time Factors , ZebrafishABSTRACT
Cell-cell and cell-matrix interactions are necessary for neuronal patterning and brain wiring during development. Matrix metalloproteinases (MMPs) are proteolytic enzymes capable of remodelling the pericellular environment and regulating signaling pathways through cleavage of a large degradome. MMPs have been suggested to affect cerebellar development, but the specific role of different MMPs in cerebellar morphogenesis remains unclear. Here, we report a role for MMP-3 in the histogenesis of the mouse cerebellar cortex. MMP-3 expression peaks during the second week of postnatal cerebellar development and is most prominently observed in Purkinje cells (PCs). In MMP-3 deficient (MMP-3(-/-)) mice, a protracted granule cell (GC) tangential migration and a delayed GC radial migration results in a thicker and persistent external granular layer, a retarded arrival of GCs in the inner granular layer, and a delayed GABAergic interneuron migration. Importantly, these neuronal migration anomalies, as well as the consequent disturbed synaptogenesis on PCs, seem to be caused by an abnormal PC dendritogenesis, which results in reduced PC dendritic trees in the adult cerebellum. Of note, these developmental and adult cerebellar defects might contribute to the aberrant motor phenotype observed in MMP-3(-/-) mice and suggest an involvement of MMP-3 in mouse cerebellar development.