RNA Editing by ADAR1 Prevents MDA5 Sensing of Endogenous dsRNA as Nonself PDF

RE S EAR CH | R E P O R T S RNA EDITING tained in vivo and were lost over time (fig. S3)....

RE S EAR CH | R E P O R T S RNA EDITING tained in vivo and were lost over time (fig. S3). The A-to-I editing activity of ADAR1 is essential for embryonic development and the maintenance RNA editing by ADAR1 prevents of hematopoiesis in vivo. We performed transcriptional profiling on MDA5 sensing of endogenous dsRNA three independent littermate Adar1+/+ and Adar1 E861A/E861A FLs. An absence of ADAR1 edit- ing resulted in the up-regulation of 383 transcripts as nonself (log2FC > 2), 258 of which are IFN-stimulated genes (ISGs) (Fig. 2A and table S1). Pathway anal- Brian J. Liddicoat,1,2 Robert Piskol,3 Alistair M. Chalk,1,2 Gokul Ramaswami,3 ysis revealed a profound enrichment of signatures activated after IFN treatment or viral infection Miyoko Higuchi,4 Jochen C. Hartner,5 Jin Billy Li,3* (Fig. 2B and table S1). Of the 50 most differentially Peter H. Seeburg,4* Carl R. Walkley1,2*† expressed genes, 44 were up-regulated by either type I IFN, type II IFN, or both (Fig. 2C and table Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of S1). The derepression of ISGs was confirmed by RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA quantitative reverse transcriptase polymerase editing, additional functions have been proposed for ADAR1. To determine the specific role of chain reaction (qRT-PCR) (Fig. 2D). Further- RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation more, we demonstrated that the transcriptional (Adar1E861A, where E861A denotes Glu861→Ala861). Adar1E861A/E861A embryos died at ~E13.5 response in Adar1E861A/E861A FLs closely parallels (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)–sensing that of complete ADAR1 deficiency (12) (fig. S4A). pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered Synthetic dsRNA sequences based on endoge- hyperediting within long dsRNA stem loops within 3′ untranslated regions of endogenous nous RNA containing adenosine but not inosine Downloaded from https://www.science.org on September 15, 2024 transcripts. Finally, embryonic death and phenotypes of Adar1E861A/E861A were rescued by stimulate ISGs and apoptosis in vitro by binding concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous to MDA5 and RIG-I (13). We defined a gene set dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA associated with the response to IU-dsRNA (inosine- response by endogenous transcripts. uracil–paired dsRNA) in human cells (13). Gene signatures of both Adar1E861A/E861A FL and Adar1−/− A denosine-to-inosine (A-to-I) editing is the genomic substitution of the single edited aden- HSCs were highly enriched for the IU-dsRNA most prevalent form of RNA base modifi- osine for guanosine, mimicking editing of the response (Fig. 2E), demonstrating a species- cation in mammals. Hundreds of thousands transcript (10). In contrast to this elegant single- conserved response to dsRNA that is constrained of A-to-I editing events have been reported substrate paradigm that describes the Adar2−/− by the presence of inosine residues. Therefore, in the human transcriptome (1–3). A-to-I phenotype, no such editing site(s) described to the deamination of adenosine in dsRNA by editing is catalyzed by ADAR (adenosine deam- date have resolved the physiological requirement ADAR1 is necessary for suppression of the IFN inase acting on RNA) enzymes, which deaminate for ADAR1. response under homeostatic conditions. Fur- genomically encoded A-to-I in double-stranded To directly determine the contribution of thermore, it suggested that the absence of A-to-I RNA (dsRNA). A-to-I editing predominantly oc- A-to-I editing to ADAR1’s biological function, we substitutions in endogenous dsRNA may initiate curs in noncoding, repetitive elements such as generated a constitutive knock-in of an editing- this response. inverted Alus and short interspersed nuclear ele- deficient ADAR1 allele in mice (Adar1E861A, where To define ADAR1-specific editing events in vivo, ments (SINEs) (3, 4). There are three mamma- E861A denotes Glu861→Ala861) (Fig. 1A and fig. we analyzed A-to-I (G) mismatches within the lian ADAR proteins: ADAR1, -2, and -3. ADAR1 S1A), homologous to the human ADAR1E912A RNA sequencing data. Across all samples, 6167 is widely expressed during embryonic and post- allele, which is catalytically inactive in vitro (11) A-to-I editing sites were identified: 5540 known natal development and is present as a predom- (also see supplementary materials and methods). (14) and 627 previously undiscovered (Fig. 3A). inantly nuclear, constitutive ADAR1p110 isoform In E12.5 whole embryos, ADAR1p110 protein was Strain-specific single-nucleotide polymorphisms expressed in all tissues and an additional in- detected in Adar1E861A/E861A samples at compa- were excluded, and only A-to-I mismatch fre- terferon (IFN)–inducible ADAR1p150 isoform rable levels to Adar1+/+ and Adar1E861A/+ controls quencies that differed significantly between geno- that is found in both the nucleus and the cyto- (Fig. 1B). Additionally, Adar1E861A/E861A embryos types were considered (Fig. 3B). Using these plasm (5). Adar1−/− (null for both isoforms) and had elevated expression of ADAR1p150 (Fig. 1B). criteria, 673 A-to-I editing sites were defined Adar1p150−/− mice die in utero at E11.5 (embry- Adar1E861A/+ heterozygous mice were normal, (Fig. 3A). Of these, 666 sites had reduced editing onic day 11.5) to E12.5, due to failed erythropoi- were present at the expected Mendelian ratio, in mutants, including hyperedited loci, which had esis and fetal liver (FL) disintegration (6–8). and had no discernible phenotype. No viable no detectable editing (Fig. 3B and tables S2 and Only ADAR1 and ADAR2 demonstrate editing Adar1E861A/E861A pups were born from Adar1E861A/+ S3). This confirmed that the Adar1E861A allele is activity in vitro. The AMPA receptor GluA2 pre- intercrosses and were found to be dying at ~E13.5 catalytically inactive and the majority of FL A- mRNA is edited by ADAR2 in the brain, con- (Fig. 1C). Adar1E861A/E861A yolk sacs were pale, to-I editing is ADAR1 dependent. We validated verting a glutamine residue to an arginine in a and embryos appeared developmentally delayed 281 of the identified A-to-I sites on independent functionally critical position (9). Adar2−/− mice compared with controls (Fig. 1D), despite being Adar1E861A/E861A and Adar1+/+ FL samples (15). die from seizures and were rescued by the normal at E12.5 (fig. S1, D and E). At E13.5, All tested sites were confirmed as differentially Adar1E861A/E861A FL was smaller, with eightfold edited in both FL and mouse embryonic fibro- fewer viable cells (Fig. 1D and fig. S1E). We blasts (MEFs) (table S4). Sanger sequencing 1 St. Vincent’s Institute of Medical Research, Fitzroy, Victoria observed a failure in erythropoiesis with a severe validated ADAR1-specific BlcapY2C, Mad2l1, Rbbp4, 3065, Australia. 2Department of Medicine, St. Vincent’s Hospital, University of Melbourne, Fitzroy, Victoria 3065, loss of erythroblast populations (Fig. 1, E and F) and Klf1 editing sites from independent FL sam- Australia. 3Department of Genetics, Stanford University, and increased cell death (Fig. 1G), also apparent ples (Fig. 3C). Stanford, CA 94305, USA. 4Department of Molecular in other hematopoietic populations (fig. S2). ADAR1 is required for erythropoiesis (Fig. 1, Neurobiology, Max Planck Institute for Medical Research, Consistent with our previous analysis of the E to G). Interestingly, 40% (264 of 666) of the 69120 Heidelberg, Germany. 5Taconic Biosciences, 51063 Cologne, Germany. ADAR1 conditional allele (12), adult hemato- ADAR1-specific editing sites were within hyper- *These authors contributed equally to this work. †Corresponding poietic stem cells (HSCs) expressing only the edited clusters of only three genes—Klf1, Oip5, and author. E-mail: [email protected] catalytically inactive ADAR1 could not be main- Optn—which have peak or restricted expression in SCIENCE sciencemag.org 4 SEPTEMBER 2015 VOL 349 ISSUE 6252 1115 R ES E A RC H | R E PO R TS the erythroid lineage (table S3). Seventy, 61, and suming wobble base paring. In both cases, the genitor cells expressing only the Adar1E861A allele 133 ADAR1-specific A-to-I hyperedited loci were predicted dsRNA structures have higher free- to control levels, using two independent MDA5 within long 3′ untranslated regions (3′UTRs) of energy states in the presence of A-to-I or A-to-G shRNAs (fig. S8). Klf1, Oip5, and Optn, respectively. An example substitutions (fig. S7, A to F). Therefore, ex- Based on the in vitro rescue, we crossed of hyperediting in Klf1 is depicted in Fig. 3D. An tensive I-U mismatches would be predicted to Adar1 E861A/E861A to MDA5−/− (Ifih1−/−) mice. Ini- absence of ADAR1 editing of these loci provides destabilize perfect dsRNA stem loops within tially we assessed embryos at E13.5, a time a possible link to the FL failure in Adar1E861A/E861A hyperedited 3′UTRs (Fig. 3E and fig. S7, A to F). point when Adar1E861A/E861A embryos are no longer embryos. Thus, we hypothesized that ADAR1 editing re- viable (Fig. 1C). Adar1E861A/E861AIfih1−/− double mu- Modeling of the predicted secondary structure modeled the secondary structure of endogenous tant yolk sac, embryo, and FL were comparable of the hyperedited 3′UTRs showed that in the RNA to abrogate the formation of long matched to controls and were present at the expected absence of editing there was the potential for dsRNAs. ratio (Fig. 4D and fig. S9A). Erythropoiesis was long perfect dsRNA segments to be formed through The transcriptional signatures in the Adar1E861A/E861A completely rescued, and FL ISGs were equivalent duplexing of repetitive regions (Fig. 3E and fig. FL resembled that of RIG-I and MDA5 activa- to those of control littermates (Fig. 4, E to G). S7, A to C). The thermodynamics of inosine base tion (13). We postulated that in the absence of Most surprisingly, viable Adar1E861A/E861AIfih1−/− pairing is not defined. Therefore, we modeled ADAR1 editing, endogenous dsRNAs—such as the mutants that survived past weaning have been secondary structures for hyperedited substrates 3′UTRs of Klf1, Oip5, and Optn—could be bound identified (fig. S9B). The viable double mutants in two ways: (i) by predicting secondary struc- by MDA5 and/or RIG-I and could activate the are outwardly healthy, albeit slightly smaller than tures of Klf1 (Fig. 3E, left), Optn, and Oip5 (fig. cellular dsRNA response. Short hairpin RNA littermate controls, and have not demonstrated S7) 3′UTRs with inosine in place of adenosine, (shRNA) knockdown of MDA5 rescued prolifer- additional phenotypes to date (Fig. 4H). The res- assuming no base pairing, or (ii) by replacing ation and viability (Fig. 4, A to C) and suppressed cue of both developmental and adult viability adenosine with guanosine (Fig. 3E, middle), as- ISG induction in hematopoietic stem and pro- demonstrates that MDA5 is the primary sensor Downloaded from https://www.science.org on September 15, 2024 Fig. 1. Adar1E861A/E861A embryos die in utero. (A) Schematic of Adar1E861A knock-in allele. (B) ADAR1 protein expression in whole E12.5 embryos of the indicated genotypes. (C) Survival data at the indicated stages. (D) Images of viable E13.5 yolk sacs, embryos, and FL. Scale: yolk sac and embryo, 1 cm; FL, 2 mm. Representative (E) fluorescence-activated cell sorting (FACS) profiles, (F) cell numbers, and (G) frequency of 7AAD+ FL erythroblasts at E13.5. Results are mean ± SEM (+/+, n = 5 embryos; E861A/+, n = 18; E861A/E861A, n = 3). **P < 0.005 and ***P < 0.0005 compared with Adar1+/+. R2 to R5 denote erythroblast populations. 1116 4 SEPTEMBER 2015 VOL 349 ISSUE 6252 sciencemag.org SCIENCE  RE S EAR CH | R E P O R T S of endogenous dsRNA in the absence of ADAR1 transcriptional consequences of complete ADAR1 nonediting functions of ADAR1. We believe the editing. deficiency and the specific loss of A-to-I editing difference in survival is accounted for by the Unlike ADAR2, the primary role of ADAR1 has are markedly similar, demonstrating that RNA catalytically inactive ADAR1 retaining the ability not been clearly defined. Although it was as- editing is the primary and physiologically most to sequester immunogenic dsRNA (16). ADAR1 sumed that RNA editing was its central function, important function of ADAR1. Adar1 E861A/E861A sequestration is not sufficient, however, to com- additional RNA editing–independent roles have embryos die ~1 to 1.5 days later than Adar1−/− pletely prevent MDA5 recognition of unedited been proposed. The murine phenotypes and embryos, suggesting limited contributions from dsRNA and subsequent signaling. Therefore, the Fig. 2. Absence of editing transcription- ally phenocopies loss of ADAR1. (A) MA plot comparing gene expression in WT and E861A E12.5 FL. Red dots, differentially expressed genes; blue dots, differentially expressed ISGs. (B) QuSAGE analysis of the top 100 differen- tial pathway signatures Downloaded from https://www.science.org on September 15, 2024 ranked by fold enrich- ment and P value. (C) Heat map of the 50 most differentially expressed genes. Black dots indicate known ISGs. (D) qRT-PCR of ISGs in E861A com- pared with controls in FL and MEFs. Results are mean T SEM (n = 3). *P < 0.05, **P < 0.005, and ***P < 0.0005 compared with Adar1+/+. (E) IU-dsRNA response gene set in E861A compared with WT samples (left) and Adar1−/− HSCs com- pared with controls (right). SCIENCE sciencemag.org 4 SEPTEMBER 2015 VOL 349 ISSUE 6252 1117 R ES E A RC H | R E PO R TS Downloaded from https://www.science.org on September 15, 2024 Fig. 3. Defining the ADAR1 FL editome. (A) Summary of A-to-I editing site of editing sites. Red arrows highlight edited adenosine. (D) Integrative Genomics analysis in FL. SNPs, single-nucleotide polymorphisms; ANOVA, analysis of Viewer image of Klf1 in WT E12.5 FL and predicted secondary structure of 3′UTR. variance; ncRNA, noncoding RNA. (B) Mean editing difference for sites with The red line denotes the region depicted in (E). (E) Predicted secondary structure >20 reads (left, n = 1634). Mean editing frequency of differentially edited sites of a 212–base pair dsRNA stem loop from the 3′UTR of Klf1, with inosine (IU-dsRNA, between E861A and controls (right, n = 673). (C) Genomic DNA (gDNA) (bottom) left) and guanosine (GU-dsRNA, middle) in place of adenosine at the edited and complementary DNA (cDNA) (top and middle) Sanger sequencing validation sites. Right, predicted secondary structure with no A-to-I editing. 1118 4 SEPTEMBER 2015 VOL 349 ISSUE 6252 sciencemag.org SCIENCE  RE S EAR CH | R E P O R T S extended survival of the editing-deficient animals of self-dsRNA (18). Thus, the physiological func- of Klf1, Optn, and Oip5) generates multiple I-U reflects a delay rather than a fundamental differ- tion of ADAR1 is probably conserved in mammals. mismatches that act to prevent MDA5 oligomer- ence in the presentation of the same phenotype. Approximately half of the mammalian genome ization (21). In the absence of ADAR1 editing, The data from the Adar1E861A mutants are is composed of noncoding retrotransposons such long dsRNA stem loops can form that activate consistent with the type I interferonopathies as SINEs and Alus (19, 20), which typically form MDA5 (fig. S10). However, we cannot rule out an of Aicardi-Goutières syndrome (AGS) patients dsRNA duplexes. Retrotransposons are subjected alternate possibility that edited substrates pref- bearing ADAR1 mutations (17, 18). Editing of to extensive A-to-I RNA editing (3, 4). The location erentially bind MDA5 to prevent its activation endogenous transcripts in ADAR1-mutant AGS of repetitive elements may determine their immu- by other (unidentified and nonedited) dsRNA patients is probably reduced, leading to retention nogenicity. Retrotransposons located within in- transcripts (13). of paired endogenous dsRNA that can be sensed trons do not persist in the cytosol and therefore Concurrent ablation of MAVS, the downstream by MDA5. Consistent with this, gain-of-function cannot activate MDA5. Repetitive elements in adaptor of MDA5 and RIG-I, rescues ADAR1-null mutations of MDA5 have been identified in non– 3′UTRs, though rare (4), can be retained and mice to birth (22), demonstrating a role for ADAR1-mutant AGS patients (18). AGS MDA5 form duplexes, harboring the potential for recog- ADAR1 in the suppression of the RLR pathway. mutations caused higher-affinity binding to self- nition by MDA5. We propose that hyperediting Our study specifically identifies the critical cyto- dsRNA, resulting in the inappropriate detection of self-dsRNA by ADAR1 (such as the 3′UTRs solic sensor upstream of MAVS and demonstrates Downloaded from https://www.science.org on September 15, 2024 Fig. 4. Loss of MDA5 rescues Adar1E861A/E861A viability. (A) LKS+ cells isolated embryo, and FL of the indicated genotype (all Ifih1−/−). Scale: yolk sac and em- from Rosa26CreERT2 Adar1fl/+ (D/+) and Rosa26CreERT2 Adar1fl/E861A (D/E861A) bryo, 5 mm; FL, 1.6 mm. Representative (E) FACS profiles and (F) numbers of FL infected with pLKO.1 empty vector, shGFP, or (B) two independent shMDA5 erythrocytes at E13.5. (G) E13.5 FL qRT-PCR of ISGs. Results are mean T SEM [shMDA5(1) and shMDA5(2)] were cultured for 8 days. Results are mean T SEM (+/+, n = 2; E861A/+, n = 8; E861A/E861A, n = 4). *P < 0.05 compared with (n = 3). **P < 0.005 and ***P < 0.0005 compared with D/+. n.s, not signifi- Adar1+/+Ifih1−/− controls. (H) Photo of an Adar1E861A/E861A Ifih1−/− mouse and cant. (C) Analysis of apoptosis on day 8. (D) Images of viable E13.5 yolk sac, an Adar1E861A/+Ifih1+/− littermate at 26 days of age. SCIENCE sciencemag.org 4 SEPTEMBER 2015 VOL 349 ISSUE 6252 1119 R ES E A RC H | R E PO R TS that the Adar1E861A/E861A phenotype and, by ex- DNA SEGREGATION tension, Adar1−/− can be ascribed to the lack of editing of multiple substrates, resulting in the inappropriate activation of MDA5. We speculate that these unedited transcripts are sensed as Structures of archaeal DNA nonself by MDA5 and activate innate immune signaling. ADAR1’s primary physiological function segregation machinery reveal is to edit endogenous dsRNA to prevent sensing of endogenous dsRNA as nonself by MDA5. bacterial and eukaryotic linkages RE FE RENCES AND N OT ES Maria A. Schumacher,1* Nam K. Tonthat,1 Jeehyun Lee,1 1. P. Danecek et al., Genome Biol. 13, R26 (2012). 2. J. B. Li et al., Science 324, 1210–1213 (2009). Fernando A. Rodriguez-Castañeda,2 Naga Babu Chinnam,1 Anne K. Kalliomaa-Sanford,2 3. G. Ramaswami et al., Nat. Methods 9, 579–581 (2012). Irene W. Ng,2 Madhuri T. Barge,2 Porsha L. R. Shaw,1 Daniela Barillà2* 4. Y. Neeman, E. Y. Levanon, M. F. Jantsch, E. Eisenberg, RNA 12, 1802–1809 (2006). 5. J. B. Patterson, C. E. Samuel, Mol. Cell. Biol. 15, 5376–5388 Although recent studies have provided a wealth of information about archaeal biology, (1995). nothing is known about the molecular basis of DNA segregation in these organisms. Here, 6. J. C. Hartner et al., J. Biol. Chem. 279, 4894–4902 (2004). we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 7. Q. Wang et al., J. Biol. Chem. 279, 4952–4961 (2004). 8. S. V. Ward et al., Proc. Natl. Acad. Sci. U.S.A. 108, 331–336 partition system. This system uses three proteins: ParA; an atypical ParB adaptor; and a (2011). centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a 9. M. Higuchi et al., Cell 75, 1361–1370 (1993). DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, 10. M. Higuchi et al., Nature 406, 78–81 (2000). 11. F. Lai, R. Drakas, K. Nishikura, J. Biol. Chem. 270, 17098–17105 which contains a bacteria-like Walker motif. The C domain of ParB harbors structural (1995). similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system Downloaded from https://www.science.org on September 15, 2024 12. J. C. Hartner, C. R. Walkley, J. Lu, S. H. Orkin, Nat. Immunol. 10, combines bacteria-like and eukarya-like components, which suggests the possible 109–115 (2009). conservation of DNA segregation principles across the three domains of life. 13. P. Vitali, A. D. Scadden, Nat. Struct. Mol. Biol. 17, 1043–1050 D (2010). 14. G. Ramaswami, J. B. Li, Nucleic Acids Res. 42, D109–D113 NA segregation or partition is an essential similarity to any characterized partition protein. (2014). biological process ensuring faithful ge- The organization of the orf44-parB-parA genes 15. R. Zhang et al., Nat. Methods 11, 51–54 (2014). 16. M. C. Washburn et al., Cell Reports 6, 599–607 (2014). nomic transmission. The best-understood (fig. S1A) is reminiscent of bacterial partition 17. G. I. Rice et al., Nat. Genet. 44, 1243–1248 (2012). segregation systems at the molecular level cassettes; however, the cluster is tricistronic, un- 18. G. I. Rice et al., Nat. Genet. 46, 503–509 (2014). are those used by bacterial plasmids. like typical bicistronic bacterial systems (1–4). 19. E. S. Lander et al., Nature 409, 860–921 (2001). These simplified systems consist of a DNA cen- Hypothesizing that the pNOB8 centromere 20. Mouse Genome Sequencing Consortium, Nature 420, 520–562 (2002). tromere; ParA nucleoside triphosphatase (NTPase); may be located either 5′ or 3′ of the cassette as 21. B. Wu et al., Cell 152, 276–289 (2013). and centromere-binding protein (CBP), ParB (1–6). in bacteria, we analyzed pNOB8 protein binding 22. N. M. Mannion et al., Cell Reports 9, 1482–1494 The most common partition apparatuses use to these regions. Unlike its bacterial counterparts, (2014). Walker-box NTPases (7). The segregation sys- pNOB8 ParB only bound DNA nonspecifically ACKN OW LEDG MEN TS tems that have been identified on bacterial chro- (fig. S1B). Orf44, however, bound the upstream We thank V. Sankaran, S. Orkin, L. Purton, and J. Heierhorst for mosomes also harbor Walker NTPases, although DNA with high affinity [apparent dissociation discussion and SVH BioResources Centre for animal care. Data their mechanisms are less clear (1–6). In con- constant (Kdapp) = ~50 nM] (Fig. 1A). Therefore, sets described in the paper are deposited in Gene Expression trast to bacterial partition, eukaryotic partition we named this CBP, archaeal segregation pro- Omnibus (accession number GSE58917). This work was supported is highly complex. However, the linchpin in eu- tein A (AspA). Deoxyribonuclease I (DNase I) by the Leukaemia Foundation (C.R.W.), a Leukaemia Foundation Ph.D. scholarship (B.J.L), National Health and Medical Research karyotic segregation is the histone protein, CenpA, footprinting showed that AspA interacted with Council (NHMRC) Project Grant 1021216, NHMRC Career which is deposited in place of histone H3 at DNA a 23–base pair (bp) palindrome in the upstream Development Award 559016 (C.R.W.), a German Academic centromeres and dictates assembly of the segre- region and that increasing AspA concentrations Exchange Service Postdoctoral Fellowship (R.P.), a Stanford gation machinery (8–11). Although some pro- led to spreading around this site (Fig. 1B). Hence, University Dean’s Fellowship (R.P.), Stanford Genome Training Program (NIH grant T32 HG000044) and Stanford Graduate gress has been made in understanding DNA like bacterial ParB proteins, AspA spreads non- Fellowship (G.R.), NIH grant R01GM102484 (J.B.L.), the Ellison segregation in eukarya and bacteria, virtually specifically to DNA adjacent to its centromere Medical Foundation (J.B.L.), and the Stanford University nothing is known about the molecular process to form an extended “partition-complex.” Although Department of Genetics (J.B.L.). This work was also supported in of segregation in archaea, the third domain of poorly characterized, higher-order partition- part by the Victorian State Government Operational Infrastructure Support Scheme (to St. Vincent’s Institute of Medical Research). C. life (12–14). complexes are central to segregation, as they R.W. was the Leukaemia Foundation Phillip Desbrow Senior To gain insight into the underpinnings of mediate stabilizing, dynamic interactions with Research Fellow. J.C.H. is an employee of Taconic Biosciences. archaeal segregation, we performed a molecular ParA assemblages (1–4). Indeed, the CBP-NTPase Taconic Biosciences had no role in the preparation and content of dissection of the proteins encoded on the plas- interaction is key to the partition process. Bio- this Report. All other authors declare no conflicts of interest. Author contributions: M.H., P.H.S., and J.C.H. generated the knock- mid pNOB8 partition cassette harbored in Sulfo- chemical experiments, however, showed that in mouse line; B.J.L., R.P., A.M.C., G.R., J.C.H., J.B.L., and C.R.W. lobus NOB8H2 (15). This cassette contains three AspA does not bind pNOB8 ParA. Rather, ParB performed experiments and analyzed and interpreted data; M.H., open reading frames (ORFs): orf44, orf45, and bound both AspA [dissociation constant (Kd = P.H.S., and J.C.H. provided intellectual input and conceptual orf46. orf46 and orf45 encode 315- and 470- 12 mM)] and ParA (Kd = 17 mM), which indicated advice; and B.J.L. and C.R.W. wrote the manuscript. residue proteins, respectively, which show 33 to that it acts as an adaptor (fig. S1C). Thus, the SUPPLEMENTARY MATERIALS 37% and 42 to 58% sequence similarity to bac- pNOB8 partition system is composed of the www.sciencemag.org/content/349/6252/1115/suppl/DC1 terial ParA and ParB proteins. orf44 generates CBP AspA, adaptor ParB, and ParA NTPase. Materials and Methods a 93-residue protein that shows no sequence This finding prompted us to perform BLAST Supplementary Text searches for the occurrence of aspA-parB-parA Figs. S1 to S10 1 Tables S1 to S4 Department of Biochemistry, Duke University School of cassettes on archaeal genomes. The results re- Medicine, 243 Nanaline H. Duke, Box 3711, Durham, NC vealed that this tripartite cluster of genes is wide- References (23–41) 27710, USA. 2Department of Biology, University of York, York 1 June 2015; accepted 13 July 2015 YO10 5DD, UK. spread across different crenarchaeal genera and is Published online 23 July 2015 *Corresponding author. E-mail: [email protected] harbored on both chromosomes and plasmids 10.1126/science.aac7049 (M.A.S.); [email protected] (D.B.) (fig. S2). 1120 4 SEPTEMBER 2015 VOL 349 ISSUE 6252 sciencemag.org SCIENCE