Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq

Ribonucleotides are the most common non-canonical nucleotides incorporated into the genome of replicating cells. They are efficiently removed by ribonucleotide excision repair initiated by Ribonuclease (RNase) H2 cleavage. In the absence of RNase H2, such embedded ribonucleotides can be used to track DNA polymerase activity in vivo. To determine their precise location in Saccharomyces cerevisiae we developed embedded Ribonucleotide Sequencing (emRiboSeq), which uses recombinant RNase H2 to selectively create ligatable 3’-hydroxyl groups, in contrast to alternative methods that utilize alkaline hydrolysis. EmRiboSeq allows reproducible, strand-specific and potentially quantitative detection of embedded ribonucleotides at single-nucleotide resolution. This protocol can be adapted for the genome-wide mapping of other non-canonical bases by replacing RNase H2 with specific nicking endonucleases, a method we term Endonuclease Sequencing (EndoSeq). With the protocol taking <5 days to complete, these methods allow the in vivo study of DNA replication and repair, including the identification of replication origins and termination regions

RNase H2, mutated in Aicardi-Goutières syndrome, promotes LINE-1 retrotransposition

Long INterspersed Element class 1 (LINE-1) elements are a type of abundant retrotransposons active in mammalian genomes. An average human genome contains ~100 retrotransposition-competent LINE-1s, whose activity is influenced by the combined action of cellular repressors and activators. TREX1, SAMHD1 and ADAR1 are known LINE-1 repressors and when mutated cause the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). Mutations in RNase H2 are the most common cause of AGS, and its activity was proposed to similarly control LINE-1 retrotransposition. It has therefore been suggested that increased LINE-1 activity may be the cause of aberrant innate immune activation in AGS. Here, we establish that, contrary to expectations, RNase H2 is required for efficient LINE-1 retrotransposition. As RNase H1 overexpression partially rescues the defect in RNase H2 null cells, we propose a model in which RNase H2 degrades the LINE-1 RNA after reverse transcription, allowing retrotransposition to be completed. This also explains how LINE-1 elements can retrotranspose efficiently without their own RNase H activity. Our findings appear to be at odds with LINE-1-derived nucleic acids driving autoinflammation in AGS.M.B.-G. is funded by a “Formacion Profesorado Universitario” (FPU) PhD fellowship from the Government of Spain (MINECO, Ref FPU15/03294), and this paper is part of her thesis project (“Epigenetic control of the mobility of a human retrotransposon”). R.V.-A. is funded by a PFIS Fellowship from the Government of Spain (ISCiii, FI16/00413). O.M. is funded by an EMBO Long-Term Fellowship (ALTF 7-2015), the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409) and the Swiss National Science Foundation (P2ZHP3_158709). S.R.H. is funded by the Government of Spain (MINECO, RYC-2016-21395 and SAF2015-71589-P). A.P.J’s laboratory is supported by the UK Medical Research Council (MRC University Unit grant U127527202). J.L.G.P’s laboratory is supported by CICEFEDER- P12-CTS-2256, Plan Nacional de I+D+I 2008-2011 and 2013-2016 (FISFEDER- PI14/02152), PCIN-2014-115-ERA-NET NEURON II, the European Research Council (ERC-Consolidator ERC-STG-2012-233764), by an International Early Career Scientist grant from the Howard Hughes Medical Institute (IECS-55007420), by The Wellcome Trust-University of Edinburgh Institutional Strategic Support Fund (ISFF2) and by a private donation from Ms Francisca Serrano (Trading y Bolsa para Torpes, Granada, Spain)

The Lsm2-8 complex determines nuclear localization of the spliceosomal U6 snRNA

Lsm proteins are ubiquitous, multifunctional proteins that are involved in the processing and/or turnover of many, if not all, RNAs in eukaryotes. They generally interact only transiently with their substrate RNAs, in keeping with their likely roles as RNA chaperones. The spliceosomal U6 snRNA is an exception, being stably associated with the Lsm2-8 complex. The U6 snRNA is generally considered to be intrinsically nuclear but the mechanism of its nuclear retention has not been demonstrated, although La protein has been implicated. We show here that the complete Lsm2-8 complex is required for nuclear accumulation of U6 snRNA in yeast. Therefore, just as Sm proteins effect nuclear localization of the other spliceosomal snRNPs, the Lsm proteins mediate U6 snRNP localization except that nuclear retention is the likely mechanism for the U6 snRNP. La protein, which binds only transiently to the nascent U6 transcript, has a smaller, apparently indirect, effect on U6 localization that is compatible with its proposed role as a chaperone in facilitating U6 snRNP assembly

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.This work was supported by funding from the Medical Research Council and the European Research Council (ERC, 281847) (A.P.J.), the Lister Institute for Preventative Medicine (A.P.J. and G.S.S.), Medical Research Scotland (L.S.B.), German Federal Ministry of Education and Research (BMBF, 01GM1404) and E-RARE network EuroMicro (B.W), Wellcome Trust (M. Hurles), CMMC (P.N.), Cancer Research UK (C17183/A13030) (G.S.S. and M.R.H), Swiss National Science Foundation (P2ZHP3_158709) (O.M.), AIRC (12710) and ERC/EU FP7 (CIG_303806) (S.S.), Cancer Research UK (C6/A11224) and ERC/EU FP7 (HEALTH-F2- 2010-259893) (A.N.B. and S.P.J.).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.345

Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response

Aicardi–Goutières syndrome (AGS) provides a monogenic model of nucleic acid‐mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of either RNA:DNA hybrid or genome‐embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid‐sensing pathway. Here, we establish Rnaseh2b (A174T/A174T) knock‐in mice as a subclinical model of disease, identifying significant interferon‐stimulated gene (ISG) transcript upregulation that recapitulates the ISG signature seen in AGS patients. The inflammatory response is dependent on the nucleic acid sensor cyclic GMP‐AMP synthase (cGAS) and its adaptor STING and is associated with reduced cellular ribonucleotide excision repair activity and increased DNA damage. This suggests that cGAS/STING is a key nucleic acid‐sensing pathway relevant to AGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood‐onset interferonopathy and adult systemic autoimmune disorders

PCNA directs type 2 RNase H activity on DNA replication and repair substrates

Ribonuclease H2 is the major nuclear enzyme degrading cellular RNA/DNA hybrids in eukaryotes and the sole nuclease known to be able to hydrolyze ribonucleotides misincorporated during genomic replication. Mutation in RNASEH2 causes Aicardi–Goutières syndrome, an auto-inflammatory disorder that may arise from nucleic acid byproducts generated during DNA replication. Here, we report the crystal structures of Archaeoglobus fulgidus RNase HII in complex with PCNA, and human PCNA bound to a C-terminal peptide of RNASEH2B. In the archaeal structure, three binding modes are observed as the enzyme rotates about a flexible hinge while anchored to PCNA by its PIP-box motif. PCNA binding promotes RNase HII activity in a hinge-dependent manner. It enhances both cleavage of ribonucleotides misincorporated in DNA duplexes, and the comprehensive hydrolysis of RNA primers formed during Okazaki fragment maturation. In addition, PCNA imposes strand specificity on enzyme function, and by localizing RNase H2 and not RNase H1 to nuclear replication foci in vivo it ensures that RNase H2 is the dominant RNase H activity during nuclear replication. Our findings provide insights into how type 2 RNase H activity is directed during genome replication and repair, and suggest a mechanism by which RNase H2 may suppress generation of immunostimulatory nucleic acids

DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency.

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins