Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats

R-loops have been described at immunoglobulin class switch sequences, prokaryotic and mitochondrial replication origins, and disease-associated (CAG)n and (GAA)n trinucleotide repeats. The determinants of trinucleotide R-loop formation are unclear. Trinucleotide repeat expansions cause diseases including DM1 (CTG)n, SCA1 (CAG)n, FRAXA (CGG)n, FRAXE (CCG)n and FRDA (GAA)n. Bidirectional convergent transcription across these disease repeats can occur. We find R-loops formed when CTG or CGG and their complementary strands CAG or CCG were transcribed; GAA transcription, but not TTC, yielded R-loops. R-loop formation was sensitive to DNA supercoiling, repeat length, insensitive to repeat interruptions, and formed by extension of RNA:DNA hybrids in the RNA polymerase. R-loops arose by transcription in one direction followed by transcription in the opposite direction, and during simultaneous convergent bidirectional transcription of the same repeat forming double R-loop structures. Since each transcribed disease repeat formed R-loops suggests they may have biological functions

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

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

Analysis of the Formation, Binding and Processing of Alternative Nucleic Acid Structures in Disease-Associated Repeat Sequences

Expansion of gene-specific tandem repeat sequences is the causative mutation of a growing list of neurological, neuromuscular and neurodegenerative diseases, of which there are currently over 40. The cause of the initial tandem repeat expansion mutations, the molecular mechanisms driving ongoing repeat tract instability and the causes of pathogenesis are not well understood for many of these diseases. The formation of alternative secondary nucleic acid structures in the DNA and RNA of expanded tandem repeat sequences is thought to drive repeat instability and pathogenesis by impairing normal DNA and RNA metabolic processes. My thesis involves characterization of three such alternative secondary structures that could potentially form in the DNA or RNA of disease-associated tandem repeat sequences and assessment of their processing and binding to candidate structure-specific proteins. In DNA, I assessed how the specific configuration of slipped-out 3-way junction structures formed in disease-associated (CAG)*(CTG) tandem repeats can influence protein binding and cleavage by structure-specific DNA repair proteins in vitro. Whereas previous studies focused upon slipped-DNA structures as a static entity, we show that slipped-DNA junctions exist in multiple interchanging conformations and the specific conformation of the junction can influence how it gets processed. These findings support that the junction conformation is an important modifier of disease-associated (CAG)*(CTG) instability. To further extend our understanding of nucleic acid structure in tandem repeat disease, I analyzed the structural and sequence determinants governing RNA:DNA hybrid formation and processing at various trinucleotide repeats. By using a well-established in vitro transcription assay, I demonstrated that stable R-loop formation occurs in all disease-associated trinucleotide repeats. I also identified novel double-R-loop structures formed when repeats are simultaneously bidirectionally transcribed, as occurs at many disease-associated trinucleotide repeat-containing genes. I went on to establish a novel R-loop processing assay using a cell-free extract system and demonstrated that R-loops and particularly double-R-loops can increase the instability of (CAG)*(CTG) repeats. Finally, I identified extremely stable RNA G-quadruplex structures in the amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD)-associated (GGGGCC)n repeat in vitro, using RNA oligonucleotide models and assessed their ability to bind structure-specific RNA binding proteins. This led to the identification of the ASF/SF2 essential splicing factor as a candidate protein interactor that may have relevance to ALS-FTD. Taken together, my findings demonstrate unusual nucleic acid structure formation by disease-associated tandem repeats in the DNA, the RNA and during transcription when the DNA interacts with the RNA. These secondary structures can be differentially bound and/or processed by structure-specific proteins. Understanding the secondary structures formed by disease-associated repeat sequences and identifying the proteins that interact with them expands the potential therapeutics that can be developed to modulate pathogenesis and also expands our understanding of the normal biological roles of repeat sequences in the genome.Ph.D

RAN Translation: Fragile X in the Running

In this issue of Neuron, Todd et al. (2013) reveal that noncanonical repeat associated non-AUG (RAN) translation occurs on nonexpanded (CGG)30–50 and premutation (CGG)59–160 repeats, associated with the FMR1 gene, suggesting that the polyglycine and polyalanine products might have natural and pathogenic roles

Mitigating RNA Toxicity in Myotonic Dystrophy using Small Molecules

This review, one in a series on myotonic dystrophy (DM), is focused on the development and potential use of small molecules as therapeutics for DM. The complex mechanisms and pathogenesis of DM are covered in the associated reviews. Here, we examine the various small molecule approaches taken to target the DNA, RNA, and proteins that contribute to disease onset and progression in myotonic dystrophy type 1 (DM1) and 2 (DM2)

Interconverting Conformations of Slipped-DNA Junctions Formed by Trinucleotide Repeats Affect Repair Outcome

Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-way slipped-DNA junction, at which the excess repeats slip out from the duplex, is a poorly understood feature common to these mutagenic intermediates. Here, we reveal that slipped junctions can assume a surprising number of interconverting conformations where the strand opposite the slip-out either is fully base paired or has one or two unpaired nucleotides. These unpaired nucleotides can also arise opposite either of the nonslipped junction arms. Junction conformation can affect binding by various structure-specific DNA repair proteins and can also alter correct nick-directed repair levels. Junctions that have the potential to contain unpaired nucleotides are repaired with a significantly higher efficiency than constrained fully paired junctions. Surprisingly, certain junction conformations are aberrantly repaired to expansion mutations: misdirection of repair to the non-nicked strand opposite the slip-out leads to integration of the excess slipped-out repeats rather than their excision. Thus, slipped-junction structure can determine whether repair attempts lead to correction or expansion mutations