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1.
Hum Mol Genet ; 2024 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-39180495

RESUMEN

Myotonic dystrophy type 1 (DM1) is a heterogeneous multisystemic disease caused by a CTG repeat expansion in DMPK. Transcription of the expanded allele produces toxic CUG repeat RNA that sequesters the MBNL family of alternative splicing (AS) regulators into ribonuclear foci, leading to pathogenic mis-splicing. To identify genetic modifiers of toxic CUG RNA levels and the spliceopathy, we performed a genome-scale siRNA screen using an established HeLa DM1 repeat-selective screening platform. We unexpectedly identified core spliceosomal proteins as a new class of modifiers that rescue the spliceopathy in DM1. Modest knockdown of one of our top hits, SNRPD2, in DM1 fibroblasts and myoblasts, significantly reduces DMPK expression and partially rescues MBNL-regulated AS dysfunction. While the focus on the DM1 spliceopathy has centered around the MBNL proteins, our work reveals an unappreciated role for MBNL:spliceosomal protein stoichiometry in modulating the spliceopathy, revealing new biological and therapeutic avenues for DM1.

2.
Nucleic Acids Res ; 50(16): 9306-9318, 2022 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-35979951

RESUMEN

Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.


Asunto(s)
Inosina Trifosfato , ARN , Humanos , Animales , Ratones , Ratas , Inosina Trifosfato/metabolismo , Pirofosfatasas/genética , Inosina , Nucleótidos
3.
Proc Natl Acad Sci U S A ; 116(42): 20991-21000, 2019 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-31570586

RESUMEN

A CTG repeat expansion in the DMPK gene is the causative mutation of myotonic dystrophy type 1 (DM1). Transcription of the expanded CTG repeat produces toxic gain-of-function CUG RNA, leading to disease symptoms. A screening platform that targets production or stability of the toxic CUG RNA in a selective manner has the potential to provide new biological and therapeutic insights. A DM1 HeLa cell model was generated that stably expresses a toxic r(CUG)480 and an analogous r(CUG)0 control from DMPK and was used to measure the ratio-metric level of r(CUG)480 versus r(CUG)0. This DM1 HeLa model recapitulates pathogenic hallmarks of DM1, including CUG ribonuclear foci and missplicing of pre-mRNA targets of the muscleblind (MBNL) alternative splicing factors. Repeat-selective screening using this cell line led to the unexpected identification of multiple microtubule inhibitors as hits that selectively reduce r(CUG)480 levels and partially rescue MBNL-dependent missplicing. These results were validated by using the Food and Drug Administration-approved clinical microtubule inhibitor colchicine in DM1 mouse and primary patient cell models. The mechanism of action was found to involve selective reduced transcription of the CTG expansion that we hypothesize to involve the LINC (linker of nucleoskeleton and cytoskeleton) complex. The unanticipated identification of microtubule inhibitors as selective modulators of toxic CUG RNA opens research directions for this form of muscular dystrophy and may shed light on the biology of CTG repeat expansion and inform therapeutic avenues. This approach has the potential to identify modulators of expanded repeat-containing gene expression for over 30 microsatellite expansion disorders.


Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Microtúbulos/efectos de los fármacos , Distrofia Miotónica/genética , ARN/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Expansión de Repetición de Trinucleótido/efectos de los fármacos , Animales , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Microtúbulos/genética , Microtúbulos/metabolismo , Distrofia Miotónica/enzimología , Proteína Quinasa de Distrofia Miotónica/genética , Proteína Quinasa de Distrofia Miotónica/metabolismo , ARN/química , ARN/metabolismo
4.
PLoS Genet ; 15(3): e1007605, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30856165

RESUMEN

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.


Asunto(s)
Cardiomiopatía Dilatada/enzimología , Cardiomiopatía Dilatada/genética , Catarata/enzimología , Catarata/genética , Hipogonadismo/enzimología , Hipogonadismo/genética , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/genética , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Pirofosfatasas/deficiencia , Animales , Secuencia de Bases , Preescolar , Análisis Mutacional de ADN , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Femenino , Homocigoto , Humanos , Inosina/metabolismo , Masculino , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/enzimología , Mutación , Linaje , Pirofosfatasas/genética , ARN/genética , ARN/metabolismo , Secuenciación del Exoma
5.
EMBO J ; 35(8): 831-44, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26903602

RESUMEN

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 eitherRNA:DNAhybrid or genome-embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid-sensing pathway. Here, we establishRnaseh2b(A174T/A174T)knock-in mice as a subclinical model of disease, identifying significant interferon-stimulated gene (ISG) transcript upregulation that recapitulates theISGsignature seen inAGSpatients. The inflammatory response is dependent on the nucleic acid sensor cyclicGMP-AMPsynthase (cGAS) and its adaptorSTINGand is associated with reduced cellular ribonucleotide excision repair activity and increasedDNAdamage. This suggests thatcGAS/STINGis a key nucleic acid-sensing pathway relevant toAGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood-onset interferonopathy and adult systemic autoimmune disorders.


Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/genética , Inmunidad Innata/genética , Proteínas de la Membrana/inmunología , Mutación Missense , Malformaciones del Sistema Nervioso/genética , Nucleotidiltransferasas/inmunología , Ribonucleasa H/genética , Ribonucleasas/genética , Animales , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Autoinmunidad/genética , Daño del ADN , Regulación de la Expresión Génica , Humanos , Interferones/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Malformaciones del Sistema Nervioso/inmunología , Malformaciones del Sistema Nervioso/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Ribonucleasa H/metabolismo
6.
Nucleic Acids Res ; 45(22): 12808-12815, 2017 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-29106596

RESUMEN

All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent misincorporation occurs in tissues, or whether this plays a role in human disease. Here, we show that mtDNA of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the high ratio of ribonucleotide to deoxynucleotide triphosphates in tissues, and that riboadenosines account for three-quarters of them. The pattern of embedded ribonucleotides changes in a mouse model of Mpv17 deficiency, which displays a marked increase in rGMPs in mtDNA. However, while the mitochondrial dGTP is low in the Mpv17-/- liver, the brain shows no change in the overall dGTP pool, leading us to suggest that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to distort the mtDNA and impede its replication, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of Mpv17-/- mice. These findings suggest aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology.


Asunto(s)
ADN Mitocondrial/genética , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Ribonucleótidos/genética , Eliminación de Secuencia , Animales , Secuencia de Bases , Encéfalo/metabolismo , Línea Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Hígado/metabolismo , Proteínas de la Membrana/deficiencia , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/deficiencia
7.
Int J Mol Sci ; 20(16)2019 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-31426500

RESUMEN

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).


Asunto(s)
Distrofia Miotónica/tratamiento farmacológico , ARN Mensajero/antagonistas & inhibidores , Animales , Humanos , Distrofia Miotónica/metabolismo , Distrofia Miotónica/terapia
8.
Nucleic Acids Res ; 42(16): 10473-87, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25147206

RESUMEN

R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats.


Asunto(s)
Expansión de las Repeticiones de ADN , Inestabilidad Genómica , Proteínas/genética , Expansión de Repetición de Trinucleótido , Proteína C9orf72 , Línea Celular Tumoral , ADN/química , ADN/metabolismo , Células HeLa , Humanos , Neuronas/metabolismo , ARN/química , ARN/metabolismo , Ribonucleasa H/metabolismo
9.
J Biol Chem ; 289(8): 4653-9, 2014 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-24371143

RESUMEN

Certain DNA and RNA sequences can form G-quadruplexes, which can affect genetic instability, promoter activity, RNA splicing, RNA stability, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia can be caused by expansion of a (GGGGCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n- and r(GGCCCC)n-containing transcripts aggregate in nuclear foci, possibly sequestering repeat-binding proteins such as ASF/SF2 and hnRNPA1, suggesting a toxic RNA pathogenesis, as occurs in myotonic dystrophy. Furthermore, the C9orf72 repeat RNA was recently demonstrated to undergo the noncanonical repeat-associated non-AUG translation (RAN translation) into pathologic dipeptide repeats in patient brains, a process that is thought to depend upon RNA structure. We previously demonstrated that the r(GGGGCC)n RNA forms repeat tract length-dependent G-quadruplex structures that bind the ASF/SF2 protein. Here we show that the cationic porphyrin (5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin (TMPyP4)), which can bind some G-quadruplex-forming sequences, can bind and distort the G-quadruplex formed by r(GGGGCC)8, and this ablates the interaction of either hnRNPA1 or ASF/SF2 with the repeat. These findings provide proof of concept that nucleic acid binding small molecules, such as TMPyP4, can distort the secondary structure of the C9orf72 repeat, which may beneficially disrupt protein interactions, which may ablate either protein sequestration and/or RAN translation into potentially toxic dipeptides. Disruption of secondary structure formation of the C9orf72 RNA repeats may be a viable therapeutic avenue, as well as a means to test the role of RNA structure upon RAN translation.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/genética , G-Cuádruplex/efectos de los fármacos , Porfirinas/farmacología , Proteínas/genética , Proteínas de Unión al ARN/metabolismo , Secuencia de Bases , Proteína C9orf72 , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Porfirinas/química , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Factores de Empalme Serina-Arginina
10.
J Biol Chem ; 288(14): 9860-9866, 2013 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-23423380

RESUMEN

Certain DNA and RNA sequences can form G-quadruplexes, which can affect promoter activity, genetic instability, RNA splicing, translation, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia were recently shown to be caused by expansion of a (GGGGCC)n·(GGCCCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n-containing transcripts aggregate in nuclear foci possibly sequestering repeat-binding proteins, suggesting a toxic RNA pathogenesis. We demonstrate that the r(GGGGCC)n RNA but not the C-rich r(GGCCCC)n RNA forms extremely stable uni- and multimolecular parallel G-quadruplex structures (up to 95 °C). Multimolecular G-quadruplex formation is influenced by repeat number and RNA concentration. MBNL1, a splicing factor that is sequestered in myotonic dystrophy patients by binding to expanded r(CUG)n repeat hairpins, does not bind the C9orf72 repeats, but the splicing factor ASF/SF2 can bind the r(GGGGCC)n repeat. Because multimolecular G-quadruplexes are enhanced by repeat length, RNA-RNA interactions facilitated by G-quadruplex formation at expanded repeats might influence transcript aggregation and foci formation in amyotrophic lateral sclerosis-frontotemporal dementia cells. Tract length-dependent G-quadruplex formation by the C9orf72 RNA should be considered when assessing the role of this repeat in C9orf72 gene activity, protein binding, transcript foci formation, and translation of the C9orf72 product, including the noncanonical repeat-associated non-ATG translation (RAN translation) into pathologic dipeptide repeats, as well as any oligonucleotide repeat-based therapy.


Asunto(s)
G-Cuádruplex , Proteínas/química , Proteínas/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72 , Dicroismo Circular , Expansión de las Repeticiones de ADN , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Humanos , Iones , Enfermedades Neurodegenerativas/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/genética , Péptidos/química , Unión Proteica , Isoformas de Proteínas , ARN/química , ARN/genética , Empalme del ARN
11.
ACS Chem Biol ; 19(2): 348-356, 2024 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-38252964

RESUMEN

A-to-I editing catalyzed by adenosine deaminase acting on RNAs impacts numerous physiological and biochemical processes that are essential for cellular functions and is a big contributor to the infectivity of certain RNA viruses. The outcome of this deamination leads to changes in the eukaryotic transcriptome functionally resembling A-G transitions since inosine preferentially pairs with cytosine. Moreover, hyper-editing or multiple A to G transitions in clusters were detected in measles virus. Inosine modifications either directly on viral RNA or on cellular RNA can have antiviral or pro-viral repercussions. While many of the significant roles of inosine in cellular RNAs are well understood, the effects of hyper-editing of A to I on viral polymerase activity during RNA replication remain elusive. Moreover, biological strategies such as molecular cloning and RNA-seq for transcriptomic interrogation rely on RT-polymerase chain reaction with little to no emphasis placed on the first step, reverse transcription, which may reshape the sequencing results when hypermodification is present. In this study, we systematically explore the influence of inosine modification, varying the number and position of inosines, on decoding outcomes using three different reverse transcriptases (RTs) followed by standard Sanger sequencing. We find that inosine alone or in clusters can differentially affect the RT activity. To gain structural insights into the accommodation of inosine in the polymerase site of HIV-1 reverse transcriptase (HIV-1-RT) and how this structural context affects the base pairing rules for inosine, we performed molecular dynamics simulations of the HIV-1-RT. The simulations highlight the importance of the protein-nucleotide interaction as a critical factor in deciphering the base pairing behavior of inosine clusters. This effort sets the groundwork for decrypting the physiological significance of inosine and linking the fidelity of reverse transcriptase and the possible diverse transcription outcomes of cellular RNAs and/or viral RNAs where hyper-edited inosines are present in the transcripts.


Asunto(s)
ARN Viral , Transcripción Reversa , Emparejamiento Base , ARN Viral/genética , Inosina/análisis , Inosina/química , Inosina/genética , Adenosina Desaminasa/genética
12.
bioRxiv ; 2024 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-38014227

RESUMEN

Synthetic DNA motifs form the basis of nucleic acid nanotechnology, and their biochemical and biophysical properties determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.

13.
Nat Commun ; 15(1): 6636, 2024 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-39107287

RESUMEN

Synthetic DNA motifs form the basis of nucleic acid nanotechnology. The biochemical and biophysical properties of these motifs determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.


Asunto(s)
ADN , Conformación de Ácido Nucleico , Termodinámica , ADN/química , ADN/metabolismo , Magnesio/química , Magnesio/metabolismo , Nanotecnología/métodos , Humanos
14.
bioRxiv ; 2024 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-38260517

RESUMEN

Alternative splicing (AS) of Exon 11 of the Insulin Receptor ( INSR ) is highly regulated and disrupted in several human disorders. To better understand INSR exon 11 AS regulation, splicing activity of an INSR exon 11 minigene reporter was measured across a gradient of the AS regulator muscleblind-like 1 protein (MBNL1). The RNA-binding protein Fox-1 (RBFOX1) was added to determine its impact on MBNL1-regulated splicing. The role of the RBFOX1 UGCAUG binding site within intron 11 was assessed across the MBNL1 gradient. Mutating the UGCAUG motif inhibited RBFOX1 regulation of exon 11 and had the unexpected effect of reducing MBNL1 regulation of this exon. Molecular dynamics simulations showed that exon 11 and the adjacent RNA adopts a dynamically stable conformation. Mutation of the RBFOX1 binding site altered RNA structure and dynamics, while a mutation that created an optimal MBNL1 binding site at the RBFOX1 site shifted the RNA back to wild type. An antisense oligonucleotide (ASO) was used to confirm the structure in this region of the pre-mRNA. This example of intronic mutations shifting pre-mRNA structure and dynamics to modulate splicing suggests RNA structure and dynamics should be taken into consideration for AS regulation and therapeutic interventions targeting pre-mRNA.

15.
bioRxiv ; 2024 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-39211226

RESUMEN

Spinocerebellar ataxias (SCAs) are a genetically heterogenous group of devastating neurodegenerative conditions for which clinical care currently focuses on managing symptoms. Across these diseases there is an unmet need for therapies that address underlying disease mechanisms. We utilised the shared CAG repeat expansion mutation causative for a large subgroup of SCAs, to develop a novel disease-gene independent and mechanism agnostic small molecule screening approach to identify compounds with therapeutic potential across multiple SCAs. Using this approach, we identified the FDA approved microtubule inhibitor Colchicine and a novel CAG-repeat binding compound that reduce expression of disease associated transcripts across SCA1, 3 and 7 patient derived fibroblast lines and the Atxn1 154Q/2Q SCA1 mouse model in a repeat selective manner. Furthermore, our lead candidate rescues dysregulated alternative splicing in Atxn1 154Q/2Q mice. This work provides the first example of small molecules capable of targeting the underlying mechanism of disease across multiple CAG SCAs.

16.
Biochemistry ; 52(5): 773-85, 2013 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-23339280

RESUMEN

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.


Asunto(s)
Reparación del ADN , ADN/química , ADN/metabolismo , Repeticiones de Trinucleótidos , Emparejamiento Base , Secuencia de Bases , ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Proteína HMGB1/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/metabolismo , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica , Factores de Transcripción/metabolismo
17.
Nucleic Acids Res ; 39(5): 1749-62, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21051337

RESUMEN

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.


Asunto(s)
ADN/química , ARN/química , Transcripción Genética , Repeticiones de Trinucleótidos , ADN/ultraestructura , ADN Superhelicoidal/química , ARN/ultraestructura
18.
Wiley Interdiscip Rev RNA ; 14(5): e1790, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37092460

RESUMEN

Inosine triphosphate pyrophosphatase (ITPase), encoded by the ITPA gene in humans, is an important enzyme that preserves the integrity of cellular nucleotide pools by hydrolyzing the noncanonical purine nucleotides (deoxy)inosine and (deoxy)xanthosine triphosphate into monophosphates and pyrophosphate. Variants in the ITPA gene can cause partial or complete ITPase deficiency. Partial ITPase deficiency is benign but clinically relevant as it is linked to altered drug responses. Complete ITPase deficiency causes a severe multisystem disorder characterized by seizures and encephalopathy that is frequently associated with fatal infantile dilated cardiomyopathy. In the absence of ITPase activity, its substrate noncanonical nucleotides have the potential to accumulate and become aberrantly incorporated into DNA and RNA. Hence, the pathophysiology of ITPase deficiency could arise from metabolic imbalance, altered DNA or RNA regulation, or from a combination of these factors. Here, we review the known functions of ITPase and highlight recent work aimed at determining the molecular basis for ITPA-associated pathogenesis which provides evidence for RNA dysfunction. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.


Asunto(s)
Nucleótidos , ARN , Humanos , Nucleótidos/metabolismo , ARN/genética , Inosina , Inosina Trifosfato , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , ADN
19.
bioRxiv ; 2023 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-36778282

RESUMEN

Myotonic dystrophy is a multisystemic neuromuscular disease caused by either a CTG repeat expansion in DMPK (DM1) or a CCTG repeat expansion in CNBP (DM2). Transcription of the expanded alleles produces toxic gain-of-function RNA that sequester the MBNL family of alternative splicing regulators into ribonuclear foci, leading to pathogenic mis-splicing. There are currently no approved treatments that target the root cause of disease which is the production of the toxic expansion RNA molecules. In this study, using our previously established HeLa DM1 repeat selective screening platform, we identified the natural product quercetin as a selective modulator of toxic RNA levels. Quercetin treatment selectively reduced toxic RNA levels and rescued MBNL dependent mis-splicing in DM1 and DM2 patient derived cell lines and in the HSALR transgenic DM1 mouse model where rescue of myotonia was also observed. Based on our data and its safety profile for use in humans, we have identified quercetin as a priority disease-targeting therapeutic lead for clinical evaluation for the treatment of DM1 and DM2.

20.
EMBO Mol Med ; 13(11): e14095, 2021 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-34632710

RESUMEN

Spinocerebellar ataxia type 8 (SCA8), a dominantly inherited neurodegenerative disorder caused by a CTG•CAG expansion, is unusual because most individuals that carry the mutation do not develop ataxia. To understand the variable penetrance of SCA8, we studied the molecular differences between highly penetrant families and more common sporadic cases (82%) using a large cohort of SCA8 families (n = 77). We show that repeat expansion mutations from individuals with multiple affected family members have CCG•CGG interruptions at a higher frequency than sporadic SCA8 cases and that the number of CCG•CGG interruptions correlates with age at onset. At the molecular level, CCG•CGG interruptions increase RNA hairpin stability, and in cell culture experiments, increase p-eIF2α and polyAla and polySer RAN protein levels. Additionally, CCG•CGG interruptions, which encode arginine interruptions in the polyGln frame, increase toxicity of the resulting proteins. In summary, SCA8 CCG•CGG interruptions increase polyAla and polySer RAN protein levels, polyGln protein toxicity, and disease penetrance and provide novel insight into the molecular differences between SCA8 families with high vs. low disease penetrance.


Asunto(s)
Degeneraciones Espinocerebelosas , Expansión de Repetición de Trinucleótido , Ataxia , Humanos , Proteínas del Tejido Nervioso/genética , Penetrancia , Proteínas , ARN Largo no Codificante/genética , Degeneraciones Espinocerebelosas/genética
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