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Nucleic Acids Research Jul 2021Transcription induced CAG repeat instability is associated with fatal neurological disorders. Genetic approaches found transcription-coupled nucleotide excision repair...
Transcription induced CAG repeat instability is associated with fatal neurological disorders. Genetic approaches found transcription-coupled nucleotide excision repair (TC-NER) factor CSB protein and TFIIS play critical roles in modulating the repeat stability. Here, we took advantage of an in vitro reconstituted yeast transcription system to investigate the underlying mechanism of RNA polymerase II (Pol II) transcriptional pausing/stalling by CAG slip-out structures and the functions of TFIIS and Rad26, the yeast ortholog of CSB, in modulating transcriptional arrest. We identified length-dependent and strand-specific mechanisms that account for CAG slip-out induced transcriptional arrest. We found substantial R-loop formation for the distal transcriptional pausing induced by template strand (TS) slip-out, but not non-template strand (NTS) slip-out. In contrast, Pol II backtracking was observed at the proximal transcriptional pausing sites induced by both NTS and TS slip-out blockage. Strikingly, we revealed that Rad26 and TFIIS can stimulate bypass of NTS CAG slip-out, but not TS slip-out induced distal pausing. Our biochemical results provide new insights into understanding the mechanism of CAG slip-out induced transcriptional pausing and functions of transcription factors in modulating transcription-coupled CAG repeat instability, which may pave the way for developing potential strategies for the treatment of repeat sequence associated human diseases.
Topics: Adenosine Triphosphatases; R-Loop Structures; RNA Polymerase II; Saccharomyces cerevisiae Proteins; Transcription Elongation, Genetic; Transcriptional Elongation Factors; Trinucleotide Repeats
PubMed: 34197619
DOI: 10.1093/nar/gkab573 -
Nucleic Acids Research Jul 2020How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs'...
How genetic defects trigger the molecular changes that cause late-onset disease is important for understanding disease progression and therapeutic development. Fuchs' endothelial corneal dystrophy (FECD) is an RNA-mediated disease caused by a trinucleotide CTG expansion in an intron within the TCF4 gene. The mutant intronic CUG RNA is present at one-two copies per cell, posing a challenge to understand how a rare RNA can cause disease. Late-onset FECD is a uniquely advantageous model for studying how RNA triggers disease because: (i) Affected tissue is routinely removed during surgery; (ii) The expanded CTG mutation is one of the most prevalent disease-causing mutations, making it possible to obtain pre-symptomatic tissue from eye bank donors to probe how gene expression changes precede disease; and (iii) The affected tissue is a homogeneous single cell monolayer, facilitating accurate transcriptome analysis. Here, we use RNA sequencing (RNAseq) to compare tissue from individuals who are pre-symptomatic (Pre_S) to tissue from patients with late stage FECD (FECD_REP). The abundance of mutant repeat intronic RNA in Pre_S and FECD_REP tissue is elevated due to increased half-life in a corneal cells. In Pre_S tissue, changes in splicing and extracellular matrix gene expression foreshadow the changes observed in advanced disease and predict the activation of the fibrosis pathway and immune system seen in late-stage patients. The absolute magnitude of splicing changes is similar in pre-symptomatic and late stage tissue. Our data identify gene candidates for early drivers of disease and biomarkers that may represent diagnostic and therapeutic targets for FECD. We conclude that changes in alternative splicing and gene expression are observable decades prior to the diagnosis of late-onset trinucleotide repeat disease.
Topics: Adult; Aged; Biomarkers; Cornea; Female; Fuchs' Endothelial Dystrophy; Gene Expression Regulation; Genetic Predisposition to Disease; Humans; Introns; Male; Middle Aged; Mutation; Organ Specificity; Sequence Analysis, RNA; Transcription Factor 4; Trinucleotide Repeat Expansion; Trinucleotide Repeats
PubMed: 32463444
DOI: 10.1093/nar/gkaa422 -
Nucleosides, Nucleotides & Nucleic Acids 2020Trinucleotide repeats are responsible for many genetic diseases. Previous studies have shown that duplex RNAs (dsRNAs) can be used to target expression of a mutant...
Trinucleotide repeats are responsible for many genetic diseases. Previous studies have shown that duplex RNAs (dsRNAs) can be used to target expression of a mutant repeat allele while leaving expression of the wild-type allele untouched, creating opportunities for allele-selective inhibition and better therapeutic outcomes. In contrast to successes with other genes, we report here that we cannot achieve allele-selective inhibition when targeting the expanded CAG repeat within Ataxin-1 (ATXN1), the cause of spinal cerebellar ataxia-1 (SCA1). The most likely explanation for this unfavorable outcome is that the mean CAG repeat number within wild-type ATXN1 is relatively high compared to other trinucleotide repeat diseases. Because the wild-type repeat number is high, it is likely that there is poor discrimination between the mutant and wild-type repeat and less opportunity for allele-selective inhibition across the entire spectrum of mutations found in SCA1 patients. Our data support the conclusion that the potential for multiple cooperative binding interactions is a critical factor governing allele-selective recognition of trinucleotide repeat genes by duplex RNAs. These results should be helpful in predicting which diseases and which patients are most likely to benefit from allele-selective targeting of expanded repeats.
Topics: Alleles; Ataxin-1; Cells, Cultured; Fibroblasts; Gene Expression Regulation; Gene Silencing; Humans; Mutation; Oligonucleotides; RNA Interference; RNA, Double-Stranded; Trinucleotide Repeats
PubMed: 31645175
DOI: 10.1080/15257770.2019.1671592 -
Current Opinion in Genetics &... Jun 2014Trinucleotide repeats (TNRs) expansion disorders are severe neurodegenerative and neuromuscular disorders that arise from inheriting a long tract (30-50 copies) of a... (Review)
Review
Trinucleotide repeats (TNRs) expansion disorders are severe neurodegenerative and neuromuscular disorders that arise from inheriting a long tract (30-50 copies) of a trinucleotide unit within or near an expressed gene (Figure 1a). The mutation is referred to as 'trinucleotide expansion' since the number of triplet units in a mutated gene is greater than the number found in the normal gene. Expansion becomes obvious once the number of repeating units passes a critical threshold length, but what happens at the threshold to render the repeating tract unstable? Here we discuss DNA-dependent and RNA-dependent models by which a particular DNA length permits a rapid transition to an unstable state.
Topics: DNA; Genetic Predisposition to Disease; Humans; Models, Genetic; Neurodegenerative Diseases; Neuromuscular Diseases; RNA; Trinucleotide Repeat Expansion; Trinucleotide Repeats
PubMed: 25282113
DOI: 10.1016/j.gde.2014.07.003 -
The Journal of Physical Chemistry. B Sep 2023Myotonic dystrophy type 1 is the most frequent form of muscular dystrophy in adults caused by an abnormal expansion of the CTG trinucleotide. Both the expanded DNA and...
Myotonic dystrophy type 1 is the most frequent form of muscular dystrophy in adults caused by an abnormal expansion of the CTG trinucleotide. Both the expanded DNA and the expanded CUG RNA transcript can fold into hairpins. Co-transcriptional formation of stable RNA·DNA hybrids can also enhance the instability of repeat tracts. We performed molecular dynamics simulations of homoduplexes associated with the disease, d(CTG) and r(CUG), and their corresponding r(CAG):d(CTG) and r(CUG):d(CAG) hybrids that can form under bidirectional transcription and of non-pathological d(GTC) and d(GUC) homoduplexes. We characterized their conformations, stability, and dynamics and found that the U·U and T·T mismatches are dynamic, favoring anti-anti conformations inside the helical core, followed by anti-syn and syn-syn conformations. For DNA, the secondary minima in the non-expanding d(GTC) helices are deeper, wider, and longer-lived than those in d(CTG), which constitutes another biophysical factor further differentiating the expanding and non-expanding sequences. The hybrid helices are closer to A-RNA, with the A-T and A-U pairs forming two stable Watson-Crick hydrogen bonds. The neutralizing ion distribution around the non-canonical pairs is also described.
Topics: Adult; Humans; Trinucleotide Repeats; Biophysics; DNA; Hydrogen Bonding; RNA
PubMed: 37681731
DOI: 10.1021/acs.jpcb.3c03538 -
Human Molecular Genetics Oct 2019Triplet repeat diseases (TRDs) are caused by pathogenic expansions of trinucleotide sequence repeats within coding and non-coding regions of different genes. They are... (Review)
Review
Triplet repeat diseases (TRDs) are caused by pathogenic expansions of trinucleotide sequence repeats within coding and non-coding regions of different genes. They are typically progressive, very disabling and frequently involve the nervous system. Currently available symptomatic therapies provide modest benefit at best. The development of interventions that interfere with the natural history of these diseases is a priority. A common pathogenic process shared by most TRDs is the presence of toxicity from the messenger RNA or protein encoded by the gene harboring the abnormal expansion. Strategies to interfere with the expression of these genes using different molecular approaches are being pursued and have reached the clinical stage. This review will summarize the significant progress made in this field in the last few years, focusing on three main areas: the discovery of biomarkers of disease progression and target engagement, advances in preclinical studies for the polyglutamine ataxias and the initial clinical application in myotonic dystrophy type 1 and Huntington's disease.
Topics: Animals; Biomarkers; Combined Modality Therapy; Disease Management; Drug Development; Genetic Predisposition to Disease; Genetic Therapy; Humans; Molecular Targeted Therapy; Nervous System Diseases; Proof of Concept Study; Translational Research, Biomedical; Trinucleotide Repeat Expansion; Trinucleotide Repeats
PubMed: 31227833
DOI: 10.1093/hmg/ddz138 -
Scientific Reports Jan 2019We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using...
We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using femtomolar amounts of amplified DNA. Here we tested the relevance of µLAS for sizing expanded trinucleotide repeats, which cause over 20 different neurological and neuromuscular disorders. Because the length of trinucleotide repeats correlates with the severity of the diseases, it is crucial to be able to size repeat tract length accurately and efficiently. Expanded trinucleotide repeats are however genetically unstable and difficult to amplify. Thus, the amount of amplified material to work with is often limited, making its analysis labor-intensive. We report the detection of heterogeneous allele lengths in 8 samples from myotonic dystrophy type 1 and Huntington disease patients with up to 750 CAG/CTG repeats in five minutes or less. The high sensitivity of the method allowed us to minimize the number of amplification cycles and thus reduce amplification artefacts without compromising the detection of the expanded allele. These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders.
Topics: Diagnostic Tests, Routine; Humans; Lab-On-A-Chip Devices; Nervous System Diseases; Neuromuscular Diseases; Sensitivity and Specificity; Trinucleotide Repeat Expansion
PubMed: 30631115
DOI: 10.1038/s41598-018-36632-5 -
Research in Microbiology 1999Microsatellites are direct tandem DNA repeats found in all genomes. A particular class of microsatellites, called trinucleotide repeats, is responsible for a number of... (Review)
Review
Microsatellites are direct tandem DNA repeats found in all genomes. A particular class of microsatellites, called trinucleotide repeats, is responsible for a number of neurological disorders in humans. We review here our current state of knowledge on trinucleotide repeat instability, and discuss the molecular mechanisms that may be involved in trinucleotide repeat expansions leading to fatal diseases in humans. We also present original data on microsatellite distribution in several microbial genomes, and on the use of microsatellites as physical markers to accurately and easily genotype yeast strains.
Topics: Bacteria; Chromosome Fragility; Chromosomes, Fungal; Eukaryotic Cells; Genetic Diseases, Inborn; Genome, Bacterial; Genome, Fungal; Genome, Human; Genotype; Humans; Microsatellite Repeats; Mycology; Prokaryotic Cells; Trinucleotide Repeats; Yeasts
PubMed: 10672999
DOI: 10.1016/s0923-2508(99)00131-x -
BioMed Research International 2013Myotonic dystrophy type 1 (DM1) is the most common adult onset muscular dystrophy, presenting as a multisystemic disorder with extremely variable clinical manifestation,... (Review)
Review
Myotonic dystrophy type 1 (DM1) is the most common adult onset muscular dystrophy, presenting as a multisystemic disorder with extremely variable clinical manifestation, from asymptomatic adults to severely affected neonates. A striking anticipation and parental-gender effect upon transmission are distinguishing genetic features in DM1 pedigrees. It is an autosomal dominant hereditary disease associated with an unstable expansion of CTG repeats in the 3'-UTR of the DMPK gene, with the number of repeats ranging from 50 to several thousand. The number of CTG repeats broadly correlates with both the age-at-onset and overall severity of the disease. Expanded DM1 alleles are characterized by a remarkable expansion-biased and gender-specific germline instability, and tissue-specific, expansion-biased, age-dependent, and individual-specific somatic instability. Mutational dynamics in male and female germline account for observed anticipation and parental-gender effect in DM1 pedigrees, while mutational dynamics in somatic tissues contribute toward the tissue-specificity and progressive nature of the disease. Genetic test is routinely used in diagnostic procedure for DM1 for symptomatic, asymptomatic, and prenatal testing, accompanied with appropriate genetic counseling and, as recommended, without predictive information about the disease course. We review molecular genetics of DM1 with focus on those issues important for genetic testing and counseling.
Topics: 3' Untranslated Regions; Age of Onset; Genetic Testing; Humans; Mutation; Myotonic Dystrophy; Pedigree; Phenotype; Trinucleotide Repeat Expansion; Trinucleotide Repeats
PubMed: 23586035
DOI: 10.1155/2013/391821 -
Genes Jun 2020Unstable repeat disorders comprise a variable group of incurable human neurological and neuromuscular diseases caused by an increase in the copy number of tandem repeats... (Review)
Review
Unstable repeat disorders comprise a variable group of incurable human neurological and neuromuscular diseases caused by an increase in the copy number of tandem repeats located in various regions of their resident genes. It has become clear that dense DNA methylation in hyperexpanded non-coding repeats induces transcriptional silencing and, subsequently, insufficient protein synthesis. However, the ramifications of this paradigm reveal a far more profound role in disease pathogenesis. This review will summarize the significant progress made in a subset of non-coding repeat diseases demonstrating the role of dense landscapes of 5-methylcytosine (5mC) as a common disease modifier. However, the emerging findings suggest context-dependent models of 5mC-mediated silencing with distinct effects of excessive DNA methylation. An in-depth understanding of the molecular mechanisms underlying this peculiar group of human diseases constitutes a prerequisite that could help to discover novel pathogenic repeat loci, as well as to determine potential therapeutic targets. In this regard, we report on a brief description of advanced strategies in DNA methylation profiling for the identification of unstable Guanine-Cytosine (GC)-rich regions and on promising examples of molecular targeted therapies for Fragile X disease (FXS) and Friedrich ataxia (FRDA) that could pave the way for the application of this technique in other hypermethylated expansion disorders.
Topics: 5-Methylcytosine; DNA Methylation; Fragile X Mental Retardation Protein; Fragile X Syndrome; Gene Silencing; Humans; Trinucleotide Repeat Expansion; Trinucleotide Repeats
PubMed: 32580525
DOI: 10.3390/genes11060684