The human genome contains over one million short tandem repeats. Expansion of a subset of these repeat tracts underlies over fifty human disorders, including common genetic causes of amyotrophic ...lateral sclerosis (ALS) and frontotemporal dementia (C9orf72), polyglutamine-associated ataxias and Huntington disease, myotonic dystrophy, and intellectual disability disorders such as Fragile X syndrome. In this Review, we discuss the four major mechanisms by which expansion of short tandem repeats causes disease: loss of function through transcription repression, RNA-mediated gain of function through gelation and sequestration of RNA-binding proteins, gain of function of canonically translated repeat-harbouring proteins, and repeat-associated non-AUG translation of toxic repeat peptides. Somatic repeat instability amplifies these mechanisms and influences both disease age of onset and tissue specificity of pathogenic features. We focus on the crosstalk between these disease mechanisms, and argue that they often synergize to drive pathogenesis. We also discuss the emerging native functions of repeat elements and how their dynamics might contribute to disease at a larger scale than currently appreciated. Lastly, we propose that lynchpins tying these disease mechanisms and native functions together offer promising therapeutic targets with potential shared applications across this class of human disorders.
To define potentially causal variants for autoimmune disease, we fine-mapped
76 rheumatoid arthritis (11,475 cases, 15,870 controls)
and type 1 diabetes loci (9,334 cases, 11,111 controls)
. After ...sequencing 799 1-kilobase regulatory (H3K4me3) regions within these loci in 568 individuals, we observed accurate imputation for 89% of common variants. We defined credible sets of ≤5 causal variants at 5 rheumatoid arthritis and 10 type 1 diabetes loci. We identified potentially causal missense variants at DNASE1L3, PTPN22, SH2B3, and TYK2, and noncoding variants at MEG3, CD28-CTLA4, and IL2RA. We also identified potential candidate causal variants at SIRPG and TNFAIP3. Using functional assays, we confirmed allele-specific protein binding and differential enhancer activity for three variants: the CD28-CTLA4 rs117701653 SNP, MEG3 rs34552516 indel, and TNFAIP3 rs35926684 indel.
Repeat-associated non-AUG (RAN) translation produces toxic polypeptides from nucleotide repeat expansions in the absence of an AUG start codon and contributes to neurodegenerative disorders such as ...ALS and fragile X-associated tremor/ataxia syndrome. How RAN translation occurs is unknown. Here we define the critical sequence and initiation factors that mediate CGG repeat RAN translation in the 5′ leader of fragile X mRNA, FMR1. Our results reveal that CGG RAN translation is 30%–40% as efficient as AUG-initiated translation, is m7G cap and eIF4E dependent, requires the eIF4A helicase, and is strongly influenced by repeat length. However, it displays a dichotomous requirement for initiation site selection between reading frames, with initiation in the +1 frame, but not the +2 frame, occurring at near-cognate start codons upstream of the repeat. These data support a model in which RAN translation at CGG repeats uses cap-dependent ribosomal scanning, yet bypasses normal requirements for start codon selection.
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•Expanded repeats stimulate CGG repeat-associated non-AUG (RAN) translation•CGG RAN translation shares initiation requirements with canonical initiation•RAN translation displays differential start codon selection across reading frames•Sequence context and repeat codon influence CGG RAN translation dynamics
AUG-independent translation of expanded nucleotide repeats produces toxic neuronal proteins. Kearse et al. show that RAN translation of CGG repeats initiates similarly to canonical translation, requiring an m7G cap and 40S ribosomal scanning. However, initiation codon selection differs across repeat reading frames and with repeat expansion.
•Fragile X Tremor Ataxia Syndrome (FXTAS) is caused by expanded CGG repeats.•Mechanisms of CGG repeat toxicity include RNA protein titration and RAN translation.•A key pathogenic event could be the ...near-cognate codon translation of CGG repeats.
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset inherited neurodegenerative disorder characterized by progressive intention tremor, gait ataxia and dementia associated with mild brain atrophy. The cause of FXTAS is a premutation expansion, of 55 to 200 CGG repeats localized within the 5′UTR of FMR1. These repeats are transcribed in the sense and antisense directions into mutants RNAs, which have increased expression in FXTAS. Furthermore, CGG sense and CCG antisense expanded repeats are translated into novel proteins despite their localization in putatively non-coding regions of the transcript. Here we focus on two proposed disease mechanisms for FXTAS: 1) RNA gain-of-function, whereby the mutant RNAs bind specific proteins and preclude their normal functions, and 2) repeat-associated non-AUG (RAN) translation, whereby translation through the CGG or CCG repeats leads to the production of toxic homopolypeptides, which in turn interfere with a variety of cellular functions. Here, we analyze the data generated to date on both of these potential molecular mechanisms and lay out a path forward for determining which factors drive FXTAS pathogenicity.
Most neurodegenerative disorders are thought to result primarily from the accumulation of misfolded proteins, which interfere with protein homeostasis in neurons. For a subset of diseases, however, ...noncoding regions of RNAs assume a primary toxic gain‐of‐function, leading to degeneration in many tissues, including the nervous system. Here we review a series of proposed mechanisms by which noncoding repeat expansions give rise to nervous system degeneration and dysfunction. These mechanisms include transcriptional alterations and the generation of antisense transcripts, sequestration of mRNA‐associated protein complexes that lead to aberrant mRNA splicing and processing, and alterations in cellular processes, including activation of abnormal signaling cascades and failure of protein quality control pathways. We place these potential mechanisms in the context of known RNA‐mediated disorders, including the myotonic dystrophies and fragile X tremor ataxia syndrome, and discuss recent results suggesting that mRNA toxicity may also play a role in some presumably protein‐mediated neurodegenerative disorders. Lastly, we comment on recent progress in therapeutic development for these RNA‐dominant diseases. ANN NEUROL 2010;67:291–300
Repeat-associated non-AUG (RAN) translation allows for unconventional initiation at disease-causing repeat expansions. As RAN translation contributes to pathogenesis in multiple neurodegenerative ...disorders, determining its mechanistic underpinnings may inform therapeutic development. Here we analyze RAN translation at G
C
repeat expansions that cause C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9RAN) and at CGG repeats that cause fragile X-associated tremor/ataxia syndrome. We find that C9RAN translation initiates through a cap- and eIF4A-dependent mechanism that utilizes a CUG start codon. C9RAN and CGG RAN are both selectively enhanced by integrated stress response (ISR) activation. ISR-enhanced RAN translation requires an eIF2α phosphorylation-dependent alteration in start codon fidelity. In parallel, both CGG and G
C
repeats trigger phosphorylated-eIF2α-dependent stress granule formation and global translational suppression. These findings support a model whereby repeat expansions elicit cellular stress conditions that favor RAN translation of toxic proteins, creating a potential feed-forward loop that contributes to neurodegeneration.
Fragile X-associated tremor ataxia syndrome (FXTAS) results from a CGG repeat expansion in the 5′ UTR of FMR1. This repeat is thought to elicit toxicity as RNA, yet disease brains contain ...ubiquitin-positive neuronal inclusions, a pathologic hallmark of protein-mediated neurodegeneration. We explain this paradox by demonstrating that CGG repeats trigger repeat-associated non-AUG-initiated (RAN) translation of a cryptic polyglycine-containing protein, FMRpolyG. FMRpolyG accumulates in ubiquitin-positive inclusions in Drosophila, cell culture, mouse disease models, and FXTAS patient brains. CGG RAN translation occurs in at least two of three possible reading frames at repeat sizes ranging from normal (25) to pathogenic (90), but inclusion formation only occurs with expanded repeats. In Drosophila, CGG repeat toxicity is suppressed by eliminating RAN translation and enhanced by increased polyglycine protein production. These studies expand the growing list of nucleotide repeat disorders in which RAN translation occurs and provide evidence that RAN translation contributes to neurodegeneration.
•CGG repeats in the 5′ UTR of FMR1 elicit AUG-independent (RAN) translation•This produces an aggregation-prone polyglycine protein found in patients•CGG RAN translation explains pathologic differences in FXTAS mice•CGG RAN translation is critical for CGG repeat toxicity in fly disease models
CGG repeat expansions underlie the neurodegenerative disorder fragile X-associated tremor ataxia syndrome. Todd et al. describe how CGG repeats trigger non-AUG-initiated translation, producing a polyglycine protein that accumulates in FXTAS brains and contributes to toxicity in model systems.
Objective
Repeat‐associated non‐AUG (RAN) translation drives production of toxic proteins from pathogenic repeat sequences in multiple untreatable neurodegenerative disorders. Fragile X‐associated ...tremor/ataxia syndrome (FXTAS) is one such condition, resulting from a CGG trinucleotide repeat expansion in the 5′ leader sequence of the FMR1 gene. RAN proteins from the CGG repeat accumulate in ubiquitinated inclusions in FXTAS patient brains and elicit toxicity. In addition to the CGG repeat, an antisense mRNA containing a CCG repeat is also transcribed from the FMR1 locus. We evaluated whether this antisense CCG repeat supports RAN translation and contributes to pathology in FXTAS patients.
Methods
We generated a series of CCG RAN translation‐specific reporters and utilized them to measure RAN translation from CCG repeats in multiple reading frames in transfected cells. We also developed antibodies against predicted CCG RAN proteins and used immunohistochemistry and immunofluorescence on FXTAS patient tissues to measure their accumulation and distribution.
Results
RAN translation from CCG repeats is supported in all 3 potential reading frames, generating polyproline, polyarginine, and polyalanine proteins, respectively. Their production occurs whether or not the natural AUG start upstream of the repeat in the proline reading frame is present. All 3 frames show greater translation at larger repeat sizes. Antibodies targeted to the antisense FMR polyproline and polyalanine proteins selectively stain nuclear and cytoplasmic aggregates in FXTAS patients and colocalize with ubiquitinated neuronal inclusions.
Interpretation
RAN translation from antisense CCG repeats generates novel proteins that accumulate in ubiquitinated inclusions in FXTAS patients. Ann Neurol 2016;80:871–881
Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the expansion of 55–200 CGG repeats in the 5′ UTR of FMR1. These expanded CGG repeats are ...transcribed and accumulate in nuclear RNA aggregates that sequester one or more RNA-binding proteins, thus impairing their functions. Here, we have identified that the double-stranded RNA-binding protein DGCR8 binds to expanded CGG repeats, resulting in the partial sequestration of DGCR8 and its partner, DROSHA, within CGG RNA aggregates. Consequently, the processing of microRNAs (miRNAs) is reduced, resulting in decreased levels of mature miRNAs in neuronal cells expressing expanded CGG repeats and in brain tissue from patients with FXTAS. Finally, overexpression of DGCR8 rescues the neuronal cell death induced by expression of expanded CGG repeats. These results support a model in which a human neurodegenerative disease originates from the alteration, in trans, of the miRNA-processing machinery.
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► DGCR8 binds to CGG RNA repeats, cause of the neurodegenerative FXTAS disease ► DGCR8 and its partner, DROSHA, are sequestered within CGG RNA aggregates ► DGCR8 rescues the neuronal cell death induced by expanded CGG RNA repeats ► MicroRNA processing is impaired in patients with FXTAS
Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by the accumulation of mutant RNAs containing expanded CGG repeats. Charlet-Berguerand and colleagues now find that DROSHA-DGCR8, the enzymatic complex that processes microRNAs, is sequestered within nuclear aggregates of CGG RNA repeats. In addition, they show that the processing of microRNA is reduced in patients with FXTAS. These data suggest a model in which a human neurodegenerative disease is linked to sequestration of the microRNA-processing machinery.
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