RESUMO
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.
Assuntos
Expansão das Repetições de DNA/genética , Doenças Neurodegenerativas/genética , Animais , Inativação Gênica , Instabilidade Genômica , Humanos , Mutação , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Especificidade de Órgãos , Biossíntese de Proteínas , Estruturas R-Loop , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Pathogenic repeat sequences underlie several human disorders, including amyotrophic lateral sclerosis, Huntington's disease, and myotonic dystrophy. Here, we speak to several researchers about how repeat sequences have been implicated in affecting all aspects of the Central Dogma of molecular biology through their effects on DNA, RNA, and protein.
Assuntos
Esclerose Lateral Amiotrófica , Doença de Huntington , Distrofia Miotônica , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteínas/genética , Doença de Huntington/genética , RNA/genética , Distrofia Miotônica/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context-namely, those with non-AUG start codons-to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.
Assuntos
Códon de Iniciação/genética , Resistência a Múltiplos Medicamentos/genética , Ribossomos/genética , Elongação da Transcrição Genética/efeitos dos fármacos , Cicloeximida/farmacologia , Fator de Iniciação Eucariótico 4G/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Células HEK293 , Células HeLa , Humanos , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.
RESUMO
A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Demência Frontotemporal , Biossíntese de Proteínas , Proteínas Ribossômicas , Expansão das Repetições de Trinucleotídeos , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Ataxia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Expansão das Repetições de DNA/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Sequência Rica em GC , Células HEK293 , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Ribossomos/metabolismo , Ribossomos/genética , Tremor , Expansão das Repetições de Trinucleotídeos/genética , Proteínas Ribossômicas/metabolismoRESUMO
Fragile Xassociated tremor/ataxia syndrome (FXTAS) is a debilitating late-onset neurodegenerative disease in premutation carriers of the expanded CGG repeat in FMR1 that presents with a spectrum of neurological manifestations, such as gait ataxia, intention tremor, and parkinsonism [P. J. Hagerman, R. J. Hagerman, Ann. N. Y. Acad. Sci. 1338, 5870 (2015); S. Jacquemont et al., JAMA 291, 460469 (2004)]. Here, we performed whole-genome sequencing (WGS) on male premutation carriers (CGG55200) and prioritized candidate variants to screen for candidate genetic modifiers using a Drosophila model of FXTAS. We found 18 genes that genetically modulate CGG-associated neurotoxicity in Drosophila, such as Prosbeta5 (PSMB5), pAbp (PABPC1L), e(y)1 (TAF9), and CG14231 (OSGEPL1). Among them, knockdown of Prosbeta5 (PSMB5) suppressed CGG-associated neurodegeneration in the fly as well as in N2A cells. Interestingly, an expression quantitative trait locus variant in PSMB5, PSMB5rs11543947-A, was found to be associated with decreased expression of PSMB5 and delayed onset of FXTAS in human FMR1 premutation carriers. Finally, we demonstrate evidence that PSMB5 knockdown results in suppression of CGG neurotoxicity via both the RAN translation and RNA-mediated toxicity mechanisms, thereby presenting a therapeutic strategy for FXTAS.
Assuntos
Ataxia , Síndrome do Cromossomo X Frágil , Complexo de Endopeptidases do Proteassoma , Tremor , Animais , Ataxia/genética , Modelos Animais de Doenças , Drosophila melanogaster , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Humanos , Masculino , Complexo de Endopeptidases do Proteassoma/genética , Tremor/genéticaRESUMO
Repeat-associated non-AUG (RAN) translation of expanded repeat-mutation mRNA produces toxic peptides in neurons of patients suffering from neurodegenerative diseases. Recent findings indicate that RAN translation in diverse model systems is not inhibited by cellular stressors that impair global translation through phosphorylation of the alpha subunit of eIF2, the essential eukaryotic translation initiation factor that brings the initiator tRNA to the 40S ribosome. Using in vitro, cell-based and Drosophila models, we examined the role of alternative ternary complex factors that may function in place of eIF2, including eIF2A, eIF2D, DENR and MCTS1. Among these factors, DENR knockdown had the greatest inhibitory effect on RAN translation of expanded GGGGCC and CGG repeat reporters and its reduction improved the survival of Drosophila expressing expanded GGGGCC repeats. Taken together, these data support a role for alternative initiation factors in RAN translation and suggest these may serve as novel therapeutic targets in neurodegenerative disease.
Assuntos
Proteínas de Drosophila , Doenças Neurodegenerativas , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Repeat associated non-AUG (RAN) translation of CGG repeats in the 5'UTR of FMR1 produces toxic proteins that contribute to fragile X-associated tremor/ataxia syndrome (FXTAS) pathogenesis. The most abundant RAN product, FMRpolyG, initiates predominantly at an ACG upstream of the repeat. Accurate FMRpolyG measurements in FXTAS patients are lacking. We used data-dependent acquisition and parallel reaction monitoring (PRM) mass spectrometry coupled with stable isotope labeled standard peptides to identify signature FMRpolyG fragments in patient samples. Following immunoprecipitation, PRM detected FMRpolyG signature peptides in transfected cells, and FXTAS tissues and cells, but not in controls. We identified two amino-terminal peptides: an ACG-initiated Ac-MEAPLPGGVR and a GUG-initiated Ac-TEAPLPGGVR, as well as evidence for RAN translation initiation within the CGG repeat itself in two reading frames. Initiation at all sites increased following cellular stress, decreased following eIF1 overexpression and was eIF4A and M7G cap-dependent. These data demonstrate that FMRpolyG is quantifiable in human samples and FMR1 RAN translation initiates via similar mechanisms for near-cognate codons and within the repeat through processes dependent on available initiation factors and cellular environment.
Assuntos
Ataxia , Síndrome do Cromossomo X Frágil , Tremor , Proteína ran de Ligação ao GTP , Ataxia/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Humanos , Peptídeos/metabolismo , Tremor/genética , Expansão das Repetições de Trinucleotídeos , Proteína ran de Ligação ao GTP/genéticaRESUMO
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 m(7)G 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.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/biossíntese , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Degeneração Neural , Biossíntese de Proteínas , RNA Mensageiro/genética , Repetições de Trinucleotídeos , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/patologia , Mudança da Fase de Leitura do Gene Ribossômico , Genes Reporter , Predisposição Genética para Doença , Células HeLa , Humanos , Neurônios/metabolismo , Neurônios/patologia , Fases de Leitura Aberta , Fenótipo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Sítio de Iniciação de Transcrição , Transfecção , Expansão das Repetições de TrinucleotídeosRESUMO
CGG repeat expansions in the FMR1 5'UTR cause the neurodegenerative disease Fragile X-associated tremor/ataxia syndrome (FXTAS). These repeats form stable RNA secondary structures that support aberrant translation in the absence of an AUG start codon (RAN translation), producing aggregate-prone peptides that accumulate within intranuclear neuronal inclusions and contribute to neurotoxicity. Here, we show that the most abundant RAN translation product, FMRpolyG, is markedly less toxic when generated from a construct with a non-repetitive alternating codon sequence in place of the CGG repeat. While exploring the mechanism of this differential toxicity, we observed a +1 translational frameshift within the CGG repeat from the arginine to glycine reading frame. Frameshifts occurred within the first few translated repeats and were triggered predominantly by RNA sequence and structural features. Short chimeric R/G peptides form aggregates distinct from those formed by either pure arginine or glycine, and these chimeras induce toxicity in cultured rodent neurons. Together, this work suggests that CGG repeats support translational frameshifting and that chimeric RAN translated peptides may contribute to CGG repeat-associated toxicity in FXTAS and related disorders.
Assuntos
Proteína do X Frágil da Deficiência Intelectual , Doenças Neurodegenerativas , Agregação Patológica de Proteínas , Repetições de Trinucleotídeos , Arginina/genética , Ataxia , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil , Glicina/genética , Humanos , Doenças Neurodegenerativas/genética , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Neurodegeneration in Fragile X-associated tremor/ataxia syndrome (FXTAS) is caused by a CGG trinucleotide repeat expansion in the 5' UTR of FMR1. Expanded CGG repeat RNAs form stable secondary structures, which in turn support repeat-associated non-AUG (RAN) translation to produce toxic peptides. The parameters that impact RAN translation initiation efficiency are not well understood. Here we used a Drosophila melanogaster model of FXTAS to evaluate the role of the eIF4G family of eukaryotic translation initiation factors (EIF4G1, EIF4GII and EIF4G2/DAP5) in modulating RAN translation and CGG repeat-associated toxicity. DAP5 knockdown robustly suppressed CGG repeat-associated toxicity and inhibited RAN translation. Furthermore, knockdown of initiation factors that preferentially associate with DAP5 (such as EIF2ß, EIF3F and EIF3G) also selectively suppressed CGG repeat-induced eye degeneration. In mammalian cellular reporter assays, DAP5 knockdown exhibited modest and cell-type specific effects on RAN translation. Taken together, these data support a role for DAP5 in CGG repeat associated toxicity possibly through modulation of RAN translation.
Assuntos
Proteínas de Drosophila , Síndrome do Cromossomo X Frágil , Animais , Drosophila/metabolismo , Tremor/genética , Drosophila melanogaster/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Expansão das Repetições de Trinucleotídeos , Ataxia/genética , Mamíferos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMO
FMR1 premutation cytosine-guanine-guanine repeat expansion alleles are relatively common mutations in the general population that are associated with a neurodegenerative disease (fragile X-associated tremor/ataxia syndrome), reproductive health problems and potentially a wide range of additional mental and general health conditions that are not yet well-characterised. The International Fragile X Premutation Registry (IFXPR) was developed to facilitate and encourage research to better understand the FMR1 premutation and its impact on human health, to facilitate clinical trial readiness by identifying and characterising diverse cohorts of individuals interested in study participation, and to build community and collaboration among carriers, family members, researchers and clinicians around the world. Here, we describe the development and content of the IFXPR, characterise its first 747 registrants from 32 countries and invite investigators to apply for recruitment support for their project(s). With larger numbers, increased diversity and potentially the future clinical characterisation of registrants, the IFXPR will contribute to a more comprehensive and accurate understanding of the fragile X premutation in human health and support treatment studies.
Assuntos
Proteína do X Frágil da Deficiência Intelectual , Doenças Neurodegenerativas , Humanos , Proteína do X Frágil da Deficiência Intelectual/genética , Expansão das Repetições de Trinucleotídeos/genética , Doenças Neurodegenerativas/genética , Sistema de Registros , GuaninaRESUMO
GGGGCC (G4C2) hexanucleotide repeat expansions in the endosomal trafficking gene C9orf72 are the most common genetic cause of ALS and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation of this expansion through near-cognate initiation codon usage and internal ribosomal entry generates toxic proteins that accumulate in patients' brains and contribute to disease pathogenesis. The helicase protein DEAH-box helicase 36 (DHX36-G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats are known to form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. Using a series of luciferase reporter assays both in cells and in vitro, we found that DHX36 depletion suppresses RAN translation in a repeat length-dependent manner, whereas overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Moreover, upregulation of RAN translation that is typically triggered by integrated stress response activation is prevented by loss of DHX36. These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/genética , RNA Helicases DEAD-box/metabolismo , Expansão das Repetições de DNA , Quadruplex G , RNA Helicases/metabolismo , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/metabolismo , Linhagem Celular Tumoral , Demência Frontotemporal/enzimologia , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Biossíntese de ProteínasRESUMO
BACKGROUND: X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder caused by the intronic insertion of a SINE-VNTR-Alu (SVA) retrotransposon carrying an (AGAGGG)n repeat expansion in the TAF1 gene. The molecular mechanisms by which this mutation causes neurodegeneration remain elusive. OBJECTIVES: We investigated whether (AGAGGG)n repeats undergo repeat-associated non-AUG (RAN) translation, a pathogenic mechanism common among repeat expansion diseases. METHODS: XDP-specific RAN translation reporter plasmids were generated, transfected in HEK293 cells, and putative dipeptide repeat proteins (DPRs) were detected by Western blotting. Immunocytochemistry was performed in COS-7 cells to determine the subcellular localization of one DPR. RESULTS: We detected putative DPRs from two reading frames, supporting the translation of poly-(Glu-Gly) and poly-(Arg-Glu) species. XDP RAN translation initiates within the (AGAGGG)n sequence and poly-(Glu-Gly) DPRs formed nuclear inclusions in transfected cells. CONCLUSIONS: In summary, our work provides the first in-vitro proof of principle that the XDP-linked (AGAGGG)n repeat expansions can undergo RAN translation. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Distúrbios Distônicos , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Células HEK293 , Distúrbios Distônicos/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Íntrons , Proteína C9orf72/genéticaRESUMO
BACKGROUND: Premutation-sized (55-200) CGG repeat expansions in the FMR1 gene cause fragile X-associated tremor/ataxia syndrome (FXTAS). Most studies of premutation carriers utilized reverse ascertainment to identify patients, leading to a selection bias for larger repeats. As shorter CGG premutation repeats are common in the population, understanding their impact on health outcomes has a potentially large public health footprint. OBJECTIVE: The study's objective was to compare an unselected group of premutation carriers (n = 35, 55-101 CGG repeats) with matched controls (n = 61, 29-39 CGG repeats) with respect to FXTAS-type signs using structured neurological assessments. METHODS: Three neurologists independently rated signs, using an adapted version of the FXTAS Rating Scale (Leehey MA, Berry-Kravis E, Goetz CG, et al. FMR1 CGG repeat length predicts motor dysfunction in premutation carriers. Neurology. 2008). This was a double-blind study, as genetic status (premutation vs. control) was known neither by the participants nor by any of the neurologists. Analyses controlled potentially confounding comorbid conditions in the electronic health record (eg, osteoarthritis and stroke) and probed the association of age with signs. RESULTS: Although there was no overall difference between carriers and controls, among individuals without any potentially confounding comorbid diagnoses, there was a statistically significant age-associated elevation in FXTAS-type signs in premutation carriers compared to controls. CONCLUSIONS: Among those who do not have other comorbid diagnoses, women who have CGG repeats at the lower end of the premutation range may be at greater risk for ataxia and parkinsonism than their age peers, although their overall risk of developing such clinical features is low. This study should provide reassurance to those who share characteristics with the present cohort. © 2021 International Parkinson and Movement Disorder Society.
Assuntos
Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Heterozigoto , Ataxia/genética , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Humanos , Tremor/genética , Expansão das Repetições de TrinucleotídeosRESUMO
A CGG trinucleotide repeat expansion in the 5' UTR of FMR1 causes the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). This repeat supports a non-canonical mode of protein synthesis known as repeat-associated, non-AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate-based screen of eukaryotic initiation factors and RNA helicases in cell-based assays and a Drosophila melanogaster model of FXTAS. We identified multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5'UTR. These include the DEAD-box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat-induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat-associated neurodegeneration.
Assuntos
Ataxia/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Tremor/metabolismo , Animais , Ataxia/genética , Células Cultivadas , RNA Helicases DEAD-box/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Fatores de Iniciação em Eucariotos/genética , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Masculino , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tremor/genéticaRESUMO
Repeat-associated non-AUG (RAN) translation is a noncanonical translation initiation event that occurs at nucleotide-repeat expansion mutations that are associated with several neurodegenerative diseases, including fragile X-associated tremor ataxia syndrome (FXTAS), ALS, and frontotemporal dementia (FTD). Translation of expanded repeats produces toxic proteins that accumulate in human brains and contribute to disease pathogenesis. Consequently, RAN translation constitutes a potentially important therapeutic target for managing multiple neurodegenerative disorders. Here, we adapted a previously developed RAN translation assay to a high-throughput format to screen 3,253 bioactive compounds for inhibition of RAN translation of expanded CGG repeats associated with FXTAS. We identified five diverse small molecules that dose-dependently inhibited CGG RAN translation, while relatively sparing canonical translation. All five compounds also inhibited RAN translation of expanded GGGGCC repeats associated with ALS and FTD. Using CD and native gel analyses, we found evidence that three of these compounds, BIX01294, CP-31398, and propidium iodide, bind directly to the repeat RNAs. These findings provide proof-of-principle supporting the development of selective small-molecule RAN translation inhibitors that act across multiple disease-causing repeats.
Assuntos
Esclerose Lateral Amiotrófica/genética , Ataxia/genética , Síndrome do Cromossomo X Frágil/genética , Tremor/genética , Expansão das Repetições de Trinucleotídeos/genética , Esclerose Lateral Amiotrófica/tratamento farmacológico , Animais , Ataxia/tratamento farmacológico , Azepinas/farmacologia , Azepinas/uso terapêutico , Células Cultivadas , Dicroísmo Circular , Expansão das Repetições de DNA/efeitos dos fármacos , Expansão das Repetições de DNA/genética , Avaliação Pré-Clínica de Medicamentos , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Células HEK293 , Humanos , Doenças Neurodegenerativas/genética , Propídio/farmacologia , Propídio/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Ratos , Tremor/tratamento farmacológico , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacosRESUMO
BACKGROUND: Upstream open reading frames (uORFs) initiate translation within mRNA 5' leaders, and have the potential to alter main coding sequence (CDS) translation on transcripts in which they reside. Ribosome profiling (RP) studies suggest that translating ribosomes are pervasive within 5' leaders across model systems. However, the significance of this observation remains unclear. To explore a role for uORF usage in a model of neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. RESULTS: Using a spectral coherence algorithm (SPECtre), we identify 4954 consistently translated uORFs across 31% of all neuroblastoma transcripts. These uORFs predominantly utilize non-AUG initiation codons and exhibit translational efficiencies (TE) comparable to annotated coding regions. On a population basis, the global impact of both AUG and non-AUG initiated uORFs on basal CDS translation were small, even when analysis is limited to conserved and consistently translated uORFs. However, uORFs did alter the translation of a subset of genes, including the Diamond-Blackfan Anemia associated ribosomal gene RPS24. With retinoic acid induced differentiation, we observed an overall positive correlation in translational shifts between uORF/CDS pairs. However, CDSs downstream of uORFs show smaller shifts in TE with differentiation relative to CDSs without a predicted uORF, suggesting that uORF translation buffers cell state dependent fluctuations in CDS translation. CONCLUSION: This work provides insights into the dynamic relationships and potential regulatory functions of uORF/CDS pairs in a model of neuronal differentiation.
Assuntos
Diferenciação Celular/genética , Neurônios/metabolismo , Fases de Leitura Aberta , Biossíntese de Proteínas , Algoritmos , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Humanos , Modelos Biológicos , Neurônios/citologia , Ribossomos/metabolismoRESUMO
Fragile X-associated tremor ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a CGG trinucleotide repeat expansion in the 5' UTR of the Fragile X gene, FMR1. FXTAS is thought to arise primarily from an RNA gain-of-function toxicity mechanism. However, recent studies demonstrate that the repeat also elicits production of a toxic polyglycine protein, FMRpolyG, via repeat-associated non-AUG (RAN)-initiated translation. Pathologically, FXTAS is characterized by ubiquitin-positive intranuclear neuronal inclusions, raising the possibility that failure of protein quality control pathways could contribute to disease pathogenesis. To test this hypothesis, we used Drosophila- and cell-based models of CGG-repeat-associated toxicity. In Drosophila, ubiquitin proteasome system (UPS) impairment led to enhancement of CGG-repeat-induced degeneration, whereas overexpression of the chaperone protein HSP70 suppressed this toxicity. In transfected mammalian cells, CGG repeat expression triggered accumulation of a UPS reporter in a length-dependent fashion. To delineate the contributions from CGG repeats as RNA from RAN translation-associated toxicity, we enhanced or impaired the production of FMRpolyG in these models. Driving expression of FMRpolyG enhanced induction of UPS impairment in cell models, while prevention of RAN translation attenuated UPS impairment in cells and suppressed the genetic interaction with UPS manipulation in Drosophila. Taken together, these findings suggest that CGG repeats induce UPS impairment at least in part through activation of RAN translation.