5 research outputs found
DDX3X and specific initiation factors modulate FMR1 repeatâassociated nonâAUGâinitiated translation
A CGG trinucleotide repeat expansion in the 5âČ UTR of FMR1 causes the neurodegenerative disorder Fragile Xâassociated tremor/ataxia syndrome (FXTAS). This repeat supports a nonâcanonical mode of protein synthesis known as repeatâassociated, nonâAUG (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 candidateâbased screen of eukaryotic initiation factors and RNA helicases in cellâbased 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 5âČUTR. These include the DEADâbox 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 repeatâinduced 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 repeatâassociated neurodegeneration.SynopsisFragile Xâassociated tremor/ataxia syndrome is caused by CGG repeatâassociated nonâAUG (RAN) translation that initiates within the 5âČUTR of FMR1. A candidateâbased screen identified several initiation factorsâDDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5âcritical for FMR1 RAN translation.Knockdown of the RNA helicase DDX3X selectively suppresses FMR1 RAN translation in Drosophila melanogaster, cultured HeLa cells, and in vitro translation assays.DDX3X knockdown reduces CGG repeatâassociated toxicity in Drosophila and mammalian neurons.Eukaryotic initiation factors that modulate RNAâRNA secondary structure (DDX3X, EIF4B, EIF4H) or start codon fidelity (EIF1, EIF5) impact FMR1 RAN translation.FXTAS is caused by CGG repeatâassociated nonâAUG (RAN) translation that initiates within the 5âČUTR of FMR1. A candidateâbased screen identified several initiation factorsâDDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5âcritical for FMR1 RAN translation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/1/embr201847498.reviewer_comments.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/2/embr201847498-sup-0001-Appendix.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/3/embr201847498_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/4/embr201847498.pd
The carboxyl termini of RAN translated GGGGCC nucleotide repeat expansions modulate toxicity in models of ALS/FTD
Abstract
An intronic hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat is thought to elicit toxicity through RNA mediated protein sequestration and repeat-associated non-AUG (RAN) translation of dipeptide repeat proteins (DPRs). We generated a series of transgenic Drosophila models expressing GGGGCC (G4C2) repeats either inside of an artificial intron within a GFP reporter or within the 5âČ untranslated region (UTR) of GFP placed in different downstream reading frames. Expression of 484 intronic repeats elicited minimal alterations in eye morphology, viability, longevity, or larval crawling but did trigger RNA foci formation, consistent with prior reports. In contrast, insertion of repeats into the 5âČ UTR elicited differential toxicity that was dependent on the reading frame of GFP relative to the repeat. Greater toxicity correlated with a short and unstructured carboxyl terminus (C-terminus) in the glycine-arginine (GR) RAN protein reading frame. This change in C-terminal sequence triggered nuclear accumulation of all three RAN DPRs. A similar differential toxicity and dependence on the GR C-terminus was observed when repeats were expressed in rodent neurons. The presence of the native C-termini across all three reading frames was partly protective. Taken together, these findings suggest that C-terminal sequences outside of the repeat region may alter the behavior and toxicity of dipeptide repeat proteins derived from GGGGCC repeats.http://deepblue.lib.umich.edu/bitstream/2027.42/173993/1/40478_2020_Article_1002.pd
DDX
A CGG trinucleotide repeat expansion in the 5âČ UTR of FMR1 causes the neurodegenerative disorder Fragile Xâassociated tremor/ataxia syndrome (FXTAS). This repeat supports a nonâcanonical mode of protein synthesis known as repeatâassociated, nonâAUG (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 candidateâbased screen of eukaryotic initiation factors and RNA helicases in cellâbased 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 5âČUTR. These include the DEADâbox 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 repeatâinduced 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 repeatâassociated neurodegeneration.SynopsisFragile Xâassociated tremor/ataxia syndrome is caused by CGG repeatâassociated nonâAUG (RAN) translation that initiates within the 5âČUTR of FMR1. A candidateâbased screen identified several initiation factorsâDDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5âcritical for FMR1 RAN translation.Knockdown of the RNA helicase DDX3X selectively suppresses FMR1 RAN translation in Drosophila melanogaster, cultured HeLa cells, and in vitro translation assays.DDX3X knockdown reduces CGG repeatâassociated toxicity in Drosophila and mammalian neurons.Eukaryotic initiation factors that modulate RNAâRNA secondary structure (DDX3X, EIF4B, EIF4H) or start codon fidelity (EIF1, EIF5) impact FMR1 RAN translation.FXTAS is caused by CGG repeatâassociated nonâAUG (RAN) translation that initiates within the 5âČUTR of FMR1. A candidateâbased screen identified several initiation factorsâDDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5âcritical for FMR1 RAN translation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/1/embr201847498.reviewer_comments.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/2/embr201847498-sup-0001-Appendix.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/3/embr201847498_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/4/embr201847498.pd
Translation of Expanded CGG Repeats into FMRpolyG Is Pathogenic and May Contribute to Fragile X Tremor Ataxia Syndrome
International audienceFragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a limited expansion of CGG repeats in the 5' UTR of FMR1. Two mechanisms are proposed to cause FXTAS: RNA gain-of-function, where CGG RNA sequesters specific proteins, and translation of CGG repeats into a polyglycine-containing protein, FMRpolyG. Here we developed transgenic mice expressing CGG repeat RNA with or without FMRpolyG. Expression of FMRpolyG is pathogenic, while the sole expression of CGG RNA is not. FMRpolyG interacts with the nuclear lamina protein LAP2ÎČ and disorganizes the nuclear lamina architecture in neurons differentiated from FXTAS iPS cells. Finally, expression of LAP2ÎČ rescues neuronal death induced by FMRpolyG. Overall, these results suggest that translation of expanded CGG repeats into FMRpolyG alters nuclear lamina architecture and drives pathogenesis in FXTAS