RAN Translation at \u3cem\u3eC9orf72\u3c/em\u3e-Associated Repeat Expansions is Selectively Enhanced by the Integrated Stress Response

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 G4C2 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 G4C2 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

DDX3X and specific initiation factors modulate FMR1 repeat‐associated non‐AUG‐initiated translation

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.SynopsisFragile X‐associated tremor/ataxia syndrome is caused by CGG repeat‐associated non‐AUG (RAN) translation that initiates within the 5′UTR of FMR1. A candidate‐based screen identified several initiation factors—DDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5—critical 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 repeat‐associated toxicity in Drosophila and mammalian neurons.Eukaryotic initiation factors that modulate RNA‐RNA secondary structure (DDX3X, EIF4B, EIF4H) or start codon fidelity (EIF1, EIF5) impact FMR1 RAN translation.FXTAS is caused by CGG repeat‐associated non‐AUG (RAN) translation that initiates within the 5′UTR of FMR1. A candidate‐based screen identified several initiation factors—DDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5—critical 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

A Native Function for RAN Translation and CGG Repeats in Regulating Fragile X Protein Synthesis

Repeat-associated non-AUG-initiated translation of expanded CGG repeats (CGG RAN) from the FMR1 5′-leader produces toxic proteins that contribute to neurodegeneration in fragile X-associated tremor/ataxia syndrome. Here we describe how unexpanded CGG repeats and their translation play conserved roles in regulating fragile X protein (FMRP) synthesis. In neurons, CGG RAN acts as an inhibitory upstream open reading frame to suppress basal FMRP production. Activation of mGluR5 receptors enhances FMRP synthesis. This enhancement requires both the CGG repeat and CGG RAN initiation sites. Using non-cleaving antisense oligonucleotides (ASOs), we selectively blocked CGG RAN. This ASO blockade enhanced endogenous FMRP expression in human neurons. In human and rodent neurons, CGG RAN-blocking ASOs suppressed repeat toxicity and prolonged survival. These findings delineate a native function for CGG repeats and RAN translation in regulating basal and activity-dependent FMRP synthesis, and they demonstrate the therapeutic potential of modulating CGG RAN translation in fragile X-associated disorders

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

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 5′ 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 5′ 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

A reexamination of sex differences in sexuality : new studies reveal old truths

Recent evidence seems to call into question long-established findings of sex differences in sexuality, such as differences in mate preferences and desires for casual sex. In this article, we place new findings in a broader evidence-based context and show that they confirm previous perspectives on human mating. A wealth of evidence from real-world studies of actual mate choice and marital dynamics supports evolutionary mate-preference predictions. Converging evidence from patterns of extradyadic sex, mate poaching, sexual fantasies, pornography consumption, postcoital regret, sociosexual attitudes, and willingness to engage in casual sex supports the view that men and women have distinct short-term mating psychologies. This article highlights the fact that good science requires a constant re-evaluation of old truths and the proper placement of new studies in broad evidentiary contexts

RAN translation at C9orf72-associated repeat expansions is selectively enhanced by the integrated stress response

A nucleotide repeat expansion in C9orf72 is a common genetic cause of neurodegenerative disorders. Here, the authors provide insight into the molecular mechanism by which this repeat undergoes Repeat-Associated Non-AUG (RAN) translation, implicating the integrated stress response and eIF2α phosphorylation

Distinct <i>C9orf72</i>-Associated Dipeptide Repeat Structures Correlate with Neuronal Toxicity

<div><p>Hexanucleotide repeat expansions in <i>C9orf72</i> are the most common inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansions elicit toxicity in part through repeat-associated non-AUG (RAN) translation of the intronic (GGGGCC)_n sequence into dipeptide repeat-containing proteins (DPRs). Little is known, however, about the structural characteristics and aggregation propensities of the dipeptide units comprising DPRs. To address this question, we synthesized dipeptide units corresponding to the three sense-strand RAN translation products, analyzed their structures by circular dichroism, electron microscopy and dye binding assays, and assessed their relative toxicity when applied to primary cortical neurons. Short, glycine-arginine (GR)3 dipeptides formed spherical aggregates and selectively reduced neuronal survival compared to glycine-alanine (GA)3 and glycine-proline (GP)3 dipeptides. Doubling peptide length had little effect on the structure of GR or GP peptides, but (GA)6 peptides formed β-sheet rich aggregates that bound thioflavin T and Congo red yet lacked the typical fibrillar morphology of amyloids. Aging of (GA)6 dipeptides increased their β-sheet content and enhanced their toxicity when applied to neurons. We also observed that the relative toxicity of each tested dipeptide was proportional to peptide internalization. Our results demonstrate that different <i>C9orf72</i>-related dipeptides exhibit distinct structural properties that correlate with their relative toxicity.</p></div

DDX

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.SynopsisFragile X‐associated tremor/ataxia syndrome is caused by CGG repeat‐associated non‐AUG (RAN) translation that initiates within the 5′UTR of FMR1. A candidate‐based screen identified several initiation factors—DDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5—critical 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 repeat‐associated toxicity in Drosophila and mammalian neurons.Eukaryotic initiation factors that modulate RNA‐RNA secondary structure (DDX3X, EIF4B, EIF4H) or start codon fidelity (EIF1, EIF5) impact FMR1 RAN translation.FXTAS is caused by CGG repeat‐associated non‐AUG (RAN) translation that initiates within the 5′UTR of FMR1. A candidate‐based screen identified several initiation factors—DDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5—critical 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