Human oncoprotein 5MP suppresses general and repeat-associated non-AUG translation via eIF3 by a common mechanism.
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ABSTRACT:
SUBMITTER: Singh CR
PROVIDER: S-EPMC8363759 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Furthermore, the initiation mechanism proceeded in a 5' cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism.
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Project description:Nucleotide repeat expansions underlie numerous human neurological disorders. Repeats can trigger toxicity through multiple pathogenic mechanisms, including RNA gain-of-function, protein gain-of-function, and protein loss-of-function pathways. Traditionally, inference of the underlying pathogenic mechanism derives from the repeat location, with dominantly inherited repeats within transcribed noncoding sequences eliciting toxicity predominantly as RNA via sequestration of specific RNA binding proteins. However, recent findings question this assumption and suggest that repeats outside of annotated open reading frames may also trigger toxicity through a novel form of protein translational initiation known as repeat-associated non-AUG (RAN) translation. To date, RAN translation has been implicated in 4 nucleotide repeat expansion disorders: spinocerebellar ataxia type 8; myotonic dystrophy type 1 with CTG•CAG repeats; C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia with GGGGCC•GGCCCC repeats; and fragile X-associated tremor/ataxia syndrome with CGG repeats. RAN translation contributes to hallmark pathological characteristics in these disorders by producing homopolymeric or dipeptide repeat proteins. Here, we review what is known about RAN translation, with an emphasis on how differences in both repeat sequence and context may confer different requirements for unconventional initiation. We then discuss how this new mechanism of translational initiation might function in normal physiology and lay out a roadmap for addressing the numerous questions that remain.
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Project description: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.
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Project description:The expansion of a hexanucleotide repeat GGGGCC (G4C2) in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 expansion leads to repeat-associated non-AUG (RAN) translation and the production of toxic dipeptide repeat (DPR) proteins, but the mechanisms of RAN translation remain enigmatic. Here, we report that the RNA helicase DHX36 is a robust positive regulator of C9orf72 RAN translation. DHX36 has a high affinity for the G4C2 repeat RNA, preferentially binds to the repeat RNA's G-quadruplex conformation, and efficiently unwinds the G4C2 G-quadruplex structures. Native DHX36 interacts with the G4C2 repeat RNA and is essential for effective RAN translation in the cell. In induced pluripotent stem cells and differentiated motor neurons derived from C9orf72-linked ALS patients, reducing DHX36 significantly decreased the levels of endogenous DPR proteins. DHX36 is also aberrantly upregulated in tissues of C9orf72-linked ALS patients. These results indicate that DHX36 facilitates C9orf72 RAN translation by resolving repeat RNA G-quadruplex structures and may be a potential target for therapeutic intervention.
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Project description:Over 50 genetic human disorders are attributed to the irregular expansion of microsatellites. These expanded microsatellite sequences can experience bidirectional transcription, leading to new reading frames. Beyond the standard AUG initiation or adjacent start codons, they are translated into proteins characterized by disease-causing amino acid repeats through repeat-associated non-AUG translation. Despite its significance, there's a discernible gap in comprehensive and objective articles on RAN translation. This study endeavors to evaluate and delineate the contemporary landscape and progress of RAN translation research via a bibliometric analysis. We sourced literature on RAN translation from the Web of Science Core Collection. Utilizing two bibliometric analysis tools, CiteSpace and VOSviewer, we gauged individual impacts and interactions by examining annual publications, journals, co-cited journals, countries/regions, institutions, authors, and co-cited authors. Following this, we assessed the co-occurrence and bursts of keywords and co-cited references to pinpoint research hotspots and trending in RAN translation. Between 2011 and 2022, 1317 authors across 359 institutions from 34 countries/regions contributed to 250 publications on RAN translation, spread across 118 academic journals. This article presents a systematic, objective, and comprehensive analysis of the current literature on RAN translation. Our findings emphasize that mechanisms related to C9orf72 ALS/FTD are pivotal topics in the realm of RAN translation, with cellular stress and the utilization of small molecule marking the trending research areas.
| S-EPMC11019168 | biostudies-literature
Project description: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.
| S-EPMC6726903 | biostudies-literature
Project description:Many human diseases are associated with the expansion of repeat sequences within the genes. It has become clear that expressed disease transcripts bearing such long repeats can undergo translation, even in the absence of a canonical AUG start codon. Termed "RAN translation" for repeat associated non-AUG translation, this process is becoming increasingly prominent as a contributor to these disorders. Here we discuss mechanisms and variables that impact translation of the repeat sequences associated with the C9orf72 gene. Expansions of a G4C2 repeat within intron 1 of this gene are associated with the motor neuron disease ALS and dementia FTD, which comprise a clinical and pathological spectrum. RAN translation of G4C2 repeat expansions has been studied in cells in culture (ex vivo) and in the fly in vivo. Cellular states that lead to RAN translation, like stress, may be critical contributors to disease progression. Greater elucidation of the mechanisms that impact this process and the factors contributing will lead to greater understanding of the repeat expansion diseases, to the potential development of novel approaches to therapeutics, and to a greater understanding of how these players impact biological processes in the absence of disease.
| S-EPMC6941355 | biostudies-literature
Project description: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.
| S-EPMC6901296 | biostudies-literature
Project description:Hexanucleotide repeat expansion in C9ORF72 is the most frequent cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we demonstrate that the repeat-associated non-AUG (RAN) translation of (GGGGCC) n -containing RNAs into poly-dipeptides can initiate in vivo without a 5'-cap. The primary RNA substrate for RAN translation of C9ORF72 sense repeats is shown to be the spliced first intron, following its excision from the initial pre-mRNA and transport to the cytoplasm. Cap-independent RAN translation is shown to be upregulated by various stress stimuli through phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF2α), the core event of an integrated stress response (ISR). Compounds inhibiting phospho-eIF2α-signaling pathways are shown to suppress RAN translation. Since the poly-dipeptides can themselves induce stress, these findings support a feedforward loop with initial repeat-mediated toxicity enhancing RAN translation and subsequent production of additional poly-dipeptides through ISR, thereby promoting progressive disease.
| S-EPMC5754368 | biostudies-literature
Project description: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 cell lines 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 elongation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.
| S-EPMC10274811 | biostudies-literature