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Phe-tRNA aminoacylation is disrupted by C4G2 repeat RNA C9orf72mutation associated with ALS and FTD

ABSTRACT: Expanded hexanucleotide GGGGCC repeat mutation in the C9orf72 gene is the main genetic cause of neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense transcripts of the repeats form nuclear RNA foci and bind various RNA binding proteins. Sequestration of these proteins is predicted to be cytotoxic. In this study we identified proteins that bind to antisense repeat RNA with RNA pull-down assay in combination with mass spectrometry. The interactions were confirmed in human brain lysates. Among interacting proteins is phenylalanine-tRNA synthetase (FARS). We confirmed interaction with FARS in C9orf72 mutation-positive patient-derived cells with RNA fluorescent in situ hybridization in combination with immunocytochemistry and with RNA-protein proximity ligation assay. We predict this interaction impacts the function of FARS as we observed decreased levels of charged Phe-tRNA in C9orf72 mutation-positive patient-derived cells compared to control. This study shows that antisense RNA interaction with FARS could influence protein synthesis and reveals so far unknown connection between RNA repeats and aminoacil tRNA synthetases in C9orf72 mutation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Xiaoke Yin

LAB HEAD: Manuel Mayr

PROVIDER: PXD024527 | Pride | 2023-09-18

REPOSITORIES: Pride

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Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA

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2023-09-18 | PXD042474 | Pride

C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD

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C9orf72 BAC Transgenic Mice Display Typical Pathologic Features of ALS/FTD

Project description:Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (~100–1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients.

2016-02-10 | GSE77720 | GEO

C9orf72 Intermediate Repeats are Associated with Corticobasal Degeneration, Increased C9orf72 Expression and Disruption of Autophagy

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RNA sequencing of human fibroblasts after SUPT4H1 siRNA treatment

Project description:Asses the transcriptome-wide changes associated with depletion of transcription elongation factor, SUPT4H1, in human fibroblasts. An expanded hexanucleotide repeat in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics (e.g., antisense oligonucleotides) are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor, Spt4, selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Inhibition of SUPT4H1, the human ortholog of Spt4, similarly decreased production of sense and antisense RNA foci as well as DPR proteins in c9ALS patient fibroblasts and neurons from patient-derived induced pluripotent stem cells, consistent with our finding that cerebellar DPR protein levels correlate with SUPT4H1 expression in c9FTD/ALS patients. Therapeutic targeting of a single factor to eliminate pathological features of c9FTD/ALS offers advantages over approaches that require targeting sense and antisense repeats separately.

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Profiling nucleo/cytoplasmic distribution of mRNAs identifies a role for the Golgi-to-ER trafficking in C9orf72 toxicity

Project description:Compelling evidence indicates that defects in nucleocytoplasmic transport contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). In particular, hexanucleotide (G4C2) repeat expansions in C9orf72, the most common cause of genetic ALS, have a widespread impact on the transport machinery that regulates the nucleocytoplasmic distribution of proteins and RNAs, thereby affecting their functions. We have previously reported that the expression of G4C2 hexanucleotide repeats in cultured human and mouse cells caused a marked accumulation of poly(A) mRNAs in cell nuclei, suggesting that mRNA trafficking is impaired by expanded C9orf72-ALS repeats. To further characterize the process, in this work we set out to systematically identify the specific mRNAs that are altered in their nucleocytoplasmic distribution in the presence of C9orf72-ALS repeats. Results from RNAseq profiling of nuclear and cytoplasmic RNA fractions obtained from cells expressing G4C231 repeats show a significant accumulation of mRNAs in the nuclei of G4C231 cells, further suggesting that nuclear retention of mRNAs is a major effect of C9orf72 expanded repeats expression. Interestingly, pathway analysis shows that mRNAs involved in ER-to-Golgi trafficking are particularly enriched among the identified mRNAs. Most importantly, functional studies in cultured cells and Drosophila indicate that the membrane trafficking route regulated by ARFGAP1, a GTPase-activating protein that associates with Golgi, is specifically affected by expanded C9orf72 repeats, as well as by C9orf72-related dipeptide repeat proteins (C9-DPRs), pointing to ER-to-Golgi vesicle-mediated transport as a target of C9orf72 toxicity.

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