Identification of proteins interacting with GFP-GA, GR, PA, PG and PR fusion proteins
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Elite
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture, Embryonic Stem Cell
DISEASE(S): Amyotrophic Lateral Sclerosis
SUBMITTER: 
Timothy Shaw
LAB HEAD: Junmin Peng
PROVIDER: PXD004938 | Pride | 2016-10-03
REPOSITORIES: Pride
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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 (~100â??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. To compare the RNA Seq profiles from the cortex and spinal cord of transgenic mice expressing unexpanded human C9orf72 (F08, n=4), expanded human C9orf72 (F112, n=3/4), and nontransgenic controls (n=4).
2016-02-10 | E-GEOD-77720 | biostudies-arrayexpress
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
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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Project description:Hexanucleotide repeat expansion in the C9ORF72 gene is the most frequent inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Poly-GR is one of the most toxic dipeptide repeat (DPR) proteins translated from the RNA repeats. It has been shown to affect protein synthesis, but how this contributes to neurodegeneration is not clear. Here, we found that poly-GR inhibits global translation by perturbing translation elongation. We identified that the transcripts with relatively slow elongation rate tend to be further stalled by poly-GR in iPSC-differentiated neurons. This increases ribosome collision and ZAKα-mediated ribotoxic stress response (RSR), which elevates the phosphorylation of p38 and promotes cell death. Knockdown of ZAKα or pharmacological inhibition of p38 can ameliorate the GR toxicity, and improve the survival of C9ORF72-ALS/FTD patient-derived iPSC-neurons. Our study reveals molecular mechanism of poly-GR mediated toxicity on global translatome, and identifies RSR as a potential therapeutic target for C9ORF72-ALS/FTD.
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Project description:Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). The nucleotide repeat expansions are translated into dipeptide repeat (DPR) proteins, which are aggregation-prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene knockout screens for suppressors and enhancers of C9orf72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA processing pathways, and in chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9orf72 DPRs in neurons, and improved survival of human induced motor neurons from C9orf72 ALS patients. Together, this work demonstrates the promise of CRISPR-Cas9 screens to define mechanisms of neurodegenerative diseases. This dataset contains the RNA-sequencing data used to support the conclusions from this study.
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Project description:Hexanucleotide expansion mutations in C9ORF72 are a cause of familial amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPR), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here we explored the molecular features of the stalling and examined whether known regulatory mechanisms of ribosome quality control (RQC) are involved in efforts to sense and resolve the stalls. The most well-known RQC mechanisms come largely from studies of mRNA lacking stop codons, whereby ribosomes become stalled at the poly-adenylate sequence in the 3`UTR. Here we find that arginine-containing DPRs lead to stalling in a length dependent manner, with lengths longer than 40 repeats to invoke strong stalling. Mutational screening of 40×Gly-Xxx DPRs finds that stalling is most pronounced where Xxx are positively charged amino acids. Through a genome-wide CRISPR screen we find that genes that regulate poly-adenylate readthrough respond differently to the readthrough of arginine-rich DPRs. Indeed, we find evidence that DPR-mediated stalling has no natural regulatory responses even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate Arg-rich DPR-mediated stalled ribosomes as posing a particular danger to cellular health and viability. This dataset contains the RNA-sequencing data used to support the conclusions from this study.
2022-08-01 | GSE193962 | GEO
Project description:Dysregulation of RNA processing contributes to neurodegenerative diseases, especially amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common cause of both FTD and ALS (C9-FTD/ALS), characterized with aberrant repeat RNA foci in the nucleus and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions in the cytoplasm. Here we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon-skipping and intron retention in human iPSC-derived neurons. Similar alternative splicing changes can be found in patient postmortem tissues. This work identified novel molecular mechanism of global RNA splicing defects by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets.
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Project description:An abnormal expansion of a GGGGCC hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform effectively curbed the expression of the GGGGCC repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci and reversed transcriptional deficits. This high-fidelity Cas13 variant possessed improved transcriptome-wide specificity compared to its native form and mediated efficient targeting in motor neuron-like cells derived from a patient with ALS. Our results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.
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Project description:A GGG GCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense repeat-containing transcripts undergo repeat associated non-AUG-initiated translation to produce five dipeptide proteins (DPRs). The polyGR and polyPR DPRs are extremely toxic when expressed in Drosophila neurons. To determine the mechanism that mediates this toxicity, we purified DPRs from the Drosophila brain and used mass spectrometry to identify the irst in vivo neuronal DPR interactome. PolyGR and polyPR interact with ribosomal proteins, and inhibit translation in both human iPSC-derived motor neurons, and adult Drosophila neurons.
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Project description:Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased, leading to accumulation of GGGGCC repeat–containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-?, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS. Transcriptome profiling from iPSC derived motor neurons compared to controls
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