RESUMO
In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation and suggests caution in the interpretation of ribosomal protein gene mutation data.
Assuntos
Mutação com Perda de Função , Fenótipo , Proteínas Ribossômicas/genética , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteostase , Receptores de Quinase C Ativada/genética , Receptores de Quinase C Ativada/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
RNA dysregulation is a newly recognized disease mechanism in amyotrophic lateral sclerosis (ALS). Here we identify Drosophila fragile X mental retardation protein (dFMRP) as a robust genetic modifier of TDP-43-dependent toxicity in a Drosophila model of ALS. We find that dFMRP overexpression (dFMRP OE) mitigates TDP-43 dependent locomotor defects and reduced lifespan in Drosophila. TDP-43 and FMRP form a complex in flies and human cells. In motor neurons, TDP-43 expression increases the association of dFMRP with stress granules and colocalizes with polyA binding protein in a variant-dependent manner. Furthermore, dFMRP dosage modulates TDP-43 solubility and molecular mobility with overexpression of dFMRP resulting in a significant reduction of TDP-43 in the aggregate fraction. Polysome fractionation experiments indicate that dFMRP OE also relieves the translation inhibition of futsch mRNA, a TDP-43 target mRNA, which regulates neuromuscular synapse architecture. Restoration of futsch translation by dFMRP OE mitigates Futsch-dependent morphological phenotypes at the neuromuscular junction including synaptic size and presence of satellite boutons. Our data suggest a model whereby dFMRP is neuroprotective by remodeling TDP-43 containing RNA granules, reducing aggregation and restoring the translation of specific mRNAs in motor neurons.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/metabolismo , RNA Mensageiro/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína do X Frágil da Deficiência Intelectual , Técnicas de Silenciamento de Genes , Humanos , Proteínas Associadas aos Microtúbulos/genética , Junção Neuromuscular/metabolismo , Neurônios/metabolismo , Neurotoxinas/metabolismo , Fenótipo , Proteínas de Ligação a RNA/metabolismo , Solubilidade , Translocação GenéticaRESUMO
Nucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Unconventional translation (RAN translation) of C9orf72 repeats generates dipeptide repeat proteins that can cause neurodegeneration. We performed a genetic screen for regulators of RAN translation and identified small ribosomal protein subunit 25 (RPS25), presenting a potential therapeutic target for C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia and other neurodegenerative diseases caused by nucleotide repeat expansions.
Assuntos
Proteína C9orf72/genética , Doenças Neurodegenerativas/genética , Proteínas Ribossômicas/genética , Animais , Expansão das Repetições de DNA/genética , Humanos , Biossíntese de ProteínasRESUMO
C9orf72 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) produced by unconventional translation of the C9orf72 repeat expansions cause neurodegeneration in cell culture and in animal models. We performed two unbiased screens in Saccharomyces cerevisiae and identified potent modifiers of DPR toxicity, including karyopherins and effectors of Ran-mediated nucleocytoplasmic transport, providing insight into potential disease mechanisms and therapeutic targets.