Protein quality control pathways have evolved to ensure the fidelity of protein synthesis and efficiently clear potentially toxic protein species. Defects in ribosome-associated quality control and its associated factors have been implicated in the accumulation of aberrant proteins and neurodegeneration. C9orf72 repeat-associated non-AUG translation has been suggested to involve inefficient translation elongation, lead to ribosomal pausing and activation of ribosome-associated quality control pathways. However, the role of the ribosome-associated quality control complex in the processing of proteins generated through this non-canonical translation is not well understood. Here we utilize reporter constructs containing the C9orf72-associated hexanucleotide repeat, ribosome-associated quality control complex deficient cell models and stain for ribosome-associated quality control markers in C9orf72-expansion carrier human tissue to understand its role in dipeptide repeat protein pathology. Our studies show that canonical ribosome-associated quality control substrates products are efficiently cleared by the ribosome-associated quality control complex in mammalian cells. Furthermore, using stalling reporter constructs, we show that repeats associated with the C9orf72-expansion induce ribosomal stalling when arginine (R)-rich dipeptide-repeat proteins are synthesized in a length-dependent manner. However, despite triggering this pathway, these arginine-rich dipeptide-repeat proteins are not efficiently processed by the core components of the ribosome-associated quality control complex (listerin, nuclear export mediator factor (NEMF) and valosin containing protein (VCP)) partly due to lack of lysine residues which precludes ubiquitination. Deficient processing by this complex may be implicated in C9orf72-expansion associated disease as dipeptide-repeat protein inclusions were observed to be predominantly devoid of ubiquitin and co-localize with NEMF in mutation carriers’ frontal cortex and cerebellum tissue. These findings suggest that impaired processing of these arginine-rich dipeptide-repeat proteins derived from repeat-associated non-AUG translation by the ribosome-associated quality control complex may contribute to protein homeostasis dysregulation observed in C9orf72-expansion amyotrophic lateral sclerosis and frontotemporal degeneration neuropathogenesis.

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