ZNF598 co-translationally titrates poly(GR) protein implicated in the pathogenesis of C9ORF72-associated ALS/FTD

Abstract

C9ORF72-derived dipeptide repeat proteins have emerged as the pathogenic cause of neurodegeneration in amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). However, the mechanisms underlying their expression are not fully understood. Here, we demonstrate that ZNF598, the rate-limiting factor for ribosome-associated quality control (RQC), co-translationally titrates the expression of C9ORF72-derived poly(GR) protein. A Drosophila genetic screen identified key RQC factors as potent modifiers of poly(GR)-induced neurodegeneration. ZNF598 overexpression in human neuroblastoma cells inhibited the nuclear accumulation of poly(GR) protein and decreased its cytotoxicity, whereas ZNF598 deletion had opposing effects. Poly(GR)-encoding sequences in the reporter RNAs caused translational stalling and generated ribosome-associated translation products, sharing molecular signatures with canonical RQC substrates. Furthermore, ZNF598 and listerin 1, the RQC E3 ubiquitin-protein ligase, promoted poly(GR) degradation via the ubiquitin-proteasome pathway. An ALS-relevant ZNF598R69C mutant displayed loss-of-function effects on poly(GR) expression, as well as on general RQC. Moreover, RQC function was impaired in C9-ALS patient-derived neurons, whereas lentiviral overexpression of ZNF598 lowered their poly(GR) expression and suppressed proapoptotic caspase-3 activation. Taken together, we propose that an adaptive nature of the RQC-relevant ZNF598 activity allows the co-translational surveillance to cope with the atypical expression of pathogenic poly(GR) protein, thereby acquiring a neuroprotective function in C9-ALS/FTD.

A nascent peptide code for translational control of mRNA stability in human cells

“…This includes Arg-Gly and Arg-Pro dipeptides from the C9ORF72 ORF, which cause amyotrophic lateral sclerosis and frontotemporal dementia 63,64 , and poly-glutamine repeats translated from CAG nucleotide repeat expansions in the mHtt gene, which cause Huntington's disease [77][78][79] . Interestingly, although the RQC pathway isn't demonstrated to act directly on these toxic repeats, expression of RQC pathway components is associated with lower disease severity in both instances 79,80 . As the destabilizing peptide sequences we identify in this study cause ribosome slowdown (Fig.…”

Arginine-rich C9ORF72 ALS Proteins Stall Ribosomes in a Manner Distinct From a Canonical Ribosome-Associated Quality Control Substrate

“…25% or less). Such lengths would be sufficient to fill the ribosome exit tunnel and stall through an electrostatic jamming mechanism as previously proposed (17)(18)(19). However, the most effective stalling required longer dipeptide lengths (>30 repeats for poly-GR (60 amino acids) and >50 repeats for poly-PR (100 amino acids)) to stall more than 50%, and indeed did so in a length-dependent manner, suggesting that emergent DPR chain is involved in stalling.…”

“…The activation of RQC pathway is important since it suggests that mechanisms exist to recognize the stalls, yet our other findings suggest that they are unable to be adequately resolved. Indeed other work has pointed to a role for RQC protein ZNF598 promoting the cleavage of poly-GR (but not poly-PR) by the ubiquitin-proteasome system while not affecting the readthrough efficiency (18). As such, the stalling may simply be overwhelming or improperly sensed by machinery that has not evolved to deal with such unnatural stalling sequences.…”

“…Recent studies from us and others showed that expression of mRNA encoding long repeats of poly-GR and poly-PR causes severe ribosomal stalling (17,18) and it was proposed that the positively charged R-rich nascent polypeptide electrostatically jams the ribosome exit tunnel (19). However, the mechanisms by which stalling occurs and whether it contributes to toxicity remain unclear.…”

Proteostasis Deregulation in Neurodegeneration and Its Link with Stress Granules: Focus on the Scaffold and Ribosomal Protein RACK1

“…Indeed, elongating polyribosomes have been shown to stall on GGGGCC (G 4 C 2 ) repeat expansion in the C9orf72 gene, known to cause FTD and ALS (C9-ALS/FTD), leading to the production of neurodegeneration-driving dipeptide repeat proteins through repeat-associated non-AUG (RAN) translation and to translation inhibition [ 40 ]. In this regard, the RQC rate-limiting factor zinc finger protein 598, E3 ubiquitin ligase (ZNF598), has been shown to have a neuroprotective function in C9-ALS/FTD, since it co-translationally regulates the expression of C9orf72 -derived protein to promote its degradation via the ubiquitin–proteasome pathway and to suppress proapoptotic caspase-3 activation, while ALS-linked mutant ZNF598R69C showed a loss of this function [ 41 ] ( Figure 2 A). In addition, the cytoplasmic residency of the RBP fused in sarcoma (FUS, also known as translocated in sarcoma, TLS) is prevalent in ALS and FTD and could contribute to the translational stalling of polyribosomes in an RNA-binding dependent manner [ 42 ].…”