The porphyrin TMPyP4 inhibits elongation during the noncanonical translation of the FTLD/ALS-associated GGGGCC repeat in the C9orf72 gene

Abstract

GGGGCC (G 4 C 2 ) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through r epeat- a ssociated n on-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G 4 C 2 repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G 4 C 2 repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G 4 C 2 repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G 4 C 2 repeat RNA. Urea-resistant interaction between G 4 C 2 repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G 4 C 2 repeat translation elongation.

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA inC9orf72-linked ALS/FTD

“…These results suggest the preferential binding of FUS to G 4 C 2 repeat RNA with the G-quadruplex structure, which is consistent with a previous report showing preferential binding of FUS to G-quadruplex structured Sc1 and DNMT RNAs (Ozdilek et al, 2017). Considering that higher-order structures, including G-quadruplex and hairpin structures, are reported to be involved in RAN translation (Mori et al, 2021; Simone et al, 2018; Wang et al, 2019; Zu et al, 2011), we next investigated the effects of FUS on the structure of G 4 C 2 repeat RNA. The circular dichroism (CD) spectrum of (G 4 C 2 ) 4 RNA in KCl buffer was found to exhibit a positive peak at approximately 260 nm and a negative peak at 240 nm (Figure 5B, black line), consistent with previous reports (Fratta et al, 2012; Hauesler et al, 2014; Reddy et al, 2013; Su et al, 2014).…”

“…The copyright holder for this preprint this version posted November 1, 2022. ; https://doi.org/10.1101/2022.11.01.514717 doi: bioRxiv preprint structure were both reported to inhibit RAN translation from the G 4 C 2 repeat RNA, resulting in reduced DPR levels (Wang et al, 2019, Mori et al, 2021. These findings are in accordance with our results showing that FUS modifies the G-quadruplex structure as well as the hairpin structure of G 4 C 2 repeat RNA as an RNA chaperone and reduces DPR production.…”

Biological basis and psychiatric symptoms in frontotemporal dementia

“…AUG codon which encodes methionine is the initiation point for conventional translation, but RAN translation is by its definition initiated at the non‐AUG codon 60 . Potential RAN translation inhibitors are currently attracting attention as potential disease‐modifying drugs in repeat expansion disorders including C9orf72 FTLD/ALS 61–64 …”

“…60 Potential RAN translation inhibitors are currently attracting attention as potential disease-modifying drugs in repeat expansion disorders including C9orf72 FTLD/ALS. [61][62][63][64] Neuronal toxicity of DPR has been shown in various disease models. [65][66][67][68][69][70][71][72][73] The poly-GA aggregates which is a most prominent feature in C9orf72 FTLD/ALS patient brain tissue, adsorbs a large number of proteasomes, which may impair intracellular proteostasis.…”

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

“…Increasing evidence suggests a potential involvement of ribosome stalling also in the development of other neurodegenerative diseases, such as FTD and ALS. 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).…”