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SFPQ Regulates the Accumulation of RNA Foci and Dipeptide Repeat Proteins from the Expanded Repeat Mutation in C9orf72 Mirjana Malnar1,2 and Boris Rogelj1,3,4,*

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SFPQ Regulates the Accumulation of RNA Foci and Dipeptide Repeat Proteins from the Expanded Repeat Mutation in C9orf72 Mirjana Malnar1,2 and Boris Rogelj1,3,4,* © 2021. Published by The Company of Biologists Ltd | Journal of Cell Science (2021) 134, jcs256602. doi:10.1242/jcs.256602 RESEARCH ARTICLE SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72 Mirjana Malnar1,2 and Boris Rogelj1,3,4,* ABSTRACT a mutation in the C9orf72 gene, which consists of expanded repeats The expanded GGGGCC repeat mutation in the C9orf72 gene is the of the hexanucleotide GGGGCC sequence in the non-coding region C9orf72 – most common genetic cause of the neurodegenerative diseases of the gene. Up to 20 25 of these repeats are present in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia healthy individuals, whereas up to several thousand repeats occur in C9orf72 (FTD). The expansion is transcribed to sense and antisense RNA, ALS and FTD patients (DeJesus-Hernandez et al., 2011; which form RNA foci and bind cellular proteins. This mechanism of Gao et al., 2017; Renton et al., 2011). These repeats form secondary action is considered cytotoxic. Translation of the expanded RNA structures, such as G-quadruplexes, hairpins and i-motifs, both at ž ̌ transcripts also leads to the accumulation of toxic dipeptide repeat the DNA and RNA level (Bo ic et al., 2020; Haeusler et al., 2014; Š proteins (DPRs). The RNA-binding protein splicing factor proline and Kovanda et al., 2016; ket et al., 2015). Three mechanisms of action C9orf72 glutamine rich (SFPQ), which is being increasingly associated with have been proposed for the mutation (Balendra and Isaacs, ALS and FTD pathology, binds to sense RNA foci. Here, we show that 2018). First, the mutation leads to haploinsufficiency of the SFPQ plays an important role in the C9orf72 mutation. C9ORF72 protein, presumably due to decreased levels of mRNA Overexpression of SFPQ resulted in higher numbers of both sense from the mutant allele (Balendra and Isaacs, 2018; Donnelly et al., and antisense RNA foci and DPRs in transfected human embryonic 2013; Tran et al., 2015; Waite et al., 2014). Second, sense and kidney (HEK) cells. Conversely, reduced SPFQ levels resulted in antisense RNA transcripts from mostly nuclear RNA foci in C9orf72 lower numbers of RNA foci and DPRs in both transfected HEK cells ALS and FTD (Balendra and Isaacs, 2018; DeJesus- and C9orf72 mutation-positive patient-derived fibroblasts and Hernandez et al., 2011; Donnelly et al., 2013; Gendron et al., 2013; lymphoblasts. Therefore, we have revealed a role of SFPQ in Lagier-Tourenne et al., 2013; Lee et al., 2013; Mizielinska et al., regulating the C9orf72 mutation that has implications for 2013; Vatovec et al., 2014; Zu et al., 2013). It has been proposed that understanding and developing novel therapeutic targets for ALS these foci sequester RNA-binding proteins and alter their function, a and FTD. process referred to as RNA toxicity (Gao et al., 2017; Haeusler et al., 2016; Lee et al., 2013; Todd and Paulson, 2010). Multiple studies This article has an associated First Person interview with the first have shown that various proteins bind to sense and antisense RNA C9orf72 author of the paper. from the mutation (DeJesus-Hernandez et al., 2011; Donnelly et al., 2013; Gendron et al., 2013; Haeusler et al., 2014; KEY WORDS: Amyotrophic lateral sclerosis, C9orf72, Dipeptide Lee et al., 2013; Mizielinska et al., 2013; Mori et al., 2013a; Swinnen repeat proteins, Frontotemporal dementia, RNA foci, SFPQ et al., 2018; Xu et al., 2013; Zhang et al., 2015; Zu et al., 2013). Third, expanded repeat sense and antisense RNA is translated to dipeptide INTRODUCTION repeat (DPR) proteins by repeat-associated non-AUG translation. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia Sense RNA translates to poly-glycine-alanine (pGA), poly-glycine- (FTD) are fatal neurodegenerative disorders. As they share clinical, arginine (pGR) and poly-glycine-proline (pGP) DPRs, and antisense genetic and pathological features, ALS and FTD are often presented RNA translates to poly-proline-glycine (pPG), poly-proline-arginine as two ends of a spectrum disorder (Abramzon et al., 2020; Gao (pPR) and poly-proline-alanine (pPA). DPRs form insoluble et al., 2017). Mutations of over 50 genes involved in RNA deposits in patient neurons and glia and exert toxic effects in metabolism, protein quality control and turnover have been disease models (Al-Sarraj et al., 2011; Ash et al., 2013; Balendra and implicated in ALS and FTD (Boylan, 2015; Mandrioli et al., Isaacs, 2018; Brasseur et al., 2020; Gao et al., 2017; Gendron et al., 2020; Ramaswami et al., 2013). The most common genetic cause is 2013; Haeusler et al., 2016; Lee et al., 2020; Mori et al., 2013b,c; Saberi et al., 2018; Suzuki et al., 2019; Zu et al., 2013). The above- mentioned mechanisms of action of the C9orf72 mutation are 1Department of Biotechnology, Jožef Stefan Institute, 1000 Ljubljana, Slovenia. predicted to act in synergy to provoke disease-relevant phenotypes, as 2 Graduate School of Biomedicine, Faculty of Medicine, University of Ljubljana, opposed to only one of the described mechanisms playing the 1000 Ljubljana, Slovenia. 3Biomedical Research Institute, 1000 Ljubljana, Slovenia. 4Faculty of Chemistry and Chemical Engineering, University of Ljubljana, predominant role (Balendra and Isaacs, 2018; Haeusler et al., 2016). 1000 Ljubljana, Slovenia. The mutation in C9orf72 and mutations in three other genes – TDP-43 SOD1 *Author for correspondence ([email protected]) (TAR DNA-binding protein 43), (superoxide dismutase) and FUS (fused in sarcoma) – account for B.R., 0000-0003-3898-1943 approximately two-thirds of familial ALS cases (Hardiman et al., This is an Open Access article distributed under the terms of the Creative Commons Attribution 2017). TDP-43 and FUS are RNA-binding proteins that play a role License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, in multiple RNA processing and metabolic pathways and form distribution and reproduction in any medium provided that the original work is properly attributed. cytoplasmic inclusions and nuclear depletion in ALS and FTD (Gao Handling Editor: Maria Carmo-Fonseca et al., 2017; Kwiatkowski et al., 2009; Ling et al., 2013; Mitchell Received 2 November 2020; Accepted 8 January 2021 et al., 2013; Neumann et al., 2009; Rogelj et al., 2012; Sreedharan Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2021) 134, jcs256602. doi:10.1242/jcs.256602 et al., 2008; Štalekar et al., 2015; Vance et al., 2009). Recent studies have indicated the involvement of another RNA-binding protein, splicing factor proline and glutamine rich (SFPQ), in ALS pathology (Ishigaki et al., 2017; Lee et al., 2015; Luisier et al., 2018). SFPQ exhibits intron retention and nuclear loss in ALS and aberrant localization to the cytoplasm and formation of insoluble structures in FTD (Lee et al., 2015; Luisier et al., 2018). These are well-known ALS and FTD pathologies previously observed for TDP-43 and FUS (Li et al., 2013). Mutations of these proteins have been linked to misregulation of gene expression in cell models and motor neurons in ALS and FTD (Lee et al., 2015; Li et al., 2013; Prpar Mihevc et al., 2016; Ramaswami et al., 2013). SFPQ is an abundant nuclear protein with various functions, including alternative splicing, DNA repair, transcriptional regulation and RNA processing and transport (Haeusler et al., 2016; Hirose et al., 2014; Lee et al., 2015; Shav-Tal and Zipori, 2002). SFPQ is one of the core proteins of paraspeckles – nuclear organelles formed by RNA–protein and protein–protein interactions on long non-coding Fig. 1. SFPQ binds to sense but not antisense C9orf72 repeat RNA. The – – RNA NEAT1_2 scaffold (Bond and Fox, 2009; Clemson et al., RNA constructs (G4C2)48 S1m, (C4G2)32 S1m and S1m were used. The immunoblot confirms SFPQ binding to G C RNA repeats, as the signal is 2009; Fox et al., 2018; Mao et al., 2011; West et al., 2016). An 4 2 29.94±4.04 relative to the control, whereas SFPQ is not bound by C4G2, as the increase in NEAT1_2 levels leads to the sequestration of more signal is 0.66±0.33 relative to the control S1m tag. The experiment was paraspeckle proteins, which in the case of SFPQ reduces its relative replicated three times in the laboratory. Error bars denote s.d. levels in the cytoplasm and changes the expression of its target genes (Fox et al., 2018). NEAT1_2 up-regulation and increased (G4C2)72 or (C4G2)32 repeats. The plasmid harboring (G4C2)72 was paraspeckle formation occur in the early phase of ALS (Nishimoto used for its ability to be translated to sense-derived DPRs. Expression et al., 2013; Shelkovnikova et al., 2018). Mutations in other level of SFPQ in SFPQ KD cells was reduced to 0.13±0.03 (mean± paraspeckle proteins also lead to their aggregation or s.d.) of the controls (Fig. 2A; Fig. S2). There was a significant mislocalization to the cytoplasm in ALS, indicating a strong reduction in both sense and antisense foci number: 10.94±1.71 involvement of these structures in the underlying mechanisms of (mean±s.d.) sense foci per cell in SFPQ KD cells compared with ALS (Fox et al., 2018; Li et al., 2013; Modic et al., 2019; Naganuma 22.78±2.74 in controls, and 12.12±4.88 antisense foci per cell in et al., 2012; Nishimura et al., 2010; Shelkovnikova et al., 2014). SFPQ KD cells compared with 22.18±8.01 in controls (Fig. 2B,C). Expanded RNA repeats from the C9orf72 gene have been shown Therefore, the foci number was reduced by approximately half. There to interact with SFPQ across studies (Haeusler et al., 2016), and we was also a significant impact on the range of sense foci per cell, as a have recently shown that sense RNA foci form paraspeckle-like higher percentage of SFPQ KD cells had <20 foci per cell, whereas a nuclear bodies by sequestering paraspeckle proteins (Bajc Česnik higher percentage of control cells had >20 foci per cell (Fig.
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