Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells

Abstract

Many RNA regulatory proteins controlling pre-mRNA splicing contain serine:arginine (SR) repeats. Here we found that these SR domains bound hydrogel droplets composed of fibrous polymers of the low-complexity domain of heterogeneous ribonucleoprotein A2 (hnRNPA2). Hydrogel binding was reversed upon phosphorylation of the SR domain by CDC2-like kinases 1 and 2 (CLK1/2). Mutated variants of the SR domains changing serine to glycine (SR-to-GR variants) also bound to hnRNPA2 hydrogels, but were not affected by CLK1/2. When expressed in mammalian cells, these variants bound nucleoli. The translation products of the sense and antisense transcripts of the expansion repeats associated with the C9ORF72 gene altered in neurodegenerative disease encode GR N and PR N repeat polypeptides. Both peptides bound to hnRNPA2 hydrogels independent of CLK1/2 activity. When applied to cultured cells, both peptides entered cells, migrated to the nucleus, bound nucleoli, and poisoned RNA biogenesis, which caused cell death.Among familial causes of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), between 25 and 40% of cases are attributed to a repeat expansion in a gene designated C9ORF72. The hexa-nucleotide repeat sequence GGGGCC normally present in 2 to 23 copies is expanded in affected patients to 700 to 1,600 copies (1, 2). The pattern of genetic inheritance of the C9ORF72 repeat expansion is dominant, and multiple lines of evidence suggest that the repeat expansion causes disease. Two theories have been advanced to explain repeat-generated toxicity. First, in situ hybridization assays have identified nuclear dots containing either sense or anti-sense repeat transcripts (3-5), leading to the idea that the nuclear-retained RNAs might themselves be toxic. More recently, equally clear evidence has been generated showing that both the sense and anti-sense transcripts of the * Corresponding author. steven.mcknight@utsouthwestern.edu. SUPPLEMENTARY MATERIALS www.sciencemag.org/cgi/content/full/science.1254917/DC1 Materials and Methods Figs. S1 to S6 Table S1 References (29)(30)(31)(32)(33)(34) HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript GGGGCC repeats associated with C9ORF72 can be translated in an ATG-independent manner known as repeat associated non-ATG (RAN) translation (6). Depending upon reading frame, the sense transcript of the repeats can be translated into glycine:alanine (GA N ), glycine:proline (GP N ), or glycine:arginine (GR N ) polymers. RAN translation of the anti-sense transcript of the GGGGCC repeats of C9ORF72 lead to the production of proline:alanine (PA N ), proline:glycine (PG N ) or proline:arginine (PR N ) polymers. These repeat-encoded polymers are expressed in disease tissue (5, 7-9). The disordered and hydrophobic nature of these polymers, at least the GA N , GP N , and PA N versions, properly predicted that they would aggregate into distinct foci within affected cells (5, 9). Another plausible explanation for repeat-generated toxicity ...

Astrocytes expressing ALS‐linked mutant FUS induce motor neuron death through release of tumor necrosis factor‐alpha

“…Furthermore, we recently showed that ALS astrocytes, including mutFUS expressing patient astrocytes, drive up‐regulation of the multidrug resistance transporter P‐Glycoprotein (P‐gp) in endothelial cells of the blood‐brain barrier (Qosa et al, 2016). Astrocytes participate in disease also through a “loss of function” [reviewed (Maragakis and Rothstein, 2006)] as demonstrated by several human and rodent studies that have observed defects and/or loss of astrocytic glutamate transporter EAAT2/GLT1 for several ALS subtypes including SOD1‐ALS (Benkler, Ben‐Zur, Barhum, & Offen, 2013; Howland et al, 2002) sALS (Lin et al, 1998), and C9orf72‐ALS (Kwon et al, 2014). In contrast, studies of astrocytic contributions in TDP‐43‐mediated ALS have shown conflicting results for both gain and loss‐of‐function toxicity (Haidet‐Phillips et al, 2013; Serio et al, 2013; Tong et al, 2013).…”

Requirement of Neuronal Ribosome Synthesis for Growth and Maintenance of the Dendritic Tree

“…Consequently, depletion of various RPs, including S6 or S14, disrupts ribosomal biogenesis without interfering with nucleolar integrity (4). Impaired ribosome production and/or ribosomal deficits are documented in various neurodegenerative diseases, including Alzheimer, Huntington, Parkinson, amyotrophic lateral sclerosis (ALS), and fronto-temporal lobe dementia (FTLD) (5)(6)(7)(8)(9)(10)(11)(12)(13). Reduced ribosomal biogenesis may also contribute to neurodevelopmental disorders.…”

Current developments in gene therapy for amyotrophic lateral sclerosis

“…A gain of toxic function hypothesis is also supported by the presence of repeat-associated non-ATG dependent translation (RAN translation) to generate dipeptide repeats (DPRs), which have been detected in C9orf72-ALS/FTD tissue [18,23]. It is known that RAN translation of the intronic GGGGCC C9orf72 repeat expansions in both sense and anti-sense directions can generate up to five different DPRs, which can be toxic [18,[23][24][25][26].…”