Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins

Abstract

Massive GGGGCC repeat expansion in the first intron of the gene C9orf72 is the most common known cause of familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Despite its intronic localization and lack of an ATG start codon, the repeat region is translated in all three reading frames into aggregating dipeptide-repeat (DPR) proteins, poly-(Gly-Ala), poly-(Gly-Pro) and poly-(Gly-Arg). We took an antibody-based approach to further validate the translation of DPR proteins. To test whether the antisense repeat RNA transcript is also translated, we raised antibodies against the predicted products, poly-(Ala-Pro) and poly-(Pro-Arg). Both antibodies stained p62-positive neuronal cytoplasmic inclusions throughout the cerebellum and hippocampus indicating that not only sense but also antisense strand repeats are translated into DPR proteins in the absence of ATG start codons. Protein products of both strands co-aggregate suggesting concurrent translation of both strands. Moreover, an antibody targeting the putative carboxyl terminus of DPR proteins can detect inclusion pathology in C9orf72 repeat expansion carriers suggesting that the non-ATG translation continues through the entire repeat and beyond. A highly sensitive monoclonal antibody against poly-(Gly-Arg), visualized abundant inclusion pathology in all cortical regions and some inclusions also in motoneurons. Together, our data show that the GGGGCC repeat is bidirectionally translated into five distinct DPR proteins that co-aggregate in the characteristic p62-positive TDP-43 negative inclusions found in FTLD/ALS cases with C9orf72 repeat expansion. Novel monoclonal antibodies against poly-(Gly-Arg) will facilitate pathological diagnosis of C9orf72 FTLD/ALS.

Emerging mechanisms of molecular pathology in ALS

“…Unexpectedly, neurons from cases with the C9orf72 intronic expansion also possess a third type of inclusion: intracytosolic aggregates composed of dipeptide repeat proteins encoded by the intronic hexanucleotide repeat that are produced through noncanonical, repeat-associated non-ATG-mediated (RAN-mediated) translation. These atypical peptides reflect translation of amino acids from all possible reading frames of the G 4 C 2 expanded domains (69)(70)(71)(72)(73). Some RAN-produced peptides, such as those containing repeated arginines, are neurotoxic in flies and mammalian neurons in vitro (74,75) Protein degradation.…”

Poly(GP), neurofilament and grey matter deficits in C9orf72 expansion carriers

“…Potential pathomechanisms include the loss of function of normal C9orf72 protein, and/or toxicity resulting from the accumulation of G 4 C 2 transcripts that form RNA foci, interact with RNA‐binding proteins, and impair RNA processing 6. Expanded G 4 C 2 transcripts also lead to the production of five dipeptide repeat (DPR) proteins through repeat‐associated non‐ATG (RAN) translation 7, 8. RAN translation of sense transcripts of the repeat produces poly(GA), poly(GR), and poly(GP), while RAN translation of antisense repeat transcripts produces poly(PA), poly(PR), and poly(GP).…”

Emerging role of RNA•DNA hybrids inC9orf72-linked neurodegeneration

“…39,[83][84][85][86] A genome-wide study in yeast found that RNADNA hybrids are enriched in genes that produce antisense transcripts and that the expression of these genes is regulated by the hybrids. 87 A recent study by Skourti-Stathaki et al reported that R-loops induce antisense transcription, which leads to RNA interference-dependent repressive histone marks over mammalian gene terminators.…”