Cryptic intronic transcriptional initiation generates efficient endogenous mRNA templates for C9orf72-associated RAN translation

Intronic GGGGCC hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Despite its intronic location, this repeat avidly supports synthesis of pathogenic dipeptide repeat (DPR) proteins via repeat-associated non-AUG (RAN) translation. However, the template RNA species that undergoes RAN translation endogenously remains unclear. Using long-read based 5 RNA ligase-mediated rapid amplification of cDNA ends (5 Repeat-RLM-RACE), we identified novel C9orf72 transcripts initiating within intron 1 in a C9BAC mouse model, patient-derived iNeurons, and iNeuron-derived polysomes. These cryptic m7G-capped mRNAs are at least partially polyadenylated and are more abundant than transcripts derived from intron retention or circular intron lariats. In RAN translation reporter assays, novel intronic template transcripts - even those with short (32 nucleotide) leaders - exhibited robust expression compared to exon-intron and repeat-containing lariat reporters. To assess endogenous lariat repeat RNA contributions to RAN translation, we enhanced endogenous lariat stability by knocking down the lariat debranching enzyme Dbr1. However, this modulation did not impact DPR production in patient-derived iNeurons. These findings identify cryptic, linear, m7G-capped intronic-initiating C9orf72 mRNAs as an endogenous template for RAN translation and DPR production, with implications for disease pathogenesis and therapeutic development. SIGNIFICANCE STATEMENTAn intronic GGGGCC repeat expansion in C9orf72 supports an unusual translational initiation process known as repeat-associated non-AUG (RAN) translation to produce toxic dipeptide repeat (DPR) proteins that contribute to neurodegeneration in ALS and FTD. How an intronic repeat RNA engages with ribosomes to support such translation is unclear. Here we identify a series of novel mRNA transcripts that initiate within the repeat-containing intron to create linear m7G-capped templates for RAN translation from GGGGCC repeats. These cryptic mRNAs are present in patient iNeurons, engage with ribosomes, and robustly support RAN translation. This finding has important implications for both our understanding of the mechanism by which RAN translation occurs and on therapeutic development in this currently untreatable class of neurodegenerative disorders.