Aberrant splicing exonizes C9ORF72 repeat expansion in ALS/FTD

A nucleotide repeat expansion (NRE) in the first annotated intron of the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While C9 NRE-containing RNAs can be translated into several toxic dipeptide repeat proteins, how an intronic NRE can access the translation machinery in the cytoplasm remains unclear. By capturing and sequencing NRE-containing RNAs from patient-derived cells, we found that C9 NRE was exonized by the usage of downstream 5ʹ splice sites and exported from the nucleus in a variety of spliced mRNA isoforms. C9ORF72 aberrant splicing was substantially elevated in both C9 NRE+ motor neurons and human brain tissues. Furthermore, NREs above the pathological threshold were sufficient to activate cryptic splice sites in reporter mRNAs. In summary, our results revealed a crucial and potentially widespread role of repeat-induced aberrant splicing in the biogenesis, localization, and translation of NRE-containing RNAs. Conventional RNA-seq analysis cannot distinguish reads from the NRE-containing transcripts from the much more abundant C9ORF72 mRNAs without NRE. To specifically enrich the NRE-containing transcripts, we adapted the previously described RNA antisense purification (RAP) method and used 5ʹ-biotinylated (CCCCGG)3 ASOs to capture (GGGGCC)n NRE-containing transcripts from nuclear and cytoplasmic RNAs of patient-derived fibroblasts for RNA-seq analysis.