Abstract
The GGGGCC hexanucleotide expansion in C9orf72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet a clear understanding of how C9 fits into the broader context of ALS/FTD pathology has remained lacking. The repetitive RNA derived from the C9 repeat is known to sequester hnRNPH, a splicing regulator, into insoluble aggregates, resulting in aberrant alternative splicing. Furthermore, hnRNPH insolubility and altered splicing of a robust set of targets have been observed to correlate in C9 and sporadic ALS/FTD patients alike, suggesting that changes along this axis are a core feature of disease pathogenesis. Here, we characterize previously uncategorized RNA splicing defects involving widespread intron retention affecting almost 2000 transcripts in C9ALS/FTD brains exhibiting a high amount of sequestered, insoluble hnRNPH. These intron retention events appear not to alter overall expression levels of the affected transcripts but rather the protein-coding regions. These retained introns affect transcripts in multiple cellular pathways predicted to be involved in C9 as well as sporadic ALS/FTD etiology, including the proteasomal and autophagy systems. The retained intron pre-mRNAs display a number of characteristics, including enrichment of hnRNPH-bound splicing enhancer motifs and a propensity for G-quadruplex (G-Q) formation, linking the defective splicing directly to high amounts of sequestered hnRNPH. Together, our results reveal previously undetected splicing defects in high insoluble hnRNPH-associated C9ALS brains, suggesting a feedback between effective RNA-binding protein dosage and protein quality control in C9, and perhaps all, ALS/FTD.