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Summary The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration., Graphical Abstract, Highlights • Longer G4C2 transcripts form neurotoxic RNA foci in cells and zebrafish • Longer G4C2 foci sequester RNA binding proteins hnRNP-H, SC35, and SF2 • G4C2 RNA foci and hnRNP-H knockdown similarly affect TARBP2 splicing • Seventy percent of G4C2 RNA foci in C9ORF72 ALS/FTD brains colocalize with hnRNP-H, In this study, Shaw and colleagues explore mechanisms underlying toxicity of the expanded G4C2 hexanucleotide intronic repeat in C9ORF72, the most common known cause of ALS and FTD. Pathologically expanded G4C2 RNA transcripts form intranuclear foci, sequester specific RNA-binding proteins, and are potently toxic in transfected cells and zebrafish embryos. One protein, hnRNP-H, is detected in 70% of foci in C9ORF72 brain tissues, and loss of hnRNP-H leads to aberrant RNA splicing that could contribute to neurodegeneration.