A repeat expansion in the C9orf72 gene is the most frequent cause of ALS. This repeat expansion is believed to be neurotoxic because of (a combination of) direct RNA toxicity, DPR toxicity or loss of function. We aimed to investigate the role and mechanism of direct RNA toxicity in an in vivo model. Injection of both sense and antisense repeat RNA constructs into zebrafish oocytes induced a motor neuronal phenotype at 30 hours post fertilization. Using dot blot, western blot and ELISA, generation of significant amounts of DPRs was excluded. Although forced expression of DPRs (in particular GR and PR) could induce a motor neuron phenotype, no GR and PR were detected in fish injected with repeat RNA. Moreover, synergic toxicity between low (not toxic) levels of the different DPRs was excluded. Additionally, interrupted repeat RNA constructs which are unable to generate DPRs were found to be neurotoxic as well. Hence, this transient zebrafish model seems to be a model for C9orf72 related direct RNA toxicity. To investigate the mechanism of this direct RNA toxicity we performed an RNA pull down to identify repeat RNA binding proteins. PURA was confirmed as binding partner of GGGGCC repeats. Direct RNA toxicity, but not DPR toxicity, was prevented by overexpression of PURA. This beneficial effect depended on its glycine-rich and PUR2 domains. PURA was found to increase the levels of the autophagy related protein p62 in both C9orf72 and control fibroblasts, and this effect was dependent on the PUR2 domain. Moreover, direct RNA toxicity in zebrafish was prevented by p62 overexpression. Interestingly, sense repeat RNA was found to induce stress granule formation in HEK cells, and PURA was found to prevent stress granule formation. This effect was dependent on the glycine-rich domain of PURA. In conclusion, we provide evidence for direct RNA toxicity of the C9orf72 repeat expansion using a transient zebrafish model. We show that the RNA binding protein PURA prevents this RNA toxicity and that this protective effect is likely mediated through modulation of autophagy and stress granule dynamics