The most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia is a G₄C₂ repeat expansion in the C9orf72 gene. This expansion gives rise to translation of aggregating dipeptide repeat (DPR) proteins, including poly-GA as the most abundant species. However, gain of toxic function effects have been attributed to either the DPRs or the pathological G₄C₂ RNA. Here, we analyzed in a cellular model the relative toxicity of DPRs and RNA. Cytoplasmic poly-GA aggregates, generated in the absence of G₄C₂ RNA, interfered with nucleocytoplasmic protein transport, but had little effect on cell viability. In contrast, nuclear poly-GA was more toxic, impairing nucleolar protein quality control and protein biosynthesis. Production of the G₄C₂ RNA strongly reduced viability independent of DPR translation and caused pronounced inhibition of nuclear mRNA export and protein biogenesis. Thus, while the toxic effects of G₄C₂ RNA predominate in the cellular model used, DPRs exert additive effects that may contribute to pathology.