General control nonderepressible 2 (GCN2) is activated by the accumulation of uncharged tRNA in response to amino acid shortage and regulates amino acid starvation response in the cytosol. Here we report the nucleolar localization of GCN2 and the association between GCN2 and small RNA transcripts. Immunofluorescence analysis revealed that GCN2 was constitutively localized to the nucleolus or recruited to the nucleolus by amino acid starvation stress. The nucleolus is the largest structure in the nucleus, where it primarily serves as the site of ribosome and RNA synthesis in addition to acting as a stress sensor through the regulation of p(53) function. We found that siRNA-mediated depletion of GCN2 increases small RNA transcripts such as tRNA and 5S rRNA, and induces the p(53) pathway activation. Derepression of these transcripts and p(53) pathway activation by GCN2 depletion was restored by depletion of B-related factor 1 (BRF1), a primary subunit of RNA polymerase III (pol III) components. These data suggest that the excess amount of small RNA transcripts following GCN2 depletion was responsible for the p(53) activation. Our findings reveal a role of GCN2 in the nucleolus that is involved in the expression of small RNA transcripts and serves as alternative stress-sensing machinery for nutrient deficiency. Thus, GCN2 may play pivotal roles in multiple protein translation checkpoints in both the nucleolus and cytosol.(C) 2017 Elsevier Inc. All rights reserved.