Nakamura Akito, Kimura Hiromichi
Oncology Drug Discovery Unit, Takeda Pharmaceutical Company Limited, Kanagawa 251-8555, Japan.
Biochem Biophys Res Commun. 2017 Apr 1;485(2):484-491. doi: 10.1016/j.bbrc.2017.02.038. Epub 2017 Feb 9.
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 p53 function. We found that siRNA-mediated depletion of GCN2 increases small RNA transcripts such as tRNA and 5S rRNA, and induces the p53 pathway activation. Derepression of these transcripts and p53 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 p53 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.
一般控制非抑制性2（GCN2）在氨基酸短缺时因未负载tRNA的积累而被激活，并调节细胞质中的氨基酸饥饿反应。在此，我们报告了GCN2的核仁定位以及GCN2与小RNA转录本之间的关联。免疫荧光分析显示，GCN2组成性定位于核仁，或在氨基酸饥饿应激下被募集到核仁。核仁是细胞核中最大的结构，除了通过调节p53功能作为应激传感器外，它主要作为核糖体和RNA合成的场所。我们发现，小干扰RNA（siRNA）介导的GCN2缺失会增加tRNA和5S rRNA等小RNA转录本，并诱导p53途径激活。通过缺失RNA聚合酶III（pol III）组分的主要亚基B相关因子1（BRF1），可恢复GCN2缺失导致的这些转录本的去抑制和p53途径激活。这些数据表明，GCN2缺失后小RNA转录本的过量是p53激活的原因。我们的研究结果揭示了GCN2在核仁中的作用，它参与小RNA转录本的表达，并作为营养缺乏的替代应激感应机制。因此，GCN2可能在核仁和细胞质中的多个蛋白质翻译检查点中发挥关键作用。
