Kim Yoosik, Yeo Jinah, Lee Jung Hyun, Cho Jun, Seo Daekwan, Kim Jong-Seo, Kim V Narry
Center for RNA Research, Institute for Basic Science, Seoul 151-742, South Korea; School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea.
Center for RNA Research, Institute for Basic Science, Seoul 151-742, South Korea; School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea.
Cell Rep. 2014 Nov 6;9(3):1061-74. doi: 10.1016/j.celrep.2014.09.039. Epub 2014 Oct 23.
TRBP functions as both a Dicer cofactor and a PKR inhibitor. However, the role of TRBP in microRNA (miRNA) biogenesis is controversial and its regulation of PKR in mitosis remains unexplored. Here, we generate TRBP knockout cells and find altered Dicer-processing sites in a subset of miRNAs but no effect on Dicer stability, miRNA abundance, or Argonaute loading. By generating PACT, another Dicer interactor, and TRBP/PACT double knockout (KO) cells, we further show that TRBP and PACT do not functionally compensate for one another and that only TRBP contributes to Dicer processing. We also report that TRBP is hyperphosphorylated by JNK in M phase when PKR is activated by cellular double-stranded RNAs (dsRNAs). Hyperphosphorylation potentiates the inhibitory activity of TRBP on PKR, suppressing PKR in M-G1 transition. By generating human TRBP KO cells, our study clarifies the role of TRBP and unveils negative feedback regulation of PKR through TRBP phosphorylation.
TRBP既作为Dicer辅因子发挥作用，又作为PKR抑制剂发挥作用。然而，TRBP在微小RNA（miRNA）生物合成中的作用存在争议，其在有丝分裂中对PKR的调节仍未得到探索。在此，我们生成了TRBP基因敲除细胞，发现一部分miRNA的Dicer加工位点发生了改变，但对Dicer稳定性、miRNA丰度或AGO蛋白装载没有影响。通过生成另一种Dicer相互作用蛋白PACT以及TRBP/PACT双基因敲除（KO）细胞，我们进一步表明TRBP和PACT在功能上不能相互补偿，只有TRBP对Dicer加工有作用。我们还报告，当细胞双链RNA（dsRNA）激活PKR时，TRBP在M期被JNK过度磷酸化。过度磷酸化增强了TRBP对PKR的抑制活性，在M-G1转换期抑制PKR。通过生成人TRBP基因敲除细胞，我们的研究阐明了TRBP的作用，并揭示了通过TRBP磷酸化对PKR的负反馈调节。
