Nuclear re-localization of Dicer in primary mouse embryonic fibroblast nuclei following DNA damage.
Nuclear re-localization of Dicer in primary mouse embryonic fibroblast nuclei following DNA damage.
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DOI：
10.1371/journal.pgen.1007151
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发表时间：
2018-03
期刊：
影响因子：
4.5
通讯作者：
Gullerova M
中科院分区：
文献类型：
--
作者：
Burger K;Gullerova M
Dicer是RNA干扰（RNAi）的关键组成部分，因其在细胞质中微RNA （mi）的生物发生中的作用而闻名。越来越多的证据表明，哺乳动物的Dicer也存在并活跃于细胞核中。我们之前已经证明，磷酸化的人类Dicer与染色质相关，以响应DNA损伤，并在细胞核中加工双链RNA。然而，Much等人最近的一项研究在小鼠原代胚胎成纤维细胞（pmef）以及成年纯合子活的和可育的HA-Dicer小鼠的生理条件下研究了内源性标记的HA-Dicer，并得出小鼠的Dicer完全是细胞质的结论。作者对一些发现提出了质疑，报道了Dicer在哺乳动物细胞核中的功能。我们重新研究了这个问题，应用亚细胞分离，超分辨率显微镜，然后是3D重建，以及使用相同的HA-Dicer PMEF细胞系的磷酸化dicer特异性抗体。我们的数据表明，小鼠HA-Dicer库的一小部分（约5%）定位于细胞核中，并在DNA损伤时磷酸化。我们认为Dicer定位是动态的，而不仅仅是细胞质的，特别是在暴露于DNA损伤的细胞中。细胞质Dicer是典型微RNA （mi）生物发生途径的关键组成部分。然而，越来越多的证据指向哺乳动物Dicer在细胞核中的定位和活动。Much等人最近的一项研究采用内源性ha标记的Dicer敲入小鼠细胞系，表明Dicer完全是细胞质的。本文对一些报道Dicer在人类细胞核中各种RNA代谢功能的研究提出了质疑。鉴于关于Dicer亚细胞定位的争议，解决这个问题是必要的。使用与Much及其同事使用的相同的细胞，我们结合超分辨率显微镜，3D重建和生化分析，表明内源性标记的HA-Dicer在生理条件下显著定位于细胞核。我们证明DNA损伤触发细胞核中磷酸化HA-Dicer的积累，证实了先前在人类细胞中的观察结果。我们的数据表明，哺乳动物对DNA损伤的反应中，核Dicer定位和功能的进化守恒。
Dicer is a key component of RNA interference (RNAi) and well-known for its role in biogenesis of micro (mi)RNA in the cytoplasm. Increasing evidence suggests that mammalian Dicer is also present and active in the nucleus. We have previously shown that phosphorylated human Dicer associates with chromatin in response to DNA damage and processes double-stranded (ds)RNA in the nucleus. However, a recent study by Much et al. investigated endogenously tagged HA-Dicer both in primary mouse embryonic fibroblasts (PMEFs) as well as adult homozygous viable and fertile HA-Dicer mice under physiological conditions and concluded that murine Dicer is exclusively cytoplasmic. The authors challenged several findings, reporting functions of Dicer in mammalian nuclei. We have re-investigated this issue by applying subcellular fractionation, super-resolution microscopy followed by 3D reconstitution, and phospho-Dicer-specific antibodies using the same HA-Dicer PMEF cell line. Our data show that a small fraction of the murine HA-Dicer pool, approximately 5%, localises in the nucleus and is phosphorylated upon DNA damage. We propose that Dicer localisation is dynamic and not exclusively cytoplasmic, particularly in cells exposed to DNA damage. Cytoplasmic Dicer is a key component of the canonical micro (mi)RNA biogenesis pathway. However, a growing body of evidence points toward localisation and activity of mammalian Dicer in the nucleus. A recent study by Much et al., employed an endogenously HA-tagged Dicer knock-in mouse cell line to show that Dicer is exclusively cytoplasmic. This paper challenges several studies reporting various RNA metabolic functions of Dicer in human nuclei. Given the controversy about Dicer’s subcellular localisation, it is essential to address this issue. Employing the same cells as used by Much and colleagues, we combined super-resolution microscopy followed by 3D reconstitution and biochemical assays to show that endogenously tagged HA-Dicer prominently localises in the nucleus under physiological conditions. We demonstrate that DNA damage triggers accumulation of phosphorylated HA-Dicer in the nucleus, confirming previous observations in human cells. Our data indicate evolutionary conservation of nuclear Dicer localisation and function in mammals in response to DNA damage.
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