Abrogation of DNA vector-based RNAi during apoptosis in mammalian cells due to caspase-mediated cleavage and inactivation of Dicer-1
Abrogation of DNA vector-based RNAi during apoptosis in mammalian cells due to caspase-mediated cleavage and inactivation of Dicer-1
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DOI：
10.1038/cdd.2009.15
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发表时间：
2009-06-01
影响因子：
12.4
通讯作者：
Shah, G. M.
中科院分区：
文献类型：
--
作者：
Ghodgaonkar, M. M.;Shah, R. G.;Shah, G. M.
RNA干扰(RNAi)被用作一种反向遗传工具来检测基因在包括细胞凋亡在内的不同细胞过程中的功能。由于关键的细胞蛋白在凋亡过程中被灭活，并且RNAi需要许多细胞蛋白的合作，我们研究了基于DNA载体的RNAi是否会在凋亡过程中继续发挥作用。从DNA载体转录的短发夹状RNA被DICER-1处理形成小干扰RNA，并被掺入RNA诱导沉默复合体(RISC)，以引导靶mRNA的序列特异性沉默。我们在此报道了三种不同基因，即聚ADP-核糖聚合酶-1、p14(ARF)和层蛋白A/C的DNA载体RNAi在细胞凋亡过程中被取消。基于DNA载体的RNAi的失败不是在AGO-2或RISC介导的RNAi的水平上，而是由于在其RNase IIIa结构域的STTD1476和CGVD(1538)位点的催化失活。采用多种方法，caspase-3被确定为主要的caspase-1裂解和失活的DICER-1。由于Dice-1也是哺乳动物细胞中形成microRNA(MiRNA)所需的常见内切酶，我们观察到成熟形式miR-16、miR-21和let-7a的水平降低。我们的结果表明，通过改变miRNA的可获得性，Dever-1的凋亡裂解和失活在控制凋亡事件中发挥了作用。《细胞死亡与分化》(2009年)16858-868；doi：10.1038/cdd.2009.15；在线发布
RNA interference (RNAi) is used as a reverse-genetic tool to examine functions of a gene in different cellular processes including apoptosis. As key cellular proteins are inactivated during apoptosis, and as RNAi requires cooperation of many cellular proteins, we examined whether DNA vector-based RNAi would continue to function during apoptosis. The short hairpin RNA transcribed from the DNA vector is processed by Dicer-1 to form small interfering RNA that is incorporated in the RNA-induced silencing complex (RISC) to guide a sequence-specific silencing of the target mRNA. We report here that DNA vector-based RNAi of three different genes, namely poly(ADP-ribose) polymerase-1, p14(ARF) and lamin A/C are abrogated during apoptosis. The failure of DNA vector-based RNAi was not at the level of Ago-2 or RISC-mediated step of RNAi but due to catalytic inactivation of Dicer-1 on specific cleavage at the STTD1476 and CGVD(1538) sites within its RNase IIIa domain. Using multiple approaches, caspase-3 was identified as the major caspase responsible for the cleavage and inactivation of Dicer-1. As Dicer-1 is also the common endonuclease required for formation of microRNA (miRNA) in mammalian cells, we observed decreased levels of mature forms of miR-16, miR-21 and let-7a. Our results suggest a role for apoptotic cleavage and inactivation of Dicer-1 in controlling apoptotic events through altered availability of miRNA. Cell Death and Differentiation (2009) 16, 858-868; doi: 10.1038/cdd.2009.15; published online 20 February 2009