Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells
Cytoplasmic and nuclear retained DMPK mRNAs are targets for RNA interference in myotonic dystrophy cells
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DOI：
10.1074/jbc.m501591200
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发表时间：
2005-04-29
影响因子：
4.8
通讯作者：
Lee, NS
中科院分区：
文献类型：
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作者：
Langlois, MA;Boniface, C;Lee, NS
小干扰 RNA (siRNA) 双链体可诱导哺乳动物细胞中靶标 RNA 的特异性切割。它们参与基因表达下调的过程称为 RNA 干扰 (RNAi)。人们普遍认为RNAi在细胞质中占主导地位。我们在这里报告了通过靶向强直性肌营养不良蛋白激酶 (DMPK) mRNA 在原代人强直性营养不良 1 型 (DM1) 细胞中细胞质和核 RNAi 现象的共存。来自人类 DM1 胎儿的杂合子 DM1 成肌细胞产生核保留的突变 DMPK 转录物，该转录物具有来自 DMPK 基因的一个等位基因的大 CUG 重复（类似于 3,200 个），以及具有 18 个 CUG 重复的野生型转录物，从而提供了要评估的核和细胞质表达谱。我们在这里首次证明了慢病毒递送的短发夹 RNA (shRNA) 靶向的内源性核保留突变 DMPK mRNA 的下调。当合成的 siRNA 通过阳离子脂质传递时，没有观察到这种核 RNAi（类似）现象，这表明 shRNA 的加工和核输入之间存在联系，或者在细胞核中加工 shRNA 的单独途径。我们对细胞质和核保留 DMPK 同时进行 RNAi 的观察对于哺乳动物细胞两个区室的转录后基因调控具有重要意义。
Small interfering RNA ( siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase ( DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats ( similar to 3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time downregulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells.