Langlois Marc-André, Boniface Christelle, Wang Gang, Alluin Jessica, Salvaterra Paul M, Puymirat Jack, Rossi John J, Lee Nan Sook
Laboratory of Human Genetics, Laval University Medical Research Centre, CHUQ, Pavillon CHUL, Ste-Foy, Quebec G1V 7P4, Canada.
J Biol Chem. 2005 Apr 29;280(17):16949-54. doi: 10.1074/jbc.M501591200. Epub 2005 Feb 18.
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 ( approximately 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 down-regulation 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.
小干扰RNA（siRNA）双链体可在哺乳动物细胞中诱导靶RNA的特异性切割。它们参与基因表达的下调被称为RNA干扰（RNAi）。人们普遍认为RNAi主要发生在细胞质中。我们在此报告，通过靶向强直性肌营养不良蛋白激酶（DMPK）mRNA，在原代人1型强直性肌营养不良（DM1）细胞中存在细胞质和细胞核RNAi现象。来自一名人类DM1胎儿的杂合子DM1成肌细胞从DMPK基因的一个等位基因产生具有大CUG重复序列（约3200个）的细胞核滞留突变DMPK转录本和具有18个CUG重复序列的野生型转录本，从而为评估细胞核和细胞质表达谱提供了条件。我们在此首次证明，用慢病毒传递的短发夹RNA（shRNA）靶向内源性细胞核滞留突变DMPK mRNA可导致其下调。当通过阳离子脂质传递合成siRNA时，未观察到这种细胞核RNAi（类）现象，这表明shRNA的加工与细胞核输入之间存在联系，或者在细胞核中存在加工shRNA的独立途径。我们观察到细胞质和细胞核滞留的DMPK同时发生RNAi，这对哺乳动物细胞两个区室中的转录后基因调控具有重要意义。
