Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
PLoS One. 2010 Aug 13;5(8):e12175. doi: 10.1371/journal.pone.0012175.
RNA silencing is a common term for pathways utilizing small RNAs as sequence-specific guides to repress gene expression. Components of the RNA silencing machinery are involved in different aspects of chromatin function in numerous organisms. However, association of RNA silencing with chromatin in mammalian cells remains unclear.
METHODOLOGY/PRINCIPAL FINDINGS: Immunostaining of mitotic chromosomes with antibodies visualizing either endogenous or ectopically expressed Dicer in mammalian cells revealed association of the protein with ribosomal DNA (rDNA) repeats. Chromatin immunoprecipitations and bisulfite sequencing experiments indicated that Dicer is associated with transcribed regions of both active and silenced genes in rDNA arrays of interphase chromosomes. Metabolic labeling of the mouse embryonic stem (ES) cells lacking Dicer did not reveal apparent defect in rRNA biogenesis though pre-rRNA synthesis in these cells was decreased, likely as a consequence of their slower growth caused by the loss of miRNAs. We analyzed in detail chromatin structure of rDNA but did not find any epigenetic changes at rDNA loci in Dicer(-/-) ES cells. Instead, we found that rDNA methylation is rather low in primary tissues, contrasting with rDNA methylation patterns in transformed cell lines.
CONCLUSION/SIGNIFICANCE: We found that Dicer, a key component of RNA silencing pathways, can be detected in association with rDNA chromatin in mammalian cells. The role of this particular localization of Dicer is not readily apparent since the enzyme is associated with rDNA genes regardless of their transcriptional activity. However, localization of Dicer to the transcribed region suggests that transcription may contribute to the Dicer deposition at rDNA chromatin. We hypothesize that Dicer functions in maintaining integrity of rDNA arrays.
RNA 沉默是一种常见的术语，用于描述利用小 RNA 作为序列特异性向导来抑制基因表达的途径。RNA 沉默机制的组成部分参与了许多生物体中染色质功能的不同方面。然而，RNA 沉默与哺乳动物细胞中染色质的关联仍然不清楚。
方法/主要发现：用抗体对有丝分裂染色体进行免疫染色，这些抗体可以识别内源性或异位表达的 Dicer，在哺乳动物细胞中发现该蛋白与核糖体 DNA（rDNA）重复序列相关联。染色质免疫沉淀和亚硫酸氢盐测序实验表明，Dicer 与间期染色体 rDNA 阵列中活跃和沉默基因的转录区域相关联。对缺乏 Dicer 的小鼠胚胎干细胞（ES）进行代谢标记，尽管这些细胞中的前 rRNA 合成减少，可能是由于失去 miRNA 导致它们生长缓慢，但并未发现 rRNA 生物发生明显缺陷。我们详细分析了 rDNA 的染色质结构，但在 Dicer(-/-)ES 细胞中未发现 rDNA 基因座的任何表观遗传变化。相反，我们发现 rDNA 甲基化在原代组织中相对较低，与转化细胞系中的 rDNA 甲基化模式形成对比。
结论/意义：我们发现 RNA 沉默途径的关键组成部分 Dicer 可以在哺乳动物细胞的 rDNA 染色质中被检测到。由于该酶与 rDNA 基因相关联，无论其转录活性如何，因此这种特定的 Dicer 定位的作用并不明显。然而，Dicer 定位到转录区域表明转录可能有助于 Dicer 在 rDNA 染色质上的沉积。我们假设 Dicer 可在维持 rDNA 阵列的完整性方面发挥作用。
