Differential regulation of microRNA stability
Differential regulation of microRNA stability
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DOI：
10.1261/rna.1851510
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发表时间：
2010-05-01
期刊：
影响因子：
4.5
通讯作者：
Kiledjian, Megerditch
中科院分区：
文献类型：
--
作者：
Bail, Sophie;Swerdel, Mavis;Kiledjian, Megerditch
MicroRNAs （miRNAs）是内源性单链RNA分子，长度约为21个核苷酸，是基因表达的基本转录后调节因子。尽管mirna生成过程中涉及的转录和加工过程已被广泛研究，但对调节单个mirna稳定性的成分知之甚少。所有rna都有不同的固有半衰期，这决定了它们的积累水平，mirna也应该遵循类似的原则。在这里，我们证明，尽管大多数miRNA似乎是稳定的，像mrna一样，miRNA在人类细胞中具有不同的稳定性。特别是，我们发现miR-382，一种有助于HIV-1前病毒潜伏期的miRNA，在细胞中是不稳定的。为了确定miR-382负责其快速衰变的区域，我们开发了一种无细胞系统，再现了观察到的基于细胞调节的miR-382周转率。该系统利用源自pre-miRNA的体外处理成熟miRNA，并跟踪处理后的miRNA的衰变。使用该系统，我们证明miR-382的不稳定性是由其种子区以外的序列驱动的，并且需要39个末端7个核苷酸，其中该区域的突变增加了RNA的稳定性。此外，外泌体3‘-5’外核糖核酸酶复合体被确定为参与miR-382衰变的主要核酸酶，Xrn1的贡献较小，Xrn2没有可检测到的贡献。这些研究为miRNA快速衰变所必需的miRNA元素提供了证据，并暗示外泌体参与了这一过程。开发一种生物化学可修正的系统来分析差异miRNA稳定性的机制是通过调节miRNA稳定性来调节基因表达的重要一步。
MicroRNAs ( miRNAs) are endogenous single-stranded RNA molecules of about 21 nucleotides in length that are fundamental post-transcriptional regulators of gene expression. Although the transcriptional and processing events involved in the generation of miRNAs have been extensively studied, very little is known pertaining to components that regulate the stability of individual miRNAs. All RNAs have distinct inherent half-lives that dictate their level of accumulation and miRNAs would be expected to follow a similar principle. Here we demonstrate that although most miRNA appear to be stable, like mRNAs, miRNAs possess differential stability in human cells. In particular, we found that miR-382, a miRNA that contributes to HIV-1 provirus latency, is unstable in cells. To determine the region of miR-382 responsible for its rapid decay, we developed a cell-free system that recapitulated the observed cell-based-regulated miR-382 turnover. The system utilizes in vitro-processed mature miRNA derived from pre-miRNA and follows the decay of the processed miRNA. Using this system, we demonstrate that instability of miR-382 is driven by sequences outside its seed region and required the 39 terminal seven nucleotides where mutations in this region increased the stability of the RNA. Moreover, the exosome 3'-5' exoribonuclease complex was identified as the primary nuclease involved in miR-382 decay with a more modest contribution by the Xrn1 and no detectable contribution by Xrn2. These studies provide evidence for an miRNA element essential for rapid miRNA decay and implicate the exosome in this process. The development of a biochemically amendable system to analyze the mechanism of differential miRNA stability provides an important step in efforts to regulate gene expression by modulating miRNA stability.