Université Bordeaux Segalen, Bordeaux, F-33076 Bordeaux, France.
Nucleic Acids Res. 2012 Feb;40(3):1356-65. doi: 10.1093/nar/gkr843. Epub 2011 Oct 18.
Besides the fact that miR-96 and miR-182 belong to the miR-182/183 cluster, their seed region (UUGGCA, nucleotides 2-7) is identical suggesting potential common properties in mRNA target recognition and cellular functions. Here, we used the mRNA encoding Glypican-3, a heparan-sulfate proteoglycan, as a model target as its short 3' untranslated region is predicted to contain one miR-96/182 site, and assessed whether it is post-transcriptionally regulated by these two microRNAs. We found that miR-96 downregulated GPC3 expression by targeting its mRNA 3'-untranslated region and interacting with the predicted site. This downregulatory effect was due to an increased mRNA degradation and depended on Argonaute-2. Despite its seed similarity with miR-96, miR-182 was unable to regulate GPC3. This differential regulation was confirmed on two other targets, FOXO1 and FN1. By site-directed mutagenesis, we demonstrated that the miRNA nucleotide 8, immediately downstream the UUGGCA seed, plays a critical role in target recognition by miR-96 and miR-182. Our data suggest that because of a base difference at miRNA position 8, these two microRNAs control a completely different set of genes and therefore are functionally independent.
除了 miR-96 和 miR-182 属于 miR-182/183 簇这一事实外，它们的种子区（UUGGCA，核苷酸 2-7）相同，这表明它们在 mRNA 靶标识别和细胞功能方面可能具有潜在的共同特性。在这里，我们使用编码 GPC3 的 mRNA 作为模型靶标，因为其短的 3'非翻译区被预测包含一个 miR-96/182 位点，并评估其是否受这两种 microRNA 的转录后调控。我们发现 miR-96 通过靶向其 mRNA 3'非翻译区并与预测的位点相互作用来下调 GPC3 的表达。这种下调作用是由于 mRNA 降解增加，并且依赖于 Argonaute-2。尽管与 miR-96 的种子相似，但 miR-182 无法调节 GPC3。这种差异调节在另外两个靶标 FOXO1 和 FN1 上得到了证实。通过定点诱变，我们证明了 miRNA 核苷酸 8，即 UUGGCA 种子下游的一个位置，在 miR-96 和 miR-182 的靶标识别中起着关键作用。我们的数据表明，由于 miRNA 位置 8 上的碱基差异，这两种 microRNA 控制着完全不同的一组基因，因此在功能上是独立的。
