Research Center on Aging, Department of Biochemistry and Molecular Biology, Peking University Health Science Center, 38 Xueyuan Road, Beijing 100083, USA.
Nucleic Acids Res. 2010 Mar;38(5):1547-58. doi: 10.1093/nar/gkp1114. Epub 2009 Dec 8.
The RNA-binding protein, HuR, associates with the HuR mRNA, but the consequences of this interaction are unknown. Here, we use human diploid fibroblasts (HDFs) and cervical carcinoma cells to study this regulatory paradigm. Ectopic overexpression of HuR potently enhanced the translation and cytoplasmic levels of endogenous HuR, but did not affect HuR mRNA levels. Inhibition of CRM1 function by Lemptomycin B or by knockdown of CRM1 greatly diminished the cytoplasmic levels of endogenous HuR mRNA and hence blocked the induction of endogenous HuR by exogenous HuR. Further studies showed that HuR interacted with the 3'-untranslated region (UTR) of HuR and that overexpression of HuR increased the cytoplasmic levels of a chimeric luciferase-HuR 3'-UTR reporter transcript, as well as luciferase activity; conversely, HuR knockdown reduced both parameters. Moreover, the loss of HuR in senescent, late-passage HDFs was accompanied by a reduced cytoplasmic presence of endogenous HuR mRNA, ectopic Luc-HuR-3'UTR reporter transcript, and luciferase activity relative to what was observed in young, early-passage cells. Our results reveal a positive feedback mechanism for the regulation of HuR, which may play an important role in the regulation of HuR during replicative senescence.
RNA 结合蛋白 HuR 与 HuR mRNA 结合，但这种相互作用的后果尚不清楚。在这里，我们使用人二倍体成纤维细胞（HDF）和宫颈癌细胞来研究这种调节模式。HuR 的异位过表达强烈增强了内源性 HuR 的翻译和细胞质水平，但不影响 HuR mRNA 水平。用 Lemptomycin B 或 CRM1 敲低抑制 CRM1 功能，大大降低了内源性 HuR mRNA 的细胞质水平，从而阻止了外源性 HuR 对内源性 HuR 的诱导。进一步的研究表明，HuR 与 HuR 的 3'-非翻译区（UTR）相互作用，HuR 的过表达增加了嵌合荧光素酶-HuR 3'-UTR 报告转录本以及荧光素酶活性的细胞质水平；相反，HuR 的敲低降低了这两个参数。此外，衰老、晚期传代 HDF 中 HuR 的丢失伴随着内源性 HuR mRNA、外源性 Luc-HuR-3'UTR 报告转录本和荧光素酶活性在细胞质中的减少，与年轻、早期传代细胞相比。我们的结果揭示了 HuR 调节的正反馈机制，这可能在复制性衰老过程中 HuR 的调节中发挥重要作用。
