An Unusually Stable G-Quadruplex within the 5′-UTR of the MT3 Matrix Metalloproteinase mRNA Represses Translation in Eukaryotic Cells
An Unusually Stable G-Quadruplex within the 5′-UTR of the MT3 Matrix Metalloproteinase mRNA Represses Translation in Eukaryotic Cells
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DOI：
10.1021/bi900498z
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发表时间：
2009-06-16
期刊：
影响因子：
2.9
通讯作者：
Basu, Soumitra
中科院分区：
文献类型：
--
作者：
Morris, Mark J.;Basu, Soumitra
MT3-MMP是一种基质金属蛋白酶，参与调节癌细胞的侵袭和转移。MT3-MMP mRNA在起始密码子上游含有20个核苷酸的G富集区（M3 Q）。在这里，我们报告说，M3 Q嘌呤序列形成一个非常稳定的分子内G-四链体结构，并在真核细胞中的报告基因的翻译具有抑制作用。通过圆二色谱（CD）光谱和RNase TI酶足迹法表明G-四链体结构的形成。当仅加入1 mM KCl导致解链温度（T-m）比不加入盐时增加约30 ℃时，证明了G-四链体的不寻常的稳定性。T-m与RNA浓度无关，表明分子内存在G-四链体结构。此外，在真核细胞中进行的双荧光素酶报告基因测定中，在MT3-MMP的整个5 '-UTR的背景下存在的M3 Q基序抑制其下游基因的活性超过一半。据我们所知，天然存在的M3 Q序列形成最稳定的分子内RNA G-四链体之一。本报告首次确定了MT3-MMP 5 '-UTR中G-四链体形成序列在真核细胞翻译调控中的功能作用。
MT3-MMP is a matrix metalloproteinase involved in the regulation of cancer cell invasion and metastasis. The MT3-MMP mRNA contains a 20-nucleotide G-rich region (M3Q) upstream of the initiation codon. Herein, we report that the M3Q purine-only sequence forms an extremely stable intramolecular G-quadruplex structure and has an inhibitory role on translation of a reporter gene in eukaryotic cells. The formation of the G-quadruplex structure was indicated by circular dichroism (CD) spectroscopy and enzymatic footprinting with RNase TI. The unusual stability of the G-quadruplex was evidenced when addition of only 1 mM KCl resulted in about a 30 degrees C increase in the melting temperature (T-m), as compared to that obtained in the absence of added salt. The T-m, was independent of the RNA concentration, suggesting an intramolecular G-quadruplex Structure. Additionally, in a dual luciferase reporter assay performed in eukaryotic cells, the M3Q motif present, in the context of the entire 5'-UTR of MT3-MMP repressed activity of its downstream gene by more than half. To the best of our knowledge, the naturally occurring M3Q sequence forms one of the most stable, intramolecular RNA G-quadruplexes reported. This report is the first to establish a functional role of a G-quadruplex forming sequence within the MT3-MMP 5'-UTR in the regulation of translation in eukaryotic cells.