Morris Mark J, Basu Soumitra
Department of Chemistry, Kent State University, Kent, Ohio 44242, USA.
Biochemistry. 2009 Jun 16;48(23):5313-9. doi: 10.1021/bi900498z.
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 T1. 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.
MT3 - 基质金属蛋白酶（MT3 - MMP）是一种参与癌细胞侵袭和转移调控的基质金属蛋白酶。MT3 - MMP信使核糖核酸（mRNA）在起始密码子上游含有一个20个核苷酸的富含鸟嘌呤区域（M3Q）。在此，我们报告M3Q仅由嘌呤组成的序列形成了一种极其稳定的分子内G - 四链体结构，并且对真核细胞中报告基因的翻译具有抑制作用。通过圆二色性（CD）光谱和核糖核酸酶T1的酶足迹法表明了G - 四链体结构的形成。当仅添加1 mM氯化钾时，与未添加盐时相比，熔解温度（T(m)）升高约30摄氏度，这证明了G - 四链体具有异常的稳定性。T(m)与RNA浓度无关，表明其为分子内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检测中，MT3 - MMP整个5' - 非翻译区（UTR）中的M3Q基序使其下游基因的活性受到超过一半的抑制。据我们所知，天然存在的M3Q序列形成了所报道的最稳定的分子内RNA G - 四链体之一。本报告首次证实了MT3 - MMP 5' - UTR内形成G - 四链体的序列在真核细胞翻译调控中的功能作用。
