Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Institute of Neuroscience, Guangzhou, 510260, China.
Neurosci Bull. 2013 Jun;29(3):348-54. doi: 10.1007/s12264-013-1341-z. Epub 2013 May 23.
LIM kinase 1 (LIMK1), a cytosolic serine/threonine kinase, regulates actin filament dynamics and reorganization and is involved in neuronal development and brain function. Abnormal expression of LIMK1 is associated with several neurological disorders. In this study, we performed a conservation analysis using Vector NTI (8.0) software. The dualluciferase reporter assay and real-time quantitative RT-PCR were used to assess the protein and mRNA levels of the reporter gene, respectively. We found that a region ranging from nt +884 to +966 in the human LIMK1 3' untranslated region (UTR) was highly conserved in the mouse Limk1 3' UTR and formed a structure containing several loops and stems. Luciferase assay showed that the relative luciferase activity of the mutated construct with the conserved region deleted, pGL4-hLIMK1-3U-M, in SH-SY5Y and HEK-293 cells was only ~60% of that of the wild-type construct pGL4-hLIMK1-3U, indicating that the conserved region is critical for the reporter gene expression. Real-time quantitative RT-PCR analysis demonstrated that the relative Luc2 mRNA levels in SH-SY5Y and HEK293 cells transfected with pGL4-hLIMK1-3U-M decreased to ~50% of that in cells transfected with pGL4-hLIMK1-3U, suggesting an important role of the conserved region in maintaining Luc2 mRNA stability. Our study suggests that the conserved region in the LIMK1 3' UTR is involved in regulating LIMK1 expression at the post-transcriptional level, which may help reveal the mechanism underlying the regulation of LIMK1 expression in the central nervous system and explore the relationship between the 3'-UTR mutant and neurological disorders.
LIM 激酶 1（LIMK1）是一种细胞质丝氨酸/苏氨酸激酶，调节肌动蛋白丝动力学和重组，参与神经元发育和大脑功能。LIMK1 的异常表达与几种神经紊乱有关。在这项研究中，我们使用 Vector NTI（8.0）软件进行了保守性分析。双荧光素酶报告基因检测和实时定量 RT-PCR 分别用于评估报告基因的蛋白和 mRNA 水平。我们发现，人 LIMK1 3'非翻译区（UTR）中的 nt +884 到 +966 区域在鼠 Limk1 3'UTR 中高度保守，形成包含几个环和茎的结构。荧光素酶报告基因检测显示，缺失保守区域的突变构建体 pGL4-hLIMK1-3U-M 的相对荧光素酶活性在 SH-SY5Y 和 HEK-293 细胞中仅约为野生型构建体 pGL4-hLIMK1-3U 的 60%，表明保守区域对报告基因表达至关重要。实时定量 RT-PCR 分析表明，转染 pGL4-hLIMK1-3U-M 的 SH-SY5Y 和 HEK293 细胞中的相对 Luc2 mRNA 水平下降至转染 pGL4-hLIMK1-3U 的细胞的约 50%，表明保守区域在维持 Luc2 mRNA 稳定性方面发挥重要作用。我们的研究表明，LIMK1 3'UTR 中的保守区域参与调节 LIMK1 的转录后表达，这可能有助于揭示中枢神经系统中 LIMK1 表达的调控机制，并探索 3'-UTR 突变体与神经紊乱之间的关系。
