A conserved region in the 3′ untranslated region of the human LIMK1 gene is critical for proper expression of LIMK1 at the post-transcriptional level
A conserved region in the 3′ untranslated region of the human LIMK1 gene is critical for proper expression of LIMK1 at the post-transcriptional level
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DOI：
10.1007/s12264-013-1341-z
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发表时间：
2013-05
影响因子：
5.6
通讯作者：
Guang-Fei Deng;Shu-Jing Liu;Xun-Sha Sun;Wei-Wen Sun;Qi-hua Zhao;W. Liao;Yong-Hong Yi;Y. Long
中科院分区：
文献类型：
--
作者：
Guang-Fei Deng;Shu-Jing Liu;Xun-Sha Sun;Wei-Wen Sun;Qi-hua Zhao;W. Liao;Yong-Hong Yi;Y. Long
LIM激酶1(LIMK1)是一种胞浆丝氨酸/苏氨酸激酶，调节肌动蛋白细丝的动力学和重组，参与神经元发育和脑功能。LIMK1的异常表达与多种神经系统疾病有关。在本研究中，我们使用VectorNTI(8.0)软件进行了保守性分析。采用双酶报告素法和实时荧光定量RT-PCR方法分别检测报告基因的蛋白和mRNA水平。我们发现，人LIMK13‘非翻译区的+884-+966区域在小鼠LIMK13’非翻译区中高度保守，并形成了一个包含多个环和茎的结构。荧光素酶分析表明，缺失保守区的突变构建体在SH-SY5Y和HEK293细胞中的相对荧光素酶活性仅为野生型构建体pGL4-hLIMK1-3U的60%，说明该保守区对报告基因的表达是关键的。实时荧光定量逆转录聚合酶链式反应显示，转染pGL4-hLIMK1-3U-M的SH-SY5Y和HEK293细胞中Luc2mRNA的相对表达水平下降到pGL4-hLIMK1-3U-M细胞的∼的50%，表明该保守区在维持Luc2mRNA的稳定性中起着重要的作用。我们的研究表明，LIMK13‘非编码区的保守区在转录后水平参与了LIMK1的表达调控，这可能有助于揭示LIMK1在中枢神经系统中表达的调控机制，并探讨3’-非编码区突变与神经系统疾病的关系。
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 humanLIMK13′ untranslated region (UTR) was highly conserved in the mouseLimk13′ 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 relativeLuc2mRNA 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 maintainingLuc2mRNA stability. Our study suggests that the conserved region in theLIMK13′ UTR is involved in regulatingLIMK1expression at the post-transcriptional level, which may help reveal the mechanism underlying the regulation ofLIMK1expression in the central nervous system and explore the relationship between the 3′-UTR mutant and neurological disorders.