title: 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.
abstract: 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.
authors.affiliation: Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Institute of Neuroscience, Guangzhou, 510260, China.
authors.forename: Guang-Fei
authors.initials: GF
authors.lastname: Deng
authors.forename: Shu-Jing
authors.initials: SJ
authors.lastname: Liu
authors.forename: Xun-Sha
authors.initials: XS
authors.lastname: Sun
authors.forename: Wei-Wen
authors.initials: WW
authors.lastname: Sun
authors.forename: Qi-Hua
authors.initials: QH
authors.lastname: Zhao
authors.forename: Wei-Ping
authors.initials: WP
authors.lastname: Liao
authors.forename: Yong-Hong
authors.initials: YH
authors.lastname: Yi
authors.forename: Yue-Sheng
authors.initials: YS
authors.lastname: Long
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002490:Central Nervous System / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D054461:Lim Kinases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics / Q000502:physiology*; D014162:Transfection / Q000379:methods
chemical_list: D020413:3' Untranslated Regions; D012333:RNA, Messenger; C515892:LIMK1 protein, human; D054461:Lim Kinases; C515893:Limk1 protein, mouse