The Role of miR-103 and miR-107 in Regulation of CDK5R1 Expression and in Cellular Migration

et al. (2011) The Role of miR-103 and miR-107 in Regulation of CDK5R1 Expression and in Cellular Migration. PLoS ONE 6(5): e20038. doi:10.1371/journal.pone.0020038 The Role of miR-103 and miR-107 in Regulation of CDK5R1 Expression and in Cellular Migration Silvia Moncini 0 Alessandro Salvi 0 Paola Zuccotti 0 Gabriella Viero 0 Alessandro Quattrone 0 Sergio 0 Barlati 0 Giuseppina De Petro 0 Marco Venturin 0 Paola Riva 0 Michael Polymenis, Texas A&M University, United States of America 0 1 Department of Biology and Genetics for Medical Sciences, University of Milan , Milan , Italy , 2 Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, University of Brescia , Brescia , Italy , 3 Centre for Integrative Biology (CIBIO), University of Trento , Trento , Italy , 4 Institute of Biophysics, National Research Council , Trento , Italy CDK5R1 encodes p35, a specific activator of the serine/threonine kinase CDK5, which plays crucial roles in CNS development and maintenance. CDK5 activity strongly depends on p35 levels and p35/CDK5 misregulation is deleterious for correct CNS function, suggesting that a tightly controlled regulation of CDK5R1 expression is needed for proper CDK5 activity. Accordingly, CDK5R1 expression was demonstrated to be controlled at both transcriptional and post-transcriptional levels, but a possible regulation through microRNAs (miRNAs) has never been investigated. We predicted, within the large CDK5R1 39UTR several miRNA target sites. Among them, we selected for functional studies miR-103 and miR-107, whose expression has shown a strong inverse correlation with p35 levels in different cell lines. A significant reduction of CDK5R1 mRNA and p35 levels was observed after transfection of SK-N-BE neuroblastoma cells with the miR-103 or miR-107 precursor (pre-miR103 or pre-miR-107). Conversely, p35 levels significantly increased following transfection of the corresponding antagonists (anti-miR-103 or anti-miR-107). Moreover, the level of CDK5R1 transcript shifts from the polysomal to the subpolysomal mRNA fraction after transfection with pre-miR-107 and, conversely, from the subpolysomal to the polysolmal mRNA fraction after transfection with anti-miR-107, suggesting a direct action on translation efficiency. We demonstrate, by means of luciferase assays, that miR-103 and miR-107 are able to directly interact with the CDK5R1 39-UTR, in correspondence of a specific target site. Finally, miR-103 and miR-107 overexpression, as well as CDK5R1 silencing, caused a reduction in SK-N-BE migration ability, indicating that these miRNAs affect neuronal migration by modulating CDK5R1 expression. These findings indicate that miR-103 and miR-107 regulate CDK5R1 expression, allowing us to hypothesize that a miRNA-mediated mechanism may influence CDK5 activity and the associated molecular pathways. - Funding: This study was supported by the Italian Ministry of the University and Research by means of the following grants: Research Project of National Interest (PRIN contract number 2007MWCEAL_003 to PR, http://prin.miur.it/), Italian Found in Basic Research (FIRB contract number RBFR0895DC to MV, http://firb.miur.it/) and by local MIUR funds of the University of Brescia (to GDP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Human CDK5R1 (Cyclin-dependent kinase 5, regulatory subunit 1, GeneID: 8851) encodes p35, a specific activator of Cyclindependent kinase 5 (CDK5, GeneID: 1774), which is a prolinedirected serine/threonine kinase. Monomeric CDK5 shows no enzymatic activity and requires association with p35 or p39 for activation [1]. While Cdk5 is expressed ubiquitously in mammalian tissues, its activator proteins are predominantly expressed in postmitotic neurons: for this reason its activity has mainly been associated with the central nervous system (CNS) development and function [2,3]. The functions of Cdk5 that have been best characterized mainly involve CNS development and maintenance, and are mediated by the phosphorylation of its several substrates [2,3]. The association of p35 with Cdk5 is required for neuronal survival and is involved in many fundamental processes during neuronal differentiation and function such as neurite outgrowth [4], axon regeneration [5], neuronal apoptosis [6], synaptic plasticity, learning and memory [7] and membrane trafficking during the outgrowth of neuronal processes [8], with a role also in secretion and dopamine signalling by controlling cytoskeletal dynamics [9]. Furthermore, Cdk5 and p35 are essential for radial migration and lamination of cortical neurons during the morphogenesis of the mammalian cerebral cortex. Cdk5 knockout mice display severe cortical lamination defects and perinatal death [10]. Similarly, Cdk5r1 KO mice show severe cortical lamination defects and suffer from adult mortality and seizures [11]. Cdk5 hyperactivation, associated to p35 overexpression and production of p25, a proteolytic fragment containing the C-terminal portion of p35 [12], has been implicated in some neurodegenerative disorders, such as Alzheimers disease (OMIM: 104300) [13], Parkinsons disease (OMIM: 168600) [14] and amyotrophic lateral sclerosis (OMIM: 105400) [15]. More recently, CDK5R1 has been indicated as a candidate for mental retardation in the NF1microdeletion syndrome (OMIM: 162200) [16]. The deleterious effects of CDK5 and CDK5R1 dysregulation during both physiological and pathological processes strongly suggest that a precise spatio-temporal regulation of CDK5R1 expression is needed for a proper activation of CDK5. It has been shown that p35 cellular level is the main limiting factor for the CDK5 kinase activity [17], but little is known about the regulation of p35 expression. Some data on the regulation of CDK5R1 transcription have been reported. TNF-a, through activation of the ERK1/2 pathway, regulates Cdk5r1 promoter activity in PC12 cells inducing a sustained and robust expression of Cdk5r1, thereby increasing Cdk5 kinase activity [18]. Moreover, IL-6 was found to affect p35 protein levels in hippocampal neurons, and IFN-c increased p35 protein levels in neuroblastoma Paju cells [19,20]. Some evidence of a regulation of p35 at post-translational level has also been reported: p35 was shown to undergo fast turnover through ubiquitylation and proteasomal degradation [21] and it has been recently reported that PKCd phosphorylates p35, preventing its degradation in cultured cortical neurons and regulating the radial migration of layer II/III cortical neurons by stabilizing p35 [22]. We recently demonstrated that the expression of CDK5R1 can be further modulated at post-transcriptional level by its 39-UTR [23]. 39-UTRs play key roles in post-transcriptional regulatory mechanisms, allowing a finely tuned spatio-temporal control of expre (...truncated)