Human Molecular Genetics Advance Access originally published online on September 9, 2009
Human Molecular Genetics 2009 18(23):4590-4602; doi:10.1093/hmg/ddp426
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CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery
1 Division of Neuroscience, San Raffaele Rett Research Center and 2 Stem Cells and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan 20132, Italy, 3 Department of Structural and Functional Biology, University of Insubria, Busto Arsizio, VA, Italy, 4 Laboratoire de Génétique et de Physiopathologie des Maladies Neuro-développementales, Université Paris Descartes, Institut Cochin, CNRS (UMR 8104), Inserm U567, 24 rue du Faubourg Saint Jacques, Paris 75014, France and 5 Service de Génétique Médicale, AP-HP, Hôpital Pitié-Salpétrière, 47-83, Boulevard de l'Hôpital 75651, Paris Cedex 13, France
* To whom correspondence should be addressed at: Stem Cells and Neurogenesis Unit, Division of Neuroscience, San Raffaele Scientific Institute, Via Olgettina 58, Milan 20132, Italy. Tel: +39 0226434616; Fax: +39 0226434621; Email: broccoli.vania{at}hsr.it
Received July 1, 2009; Accepted September 3, 2009
Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause severe neurodevelopmental disorders including infantile spasms, encephalopathy, West-syndrome and an early-onset variant of Rett syndrome. CDKL5 is a serine/threonine kinase whose involvement in Rett syndrome can be inferred by its ability to directly bind and mediate phosphorylation of MeCP2. However, it remains to be elucidated how CDKL5 exerts its function. Here, we report that CDKL5 localizes to specific nuclear foci referred to as nuclear speckles in both cell lines and tissues. These sub-nuclear structures are traditionally considered as storage/modification sites of pre-mRNA splicing factors. Interestingly, we provide evidence that CDKL5 regulates the dynamic behaviour of nuclear speckles. Indeed, CDKL5 overexpression leads to nuclear speckle disassembly, and this event is strictly dependent on its kinase activity. Conversely, its down-regulation affects nuclear speckle morphology leading to abnormally large and uneven speckles. Similar results were obtained for primary adult fibroblasts isolated from CDKL5-mutated patients. Altogether, these findings indicate that CDKL5 controls nuclear speckle morphology probably by regulating the phosphorylation state of splicing regulatory proteins. Nuclear speckles are dynamic sites that can continuously supply splicing factors to active transcription sites, where splicing occurs. Notably, we proved that CDKL5 influences alternative splicing, at least as proved in heterologous minigene assays. In conclusion, we provide evidence that CDKL5 is involved indirectly in pre-mRNA processing, by controlling splicing factor dynamics. These findings identify a biological process whose disregulation might affect neuronal maturation and activity in CDKL5-related disorders.