Division of Neuroscience, San Raffaele Rett Research Center, San Raffaele Scientific Institute, Milan 20132, Italy.
Hum Mol Genet. 2009 Dec 1;18(23):4590-602. doi: 10.1093/hmg/ddp426. Epub 2009 Sep 9.
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.
人类 X 连锁细胞周期依赖性激酶样 5（CDKL5）基因突变已被证实可导致严重的神经发育障碍，包括婴儿痉挛、脑病、West 综合征和雷特综合征的早发型变异型。CDKL5 是一种丝氨酸/苏氨酸激酶，其在雷特综合征中的作用可以通过其直接结合并介导 MeCP2 的磷酸化来推断。然而，CDKL5 如何发挥其功能仍有待阐明。在这里，我们报告称 CDKL5 定位于细胞系和组织中称为核斑点的特定核焦点。这些亚核结构传统上被认为是 pre-mRNA 剪接因子的储存/修饰位点。有趣的是，我们提供了证据表明 CDKL5 调节核斑点的动态行为。事实上，CDKL5 的过表达导致核斑点解体，这一事件严格依赖于其激酶活性。相反，其下调会影响核斑点形态，导致异常大和不均匀的斑点。从 CDKL5 突变患者分离的原代成人成纤维细胞也得到了类似的结果。总之，这些发现表明 CDKL5 通过调节剪接调节蛋白的磷酸化状态来控制核斑点形态。核斑点是动态的位点，可以不断向活跃的转录位点供应剪接因子，从而发生剪接。值得注意的是，我们证明 CDKL5 影响选择性剪接，至少在异源 minigene 测定中证明了这一点。总之，我们提供的证据表明，CDKL5 通过控制剪接因子的动力学，间接地参与了前体 mRNA 的加工。这些发现确定了一种生物过程，其失调可能会影响 CDKL5 相关疾病中神经元的成熟和活性。
