Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
J Neurosci. 2012 Aug 15;32(33):11164-75. doi: 10.1523/JNEUROSCI.2247-12.2012.
The RBP (RNA-binding protein) and Hu/ELAV family member HuD regulates mRNA metabolism of genes directly or indirectly involved in neuronal differentiation, learning and memory, and several neurological diseases. Given the important functions of HuD in a variety of processes, we set out to determine the mechanisms that promote HuD mRNA expression in neurons using a mouse model. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in developing neurons. Bioinformatic and 5'RACE (rapid amplification of cDNA ends) analyses of the 5' genomic flanking region identified eight conserved HuD leader exons (E1s), two of which are novel. Expression of all E1 variants was determined in mouse embryonic (E14.5) and adult brains. Sequential deletion of the 5' regulatory region upstream of the predominantly expressed E1c variant revealed a well conserved 400 bp DNA region that contains five E-boxes and is capable of directing HuD expression specifically in neurons. Using EMSA (electrophoretic mobility shift assay), ChIP (chromatin immunoprecipitation), and 5' regulatory region deletion and mutation analysis, we found that two of these E-boxes are targets of Neurogenin 2 (Ngn2) and that this mechanism is important for HuD mRNA induction. Together, our findings reveal that transcriptional regulation of HuD involves the use of alternate leader exons and Ngn2 mediates neuron-specific mRNA expression. To our knowledge, this is the first study to identify molecular events that positively regulate HuD mRNA expression.
RBP（RNA 结合蛋白）和 Hu/ELAV 家族成员 HuD 调节神经元分化、学习和记忆以及几种神经疾病中直接或间接涉及的基因的 mRNA 代谢。鉴于 HuD 在各种过程中的重要功能，我们着手使用小鼠模型确定促进神经元中 HuD mRNA 表达的机制。通过几种互补的方法，我们确定 HuD mRNA 的丰度主要受发育中神经元中转录控制。对 5' 基因组侧翼区的生物信息学和 5'RACE（快速扩增 cDNA 末端）分析鉴定了八个保守的 HuD 前导外显子（E1s），其中两个是新的。在小鼠胚胎（E14.5）和成年大脑中确定了所有 E1 变体的表达。在主要表达的 E1c 变体上游的 5' 调控区进行连续缺失，揭示了一个保守的 400bp DNA 区域，该区域包含五个 E 盒，能够特异性地在神经元中指导 HuD 表达。通过 EMSA（电泳迁移率变动分析）、ChIP（染色质免疫沉淀）以及 5' 调控区缺失和突变分析，我们发现这两个 E 盒是 Neurogenin 2（Ngn2）的靶标，并且该机制对于 HuD mRNA 诱导很重要。总之，我们的研究结果表明，HuD 的转录调控涉及使用替代的前导外显子，并且 Ngn2 介导神经元特异性 mRNA 表达。据我们所知，这是第一项确定正向调节 HuD mRNA 表达的分子事件的研究。
