Signaling and microRNA function in neurons

描述（由申请人提供）：microRNAs对mrna的转录后调控是动物基因调控中广泛而重要的组成部分。microrna与Argonaute （Ago）蛋白结合，结合mrna中的特定序列，抑制翻译和/或降低目标mrna的水平。单个microrna可以调控许多不同的基因，而单个mrna可以被多个microrna靶向。哺乳动物中枢神经系统（CNS）中表达大量microrna，并且microrna参与中枢神经系统中多种过程的调控，包括神经发生、细胞存活和死亡、细胞命运决定、神经元分化、轴突生长和突触功能。信号转导在中枢神经系统的发育和功能中起着重要的作用。细胞内激酶级联反应由细胞外信号激活，直接调节大多数生物过程，包括转录和翻译。然而，目前尚不清楚Ago/microRNA复合物对靶mrna的抑制是否可以通过信号通路直接调节。p38 MAP激酶/MAPKAP激酶2 （MAPKAP- k2）通路对Ago-2蛋白的磷酸化改变了其亚细胞定位，p38激酶/MAPKAP - k2信号通路调节mRNA的稳定性和翻译，以及其他生物过程。p38/MAPKAP-K2对Ago/microRNA复合物和相关蛋白的磷酸化可促进或阻止抑制复合物在特定mrna上的组装，从而调节microRNA介导的对神经元中特定靶标亚群的抑制。我们建议使用最近开发的生化纯化和与Ago-2蛋白相关的mRNA片段的高通量测序方法来鉴定通过激活或抑制初级小脑颗粒神经元p38通路调节Ago-2/microRNA结合的microRNA靶点。我们将进一步表征所选择的表现出调节microRNA结合的目标mrna。信号通路对microRNA结合和抑制的调节将显著扩大microRNA可调节的生物过程的范围，也可能解释为什么一些预测的microRNA靶点未能介导抑制。microRNA和p38信号通路都与多种神经系统疾病有关，p38在细胞应激和/或凋亡反应中起关键作用，这些生理过程也与microRNA功能相关。证明在中枢神经系统中，microRNA的功能可以通过信号快速调节，而不依赖于microRNA的表达水平，这将影响我们对神经系统中microRNA生物学的理解，以及microRNA和p38信号在神经系统疾病中的作用。

DESCRIPTION (provided by applicant): Post-transcriptional regulation of mRNAs by microRNAs is a widespread and essential component of gene regulation in animals. microRNAs, in association with Argonaute (Ago) proteins, bind to specific sequences in mRNAs to repress translation and/or reduce the level of the target mRNAs. Individual microRNAs may regulate many different genes, while individual mRNAs can be targeted by multiple microRNAs. Numerous microRNAs are expressed in the mammalian central nervous system (CNS), and microRNAs have been implicated in the regulation of a variety of processes in the CNS, including neurogenesis, cell survival and death, cell fate determination, neuronal differentiation, axon outgrowth, and synaptic function. Signal transduction plays essential roles in CNS development and function. Intracellular kinase cascades, activated by extracellular signals, directly modulate most biological processes, including transcription and translation. However, it is not known whether repression of target mRNAs by Ago/microRNA complexes can be modulated directly by signaling pathways. Phosphorylation of the Ago-2 protein by the p38 MAP kinase/MAPKAP kinase 2 (MAPKAP-K2) pathway alters its subcellular localization, and p38 kinase/ MAPKAP-K2 signaling is known to regulate mRNA stability and translation, as well as other biological processes. Phosphorylation of the Ago/microRNA complex and associated proteins by p38/MAPKAP-K2 may promote or prevent assembly of repressive complexes on specific mRNAs, leading to the modulation of microRNA-mediated repression on specific subsets of targets in neurons. We propose to use a recently developed method for biochemical purification and high-throughput sequencing of mRNA fragments associated with the Ago-2 protein to identify microRNA target sites at which Ago-2/microRNA binding is modulated by activation or inhibition of the p38 pathway in primary cerebellar granule neurons. We will further characterize selected target mRNAs that exhibit modulated microRNA binding. Modulation of microRNA binding and repression by signaling pathways would significantly expand the scope of biological processes that could be regulated by microRNAs, and may also explain why some predicted microRNA target sites fail to mediate repression. Both microRNAs and the p38 signaling pathway have been implicated in multiple diseases of the nervous system, and p38 plays critical roles during responses to cellular stress and/or apoptosis, physiological processes that are also linked to microRNA functions. Demonstrating that microRNA function can be rapidly modulated in the CNS by signaling, independent of microRNA expression levels, would impact both our understanding of microRNA biology in the nervous system, and of the role of microRNAs and p38 signaling in neurological disease. PUBLIC HEALTH RELEVANCE: Neurological diseases are serious human health problems that affect numerous individuals. The research in this proposal is directed at understanding genetic regulatory mechanisms in nerve cells. The proposed studies will determine whether two distinct mechanisms interact to control genes in nerve cells. This information should contribute to our knowledge of neural development, physiology, and cellular function, as well as our understanding of neurological diseases, and it should help to provide a basis for the development of rational interventions for treating or preventing neurological diseases.