Neuronal ciRNA characterization and impact upon channel functioning

神经元 ciRNA 特征及其对通道功能的影响

• 批准号: 9306949
• 负责人: JAMES H EBERWINE
• 金额: $ 64.27万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9306949
• 关键词: Alternative Splicing; Biological; Biological Assay; Biological Process; Brain; Bypass; Cadherins; Cell Communication; Cell Culture Techniques; Cells; Complement; Complex; Consensus; Cytoplasm; Dendrites; Dominant-Negative Mutation; Event; Foundations; Frequencies; Genes; Goals; Hippocampus (Brain); In Situ; Individual; Introns; Knowledge; Measures; Mediating; Messenger RNA; Molecular; Mouse Strains; Mus; Neurons; Nonsense-Mediated Decay; Pathway interactions; Pattern; Physiological; Population; Preparation; Process; Property; Protein Splicing; Proteins; RNA; RNA Interference; RNA Sequences; RNA Splicing; RNA-Binding Proteins; Regulation; Reporter; Reporting; Role; Slice; Small Interfering RNA; System; Tissues; Titrations; Transcript; Transfection; base; circular RNA; in vivo; insight; large-conductance calcium-activated potassium channels; mRNA Surveillance; neuronal cell body; next generation sequencing; novel; receptor; transcription factor; transcriptome; transcriptome sequencing; voltage clamp

The cellular requirement for mRNA diversity is apparent, as the evolutionarily conserved process of mRNA splicing generates mRNA and protein diversity through alternative mRNA splicing. Indeed it has been established that >90% of mammalian genes are alternatively spliced. The abundance of the alternatively spliced forms varies extensively, but a large fraction (~85%) of these alternatively spliced RNAs exist in the range of 5-15% of that particular gene's mRNA transcript population. The biological roles of alternatively spliced mRNAs are varied for example different spliced forms of channels and receptors give rise to differentially responsive proteins, spliced cadherin RNAs facilitate specific cell-cell interactions and distinct splice forms of individual transcription factors modulate distinct gene sets. With such examples of molecular diversity, there has been increased effort to characterize additional splicing events resulting in the recent discovery of three different types of alternatively spliced RNAs including 1) circular RNAs, 2) exitrons and 3) a complex population of alternatively spliced RNAs containing retained introns (ciRNAs) that was identified in the cytoplasm of cells through the use of highly sensitive NextGen sequencing on isolated neuronal dendrite RNA populations. This last class of RNAs is the topic of this proposal. The discovery of a large population of ciRNAs was unexpected, yet led to the hypothesis that they may exert a here to for unknown biological function. An example of a ciRNA that provides insight into functionality of this class of RNAs is one that comprises part of BKCa mRNA population. Preliminary evidence suggests a physiological role for the ciRNA in BK channel functioning but little is known about the intrinsic mechanisms involved and whether multiple ciRNAs that possess different retained introns for a particular RNA exert similar or distinct functions. The robust biological impact of this ciRNA isolated from dispersed cultured neurons highlights the need to identify and characterize the ciRNAs from cells in their native tissue microenvironment to explore how they may regulate the cells' natural physiological responsiveness. We propose to investigate these events in situ using our newly developed Transcriptome In Vivo Analysis (TIVA) to isolate RNA from individual dendrites resident in the live mouse brain slice. The identity of dendritically localized ciRNAs (including depolarization induced ciRNAs) will be determined by single cell RNAseq. A second goal is to start to dissect the mechanism(s) of action of ciRNAs by manipulating their expression and measuring function. While we expect to discover new ciRNAs in the course of this project, the ciRNAs encoding channels are among the most easily examined for a functional role and provide a starting point for functional assessments of this novel class of RNAs.

MRNA多样性的细胞需求是显而易见的，因为 mRNA剪接通过替代mRNA剪接产生mRNA和蛋白质多样性。确实有 已经确定> 90％的哺乳动物基因是剪接的。丰富的 或者，剪接形式变化很大，但其中很大一部分（约85％）替代地剪接 RNA存在于该特定基因mRNA转录群的5-15％的范围内。生物学 剪接的mRNA的作用有所不同 受体产生差异响应蛋白质，剪接的钙粘蛋白RNA促进特定细胞细胞 相互作用和单个转录因子的不同剪接形式调节不同的基因集。和 这种分子多样性的例子，越来越多的努力来表征其他剪接 导致最近发现三种不同类型的剪接RNA的事件，包括 1）圆形RNA，2）出口和3）含有剪接的RNA的复杂种群 保留的内含子（CIRNA）通过使用高度敏感的 对分离的神经元树突RNA种群进行NextGen测序。最后一类RNA是 该提议的主题。发现大量CIRNA是出乎意料的，但导致了 假设它们可能会在此处施加一个未知的生物学功能。一个cirna的例子 提供有关此类RNA功能的洞察力，它包括BKCA mRNA的一部分 人口。初步证据表明CIRNA在BK通道功能 但是，对所涉及的内在机制以及是否拥有多种CIRNA知之甚少 特定RNA的不同保留内含子具有相似或不同的功能。强大的生物学 从分散的培养神经元中分离出的ciRNA的影响强调了识别和 表征来自天然组织微环境中细胞的CIRNA，以探索它们如何 调节细胞的天然生理反应能力。我们建议原位调查这些事件 使用我们新开发的转录组在体内分析（TIVA）与单个分离RNA 树突居住在活小鼠脑切片中。树枝状局部CIRNA的身份（包括 去极化诱导的CIRNA）将通过单细胞RNASEQ确定。第二个目标是开始 通过操纵其表达和测量功能来剖定CIRNA的作用机制。 虽然我们希望在该项目过程中发现新的CIRNA，但CIRNA编码渠道 是最轻松的功能角色之一，并为功能提供了起点 评估这一新型RNA。