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: 9196471
• 负责人: JAMES H EBERWINE
• 金额: $ 67.73万
• 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/9196471
• 关键词: Biological; Biological Assay; Biological Process; Brain; Bypass; Cadherins; Cell Communication; Cell Culture Techniques; Cells; Complement; Complex; Consensus; Cytoplasm; Cytoplasmic Protein; Dendrites; Dominant-Negative Mutation; Event; Foundations; Frequencies; Genes; Goals; Hippocampus (Brain); In Situ; Individual; Introns; Knowledge; Life; 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.

细胞对mRNA多样性的需求是显而易见的，因为进化上保守的mRNA合成过程， mRNA剪接通过选择性mRNA剪接产生mRNA和蛋白质多样性。且是已经 已经确定，>90%的哺乳动物基因是选择性剪接的。的丰度 选择性剪接形式变化很大，但这些选择性剪接形式中的大部分（约85%） RNA存在于该特定基因的mRNA转录物群体的5-15%的范围内。生物 可变剪接的mRNA的作用是不同的，例如不同剪接形式的通道， 受体产生差异反应蛋白，剪接的钙粘蛋白RNA促进特异性细胞-细胞 单个转录因子的相互作用和不同的剪接形式调节不同的基因组。与 这些分子多样性的例子，已经有越来越多的努力来表征额外的剪接 最近发现了三种不同类型的选择性剪接RNA， 1)环状RNA，2）激子和3）复杂的可变剪接RNA群体， 通过使用高度敏感的技术在细胞质中识别出保留内含子（ciRNA） 对分离的神经元树突RNA群体进行NextGen测序。最后一类RNA是 这个提案的主题。大量ciRNA的发现是出乎意料的，但却导致了 假设它们可能发挥一种未知的生物学功能。一个ciRNA的例子， 提供了对这类RNA功能的深入了解，是一种包含BKCa mRNA部分的RNA 人口初步证据表明ciRNA在BK通道功能中的生理作用 但对于所涉及的内在机制以及是否有多个ciRNA， 特定RNA的不同保留内含子发挥相似或不同的功能。强大的生物 这种从分散的培养神经元中分离的ciRNA的影响突出了识别和 表征来自天然组织微环境中细胞的ciRNA，以探索它们如何 调节细胞的自然生理反应。我们建议在现场调查这些事件 使用我们新开发的体内转录组分析（TIVA）从个体中分离RNA， 存活小鼠脑切片中的树突。树突状定位的ciRNA的身份（包括 去极化诱导的ciRNA）将通过单细胞RNAseq测定。第二个目标是开始 通过操纵它们的表达和测量功能来剖析ciRNA的作用机制。 虽然我们希望在这个项目的过程中发现新的ciRNA，但ciRNA编码通道 是最容易检查的功能性角色之一，并为功能性 对这类新型RNA的评估。