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Neuronal ciRNA characterization and impact upon channel functioning

神经元 ciRNA 特征及其对通道功能的影响

基本信息

批准号：
9892047
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
项目类别：
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/9892047
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 single-cell RNA sequencing transcription factor transcriptome voltage clamp

项目摘要

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，因为进化上保守的过程信使核糖核酸剪接通过交替的信使核糖核酸剪接产生信使核糖核酸和蛋白质的多样性。事实上，它已经已经证实，90%的哺乳动物基因是交替剪接的。丰富的自然资源选择性剪接形式变化很大，但其中很大一部分(~85%)是选择性剪接 RNA存在于该特定基因的mRNA转录物群体的5%-15%的范围内。生物学的选择性剪接的mRNAs的作用是不同的，例如不同剪接形式的通道和受体产生差异反应蛋白，剪接的钙粘蛋白RNA促进特定的细胞-细胞单个转录因子的相互作用和不同的剪接形式调节着不同的基因集。使用这种分子多样性的例子，已经有了更多的努力来表征额外的剪接导致最近发现三种不同类型的选择性剪接RNA的事件包括 1)环状RNA，2)退出子和3)可选择性剪接的RNA的复杂群体，包括保留内含子(CiRNA)，通过使用高度敏感的基因在细胞质中鉴定分离的神经元树突RNA群体的NextGen测序。这最后一类RNA是这项提案的主题。大量ciRNA的发现是意想不到的，但导致了假设它们可能在这里发挥未知的生物功能。一个ciRNA的例子，它提供了对这类RNAs的功能的洞察，这类RNAs是BKca mRNA的一部分人口。初步证据表明ciRNA在BK通道功能中起生理学作用但对其中涉及的内在机制以及是否有多个ciRNA拥有特定RNA的不同保留内含子具有相似或不同的功能。健壮的生物这种从分散培养的神经元中分离出来的ciRNA的影响突出了识别和表征细胞在其天然组织微环境中的ciRNA，以探索它们如何调节细胞的自然生理反应。我们建议对这些事件进行现场调查利用我们新开发的活体分析转录组(TIVA)从个体中分离RNA 活的小鼠脑片上有树突。树突定位的ciRNA的同一性(包括去极化诱导的cRNAs)将由单细胞RNAseq确定。第二个目标是开始通过对ciRNA的表达和测量功能的操控，剖析了ciRNA的作用机制(S)。虽然我们希望在这个项目的过程中发现新的ciRNA，但ciRNA编码通道是最容易被检查的职能角色之一，并提供了职能角色的起点对这类新的RNA的评估。