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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：
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
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/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

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，因为在进化上保守的过程