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.
The discovery of a subclass of cytoplasmic RNAs with retained introns and the proof that selected ciRNAs are functional was unexpected. Understanding the in vivo function of ciRNAs requires their identification and characterization in cells that are in contact with other cells in their natural microenvironment. These studies will enhance our knowledge of normal and abnormal neuronal function.
