[
International Worm Meeting,
2017]
Small non-coding RNAs of 20-30 nucleotides can target both chromatin and transcripts in a sequence-specific manner to regulate gene expression. The first small regulatory RNA was discovered in C. elegans in 1993(Lee, Feinbaum, & Ambros, 1993). Recent progress in high-throughput sequencing has uncovered an astounding landscape of small RNAs. Various small RNAs have been found and can be categorized into three main classes according to their mechanism of biogenesis and the Argonaute protein to which they bind: micro RNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and endogenous small interfering RNAs (endo-siRNAs) (Czech and Hannon, 2011). Those small interference RNAs have diverse expression patterns and might regulate various developmental and physiology processes. In this study, we developed a pipeline to analyze siRNA sequence libraries. We used fastX-clipper, fastX-trimmer, then align to the WS253 reference genome using bowtie 2 program, and lastly a custom python script to keep only anti-sense reads. Our analysis across hundreds of libraries from dozen of different laboratories revealed that there is another class of siRNA that mapped to intronic regions of the gene (isiRNA). Though abundant, the function and biogenesis of these isiRNAs have yet to be described in C. elegans. We investigated the characteristics of introns that have isiRNA mapping to them, and we found that the median of these introns is 10-fold longer compared to the median of genomic introns. Through characterizing genes that have isiRNA mapping to them, we discovered that genes with alternative splicing have higher probability of having isiRNA mapping compared to genes without alternative splicing. By analyzing high-throughput sequences of co-immunoprecipation libraries we found that this isiRNA bound specifically to some endo-siRNA Argonautes but not to miRNA Argonautes. My current study is focusing on understanding the biogenesis and function of this newly identified isiRNA. Our study will broaden our understanding of the complex function and biogenesis of small non-coding RNA.
[
International Worm Meeting,
2015]
A significant portion of currently approved drugs target G Protein Coupled Receptors (GPCRs) but most have off-target effects and many of these include dental sequelae. Such side effects can arise when distinct GPCRs in one cell signal through similar second messengers. Thus, the stimulation of one GPCR affects the down-stream signaling pathway and adaptation of the cell's other GPCRs. Indeed, one key question in the field is how different GPCRs, though they activate many of the same second messengers, signal independently without cross adaptation of their signals. Understanding how this signaling is segregated may lead to the ability to develop interventions that limit crosstalk and off-target effects. My overarching goal is to understand how adaptation of GPCR pathways can be independently regulated.This question will be studied in the C. elegans model system, where signaling from multiple GPCRs can be studied in individual olfactory neurons. Our lab recently discovered that an endogenous RNAi (endo-siRNA) pathway promotes adaptation to GPCR signaling by odorants. This observation provides a framework to explain how multiple odors sensed by a single sensory neuron are adapted to independently. This may thus constitute one mechanism by which highly similar signal transduction pathways are insulated from each other in the same cell. This leads me to the hypothesis that the specificity of adaptation to G protein signaling in olfactory neurons is directed by the engagement of different endogenous RNAi biosynthetic pathways acting downstream of olfactory GPCR stimulation. I will test this hypothesis in 3 aims: In aim 1, I will use behavioral assays to identify endogenous RNAi processing factors that act downstream of GPCRs to promote adaptation specifically to either isoamyl alcohol (IAA) or butanone (BU). Preliminary data suggests that the exonuclease ERI-1 is one such factor as it is required for adaptation only to IAA. In aim 2, I will use biochemical and visual means to determine whether ERI-1 is, in fact uniquely required for adaptation to IAA and if so, by what mechanism. In aim 3, I will use high-resolution endo-siRNA sequencing of either IAA or BU adapted populations to determine if specific siRNA species are produced in adaptation to each odor. I will then determine whether any of these sequences or removal of their targets are sufficient to promote IAA adaptation.Together, these results will provide insight into how GPCR signaling can remain separated within the same cell, and how endogenous siRNA mediates this process..