Poster abstracts

Poster number 132 submitted by Leanne Kelley

Defining the mechanism and spatiotemporal impacts of a DEAD-box helicase on RNA editing

Leanne H. Kelley (Department of Biology, Indiana University), Heather A. Hundley (Department of Biology, Indiana University)

Abstract:
RNA binding proteins (RBPs) are crucial for proper gene expression in all organisms and the coordinated roles of these RBPs facilitate complex regulatory mechanisms. The RNA editing enzyme, ADAR, is responsible for the deamination of adenosine (A) to inosine (I) on doubled-stranded regions of mRNA. RNA editing introduces transcript diversity to the genome and ADARs can influence alternative splicing patterns, recode genetic information in coding regions, and modify miRNA binding sites. However, ADAR levels themselves are often not altered, suggesting that other regulators of RNA editing are critical to understanding RNA editing. In particular, emerging evidence shows that DEAD-box RNA helicases influence editing, but the underlying mechanism(s) remains unknown.
In Caenorhabditis elegans, ADR-2 is the sole A-to-I editing enzyme. Via immunoprecipitation and mass spectrometry experiments, our lab found that ADR-2 interacts with the DEAD-box helicase, GLH-1. In a pilot RNA-seq experiment with a glh-1 mutant, I found that 40% of the identified edit sites were altered in editing frequency upon the loss of GLH-1, demonstrating that GLH-1 is a novel regulator of editing. Interestingly, some sites were positively regulated by GLH-1, while others were negatively regulated, suggesting that GLH-1 may carry out different activities to influence editing. To ascertain the mechanism behind GLH-1-mediated editing regulation, I am developing the STAMP (Surveying Targets by APOBEC1-Mediated Profiling) method in C. elegans, which, combined with long-read sequencing, will identify GLH-1 and ADR-2 binding sites on a single RNA molecule. Future experiments will utilize GLH-1 domain mutants and STAMP methodology to narrow to the specific activity of GLH-1 that is responsible for editing regulation.

Keywords: RNA editing, RNA binding, helicase