Poster abstracts
Poster number 94 submitted by Brittany Stawicki
A Transcriptome Wide Study to Elucidate co-Translational and Translation-independent mRNA Decay Kinetics
Brittany Stawicki (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH), Eckhard Jankowsky (Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH)
Abstract:
Regulation of eukaryotic gene expression is controlled by changes in mRNA levels. It is known that mRNAs can be degraded co-translationally as well as independent of translation, but it is unclear whether and how these decay pathways are balanced. Current approaches to measure mRNA decay are unable to distinguish between translation-dependent and independent degradation. Therefore, we set out to directly measure co-translational and translation-independent mRNA decay kinetics on a transcriptome-wide scale. We perform SLAMseq 4-thiouridine (4sU) labeling of human HCT116 cells under pulse chase conditions, followed by Quantseq analysis of mRNA 3’ends to track metabolically labeled RNA populations over time in the overall and translating mRNA pool. This approach overcomes limitations from current metabolic labeling procedures; including high label concentrations, long labeling times, and bias introduced by affinity pulldown. While optimizing 4sU labeling and pulse-chase conditions, we noted that commonly used labeling conditions are associated with adverse effects on translation, likely distorting present decay kinetics. Our optimized conditions minimize the effects of metabolic labeling and pulse-chase on translation. We are currently sequencing and analyzing obtained NGS libraries. We anticipate our approach measuring co-translational and translation-independent mRNA decay rates to provide kinetic parameters for key stages in mRNA metabolism, building on existing data. Our approach will supply quantitative insights into the regulation of RNA metabolism and aid in establishing a foundation for systematic analyses of diseases associated with changes in RNA metabolism.
Keywords: RNA Metabolism, Decay