Poster Presentation 39th Annual Lorne Genome Conference 2018

VISUALISING THE NONSENSE: Fluorescent quantitation of the nonsense mediated mRNA decay pathway during brain development (#141)

Deepti Domingo 1 , Jozef Gecz 1 2 3 , David Bersten 1 , Murray Whitelaw 1 , Miles Wilkinson 4 , Lachlan Jolly 1 2
  1. The University of Adelaide, Adelaide, SA, Australia
  2. Robinson Research Institute, Adelaide, SA, Australia
  3. South Australian Health and Medical Research Institute, Adelaide, SA, Australia
  4. The University of California, San Diego, California, America

The nonsense mediated mRNA decay pathway (NMD) is widely known as an RNA surveillance system, and more recently, as a global regulator of the transcriptome essential for embryonic viability. During brain development NMD plays an imperative role. Genetic variants in several key components of the NMD machinery which compromise NMD activity either cause, or are implicated in neurodevelopmental disorders. Evidence suggests that NMD activity is itself dynamically regulated, and shows variability at the cellular, tissue and whole organism level. Yet, current methods to quantify NMD activity are limited to biochemical end-point assays reporting on large populations of cultured cells, and as such, fail to capture the dynamic and heterogenous nature of the pathway at single cell level, in-vivo or in real time. To overcome these limitations, we have engineered a novel fluorescence based NMD reporter transgene. The transgene is comprised of three elements, namely the selection, control and NMD responsive expression cassettes. The control and NMD responsive cassette co-express distinguishable nuclear localised fluorescent proteins, with the ratio of expression (control: NMD responsive) providing visual and quantitative real-time readout of NMD activity. In addition, the transgene is conducive to standard protein and RNA based quantitative analysis. Using these methods we have shown our NMD reporter transgene to be responsive to NMD inhibition in vitro. The selection cassette utilises Flp/Frt recombination features to permit transgene insertion into the Col1A1 locus of germ-line competent mouse ES cells, and thus the creation of NMD reporter mouse lines. This technology can provide real-time visual and quantitative tracking of endogenous NMD activity at a single cell resolution during embryonic development and into postnatal life, and will benefit our understanding of how NMD is regulated, and the biological processes in which it is employed, particularly relevant for our understanding of its role during brain development.