UC Riverside

Nonsense-mediated RNA decay (NMD) is a cell surveillance system that degrades aberrantly processed RNA transcripts often contain premature termination codons (PTCs). PTCs can arise from various genetic mutations. In addition to serving as a passive fail-safe mechanism, recent studies suggest NMD is also actively involved in alternative splicing (AS), stress responses, neurogenesis, and other biological processes. This dissertation is aimed to further investigate the relationship between NMD and other stress mechanisms.

To overcome some inherent problems of assaying endogenous NMD targets, Chapter 2 presents an easily and broadly applicable AS-NMD reporter assay to quantify cellular NMD activity, in real time. This new strategy reliably distinguishes NMD regulation from transcriptional control, AS regulation, and discloses a different sensitivity of NMD targets to NMD inhibition. It is then used to screen molecules for NMD inhibitors including thapsigargin in neuro-2A cells.

Chapter 3 shows stresses affect NMD differently and the mechanistic discovery of NMD inhibition upon thapsigargin treatment. The endoplasmic reticulum (ER) stress and polysome disassembly caused by thapsigargin suggest that an activated unfolded protein response (UPR) pathway by protein kinase RNA-like endoplasmic reticulum kinase (PERK) is required for NMD inhibition, instead of calcium signaling pathways. Such stress induced NMD inhibition can compound TDP-43 depletion proteinopathy via upregulating NMD isoforms that had been implicated in the pathogenic mechanisms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) which could be completely blocked by PERK deactivation.

In Chapter 4, a pioneer in vitro conditional NMD deficient neural progenitor cell (NPC) system is created using adeno-associated virus serotype 9 (AAV9). Significant loss in NPC viability is observed during UPF2 knockout. Candidate genes, Gadd45b and Gadd45g, knockout using CRISPR-Cas9 failed to rescue NPC survival. This finding suggests NMD activity is essential for cell viability, likely maintaining the homeostasis of NPC transcriptome during renewal processes.

In summary, AS-NMD strategy screens and reveals the pathway of NMD inhibition by ER stress and the implicated ALS proteinopathy. More importantly, the research highlights NMD interaction with stress and cell survival, especially in NPCs, that could provide future therapeutic approaches toward related genetic diseases.