A high-throughput screen for sequences mediating sensitivity to nonsense mediated decay (NMD)

The error-prone flow of information from DNA through RNA to protein necessitates quality-control mechanisms to ensure data integrity. One such mechanism is the eukaryotic nonsense-mediated mRNA decay (NMD) pathway which monitors translated transcripts for premature termination events, and once such events are identified, triggers rapid mRNA decay, thereby limiting production of potentially harmful truncated proteins. The canonical model of NMD states that premature termination codons (PTC) found at least 50 bases upstream of the last EJC induce an NMD response that degrades the transcript. Elucidating the code of the sequences that trigger NMD has so far primarily relied on minigene splicing systems using NMD-sensitive genes and few PTC-bearing variants. To understand with greater resolution what sensitizes a transcript to NMD, and how NMD sensitivity interfaces with other gene features, such as RNA modifications, we have designed a library of DNA oligos with varying features to be tested in conditions of varying NMD responsiveness. This library contains over a thousand sequence variants with variable distances between the stop codon and an intron, as well as variable sequence contexts of the stop codon, and codon usage of the translated sequence. By measuring the expression of the reporter in conditions where NMD activity is active or compromised, we aim to home in on the key features that underlie NMD sensitivity.