UC San Diego

Nonsense-Mediated Decay (NMD) is an mRNA quality control process that targets mRNAs containing premature termination codons (PTCs) for degradation and therefore prevents expression of potentially deleterious truncated proteins. The recognition of PTC is a quite complex process and is known to be influenced by the size of the 3' untranslated region (3'UTR). Hence, mRNAs with long artificial 3'UTRs are usually targeted for NMD. However, many physiological mRNAs with long 3'UTRs seem to be resistant to NMD. The focus of this investigation is to determine how these mRNAs with long 3' UTRs evade NMD. In this study, we used plasmids expressing mRNA reporters containing the long 3'UTR of selected mRNA candidates under the control of a tetracycline-dependent promoter. After transfection in mammalian cells, the stability of these mRNAs was assessed by transcriptional induction followed by rapid transcription shutoff using tetracycline. Cells were harvested at different time after transcriptional shutoff and mRNA levels were quantified by Northern Blot to determine mRNA half-lives. We identified 3'UTRs longer than 1000 nucleotides that evade NMD. We performed extensive deletion analyses to narrow down elements responsible for NMD evasion. We demonstrated that several candidates contain cis elements that are sufficient to inhibit NMD and are located within the first 200 nucleotides downstream of the PTC. Further studies are necessary to decipher the mechanism by which these cis elements promote NMD evasion. Considering the crucial role of NMD in mRNA surveillance as well as gene expression, uncovering the mechanisms that allow mRNAs to evade NMD is essential to better understand NMD regulation