Mechanistic aspects of nonsense-mediated mRNA decay in human cells

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological RNAs that encode full-length proteins. NMD results from improper translation termination at stop codons, and thus, it is a cytoplasmic and translation-dependent process. In human cells, mRNA decay inherent to NMD involves an endonucleolytic cleavage near the stop codon and exonucleolytic degradation from both 5’ and 3’ ends. This is done by a process not yet completely understood that recruits decapping and 5’-to-3’ exonuclease activities, as well as deadenylating and 3’-to-5’ exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. However, DIS3L2 exoribonuclease activity is independent of the exosome. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, however little is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, we performed its knockdown in HeLa cells and measured the mRNA levels of various natural NMD-targets. Our results show that some NMD-targets accumulate in DIS3L2-depleted cells. In addition, mRNA half-life analysis indicated that these NMD-targets are direct DIS3L2 substrates. Besides, we observed that DIS3L2 acts over full-length transcripts, being DIS3L2-mediated decay dependent on the activity of the terminal uridylyl transferases (TUTases) Zcchc6/11 (TUT7/4). Together, our findings establish the role of DIS3L2 and uridylation over human NMD-targets.