Abstract
When translating mRNAs are cleaved in protein-coding regions, 5' fragments of mRNAs are detached from stop codons (i.e., nonstop mRNAs) and protected from 3'-5' exonucleases by ribosomes stalled at the 3' termini. It has been shown in yeast that the nonstop mRNA decay (NSD) machinery triggers nonstop mRNA degradation by removing stalled ribosomes in the artificial reporter mRNAs. However, it is not known well whether NSD is involved in the degradation of endogenous nonstop mRNAs in higher eukaryotes. In this work, we addressed the question of whether 5'-nonstop-mRNA fragments generated by siRNA cleavage or nonsense-mediated-mRNA decay (NMD) are degraded by the NSD pathway in Drosophila melanogaster cells by knocking down three NSD components, Pelota (a yeast Dom34 homolog), Hbs1 and ABCE1 (a ribosome-recycling factor). We found that double, but not single, knockdown of any two of these three factors efficiently stabilized nonstop reporter mRNAs and triple knockdown of Pelota, Hbs1 and ABCE1 further stabilized nonstop mRNAs in highly ribosome-associated state. These findings demonstrated that Pelota, Hbs1 and ABCE1 are crucial for NSD in Drosophila cells as in yeast for rescuing stalled ribosomes and degrading nonstop mRNAs. To our knowledge, this is the first comprehensive report to show the involvement of the NSD machinery in the clearance of mRNA 5'-fragments produced by RNAi and NMD in eukaryotes.
