Dom34-Hbs1 plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). Here, we report a conserved function of human Pelota (hPelota) in mRNA quality control of nonstop mRNA. hPelota facilitated the expression of the nonstop products from GFP-Rz mRNA, which lacks a termination codon and a poly(A) tail, in exosome-defective mutant cells. hPelota promoted the dissociation of stalled ribosomes at the 3' end of GFP-Rz mRNA, and mutations in domain A diminished this activity. The hPelota-R45A mutant associated with ribosomes but was defective in peptide release. Finally, hPelota promoted the degradation of GFP-Rz mRNA and suppressed the sequential endonucleolytic cleavages caused by stalled ribosomes at the 3' end of mRNA in dom34∆ mutant cells.
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