A faux 3′-UTR promotes aberrant termination and triggers nonsense- mediated mRNA decay

Abstract

Nonsense-mediated messenger RNA decay (NMD) is triggered by premature translation termination^1,2,3, but the features distinguishing premature from normal termination are unknown. One model for NMD suggests that decay-inducing factors bound to mRNAs during early processing events are routinely removed by elongating ribosomes but remain associated with mRNAs when termination is premature, triggering rapid turnover⁴. Recent experiments^5,6,7 challenge this notion and suggest a model that posits that mRNA decay is activated by the intrinsically aberrant nature of premature termination^8,9. Here we use a primer extension inhibition (toeprinting) assay¹⁰ to delineate ribosome positioning and find that premature translation termination in yeast extracts is indeed aberrant. Ribosomes encountering premature UAA or UGA codons in the CAN1 mRNA fail to release and, instead, migrate to upstream AUGs. This anomaly depends on prior nonsense codon recognition and is eliminated in extracts derived from cells lacking the principal NMD factor, Upf1p, or by flanking the nonsense codon with a normal 3′-untranslated region (UTR). Tethered poly(A)-binding protein (Pab1p), used as a mimic of a normal 3′-UTR, recruits the termination factor Sup35p (eRF3) and stabilizes nonsense-containing mRNAs. These findings indicate that efficient termination and mRNA stability are dependent on a properly configured 3′-UTR.