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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 termination1,2,3, 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 experiments5,6,7 challenge this notion and suggest a model that posits that mRNA decay is activated by the intrinsically aberrant nature of premature termination8,9. 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.

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Figure 1: Toeprint analyses of initiation and premature termination in cell extracts.
Figure 2: Aberrant toeprints derived from PTCs in wild-type extracts in the presence of cycloheximide are dependent on upstream AUGs.
Figure 3: Aberrant toeprint signals are eliminated when premature termination codons (PTCs) are flanked by a normal 3′-UTR.
Figure 4: Stabilization of nonsense-containing mRNAs by tethered Pab1p.

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Acknowledgements

We thank P. Stockley, University of Leeds, for MS2 coat protein antibodies; T. Serio (Brown University) and D. C. Masison (NIH) for Sup35p antibodies; J. McCarthy (UMIST) for eIF4G and eIF4E antibodies; and M. P. Wickens (University of Wisconsin–Madison) for plasmids carrying MS2 constructs. This work was supported by grants to A.J. from the National Institutes of Health and a postdoctoral fellowship to N.A. from the Human Frontier Science Program.

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Correspondence to Allan Jacobson.

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Supplementary information

Supplementary Data

Supplementary results and figure legends (DOC 49 kb)

Supplementary Figure 1

Toeprint analyses of normal and premature termination. (JPG 106 kb)

Supplementary Figure 2

Data demonstrating that ribosomes can reinitiate translation at AUG codons upstream or downstream of the stop codon. (JPG 42 kb)

Supplementary Figure 3

Toeprint analyses in sup45-2 extracts. (JPG 43 kb)

Supplementary Figure 4

Data demonstrating that tethered Pab1p stabilizes nonsense-containing mRNAs. (JPG 49 kb)

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Amrani, N., Ganesan, R., Kervestin, S. et al. A faux 3′-UTR promotes aberrant termination and triggers nonsense- mediated mRNA decay. Nature 432, 112–118 (2004). https://doi.org/10.1038/nature03060

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