Illumina sequencing of CRAC data obtained with Upf1 in yeast

The nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic cytoplasmic surveillance pathway that degrades aberrant mRNAs carrying premature termination codons (PTC). In yeast, the long distance between the stop codon and the poly(A) tail dictates mRNA detection by NMD factors Upf1, Upf2 and Upf3, and trigger mRNA decay, independent of deadenylation. While Upf1 binds a wide range of mRNAs beyond PTC-containing substrates, the mechanism of its recruitment on non-PTC-containing mRNAs is unknown. Using Nanopore sequencing of Upf1-bound mRNAs, we discovered that known PTC-containing NMD targets have long poly(A) tails, confirming that Upf1 binding occurs rapidly prior to deadenylation. Strikingly, the largest category specifically bound to Upf1 consisted of mRNAs without PTC harboring short poly(A) tails, corresponding to well-translated and stable transcripts. We showed that the degradation of this subpopulation depends on the three Upf factors, revealing a hitherto unknown function of Upf1 and NMD cofactors in mRNA decay.无义介导的mRNA降解（nonsense-mediated mRNA decay，NMD）是一种保守的真核细胞质监视通路，可降解携带提前终止密码子（premature termination codons，PTC）的异常mRNA。在酵母中，终止密码子与poly(A)尾之间的较大间距决定了NMD因子Upf1、Upf2及Upf3对mRNA的识别，并触发不依赖脱腺苷酸化的mRNA降解过程。尽管Upf1可结合除携带PTC的底物之外的多种mRNA，但其在非PTC携带mRNA上的招募机制仍未明确。本研究通过对结合Upf1的mRNA开展纳米孔测序（Nanopore sequencing），发现已知的携带PTC的NMD靶标具有较长的poly(A)尾，证实Upf1的结合发生在脱腺苷酸化之前的早期阶段。令人瞩目的是，特异性结合Upf1的最大转录本类别为不携带PTC且poly(A)尾较短的mRNA，这类转录本对应于翻译旺盛且稳定性优异的转录产物。我们证实该亚群的降解依赖于三种Upf因子，由此揭示了Upf1与NMD辅助因子在mRNA降解过程中此前未被发现的全新功能。