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. Overall design: mRNA stranded sequencing from UPF1_HTP CRAC samples extracted from LMA3730 strain (minus UV as a negative control and plus UV).