Nanopore sequencing of Upf1-TAP immunoprecipitated transcripts in yeast

Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway known to degrade mRNAs containing premature termination codons (PTCs). A distance long enough between the stop codon and the poly(A)-binding protein (Pab1) is required for mRNA recognition by the NMD factors Upf1, Upf2 and Upf3. Using Nanopore direct RNA sequencing, we show that PTC-containing NMD targets account for only 6% of Upf1-associated RNA and have long poly(A) tails, indicating that Upf1-binding occurs prior to RNA deadenylation. Conversely, most Upf1-associated mRNAs have short poly(A) tails, lack a PTC and correspond to highly expressed genes. A short poly(A) tail is thus an important feature of NMD targets, redefining the scope of this RNA degradation pathway. We propose a model in which loss of Pab1-binding to short poly(A)-tailed mRNAs impairs translation termination and dictates the recruitment of the NMD machinery, uncovering a hitherto unknown role of NMD in the degradation of these transcripts. Overall design: Nanopore Direct RNA sequencing (SKQ_RNA002) of 3 replicates of each type : total RNA from WT (LMA 2194), upf1delta (LMA1667), Upf1-TAP upf2delta (LMA3739), Upf1-TAP upf3delta (LMA3735) and RNA Immunoprecipitated (RIP) from Upf1-TAP (LMA2194), eIF4G2-TAP(LMA3314), Upf1-TAPupf2delta (LMA3739), UPF1-TAPupf3delta (LMA3735) strains