NMD-degradome sequencing reveals ribosome-bound intermediates with 3''-end nontemplated nucleotides

Transcriptome-wide methodology is developed to capture and sequence decay intermediates deriving from direct NMD targets. Results demonstrate that mammalian-cell NMD generates decay intermediates in a process that involves the covalent addition of nontemplated U, C, G and A nucleotides to 3''-ends. Variations of this methodology demonstrate that these decay intermediates arise co-translationally. Overall design: A transcriptome-wide method that captures and sequences the decay intermediates (DIs) deriving from direct NMD targets that we call NMD-Degradome Sequencing (NMD-DegSeq). Unlike methods that identify NMD targets by their upregulation upon the downregulation of one or more NMD factors, which cannot distinguish between direct and indirect NMD targets, our method identifies direct NMD targets based on their co-immunoprecipitation (co-IP) with p-UPF1.