A unique No-Go Decay cleavage in mRNA exit-tunnel of ribosome produces 5’-OH ends phosphorylated by Rlg1

The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stalled ribosomes. An endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence. However, primary site of cleavage remains unknown. Indeed, direct evidence that two RNA fragments resulting from a precise and unique cleavage has never been obtained. We used mRNAs expressing a 3′-ribozyme to produce truncated transcripts in vivo that mimic naturally occurring truncated mRNAs, known to trigger NGD. We analysed ribosome associated NGD cleavage products at single-nucleotide resolution and show that a precise endonucleolytic cleavage event occurs within the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue. The first two stalled ribosomes are apparently not competent for mRNA cleavage. We demonstrate that NGD cleavage within the third or upstream ribosomes produces 5′-hydroxylated RNA fragments that are phosphorylated by the Rlg1/Trl1 kinase. The resulting 5′-phosphorylated RNA fragments are digested by the 5′-3′ exoribonuclease Xrn1, but surprisingly, can also be trimmed by the 5′-3′ exoribonuclease activity of Dxo1 in Xrn1 deficient cells.