No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1

The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been demonstrated. Here we use mRNAs expressing a 3′-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allows us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue and release 5′-hydroxylated RNA fragments requiring 5′-phosphorylation prior to digestion by the exoribonuclease Xrn1, or alternatively by Dxo1. Finally, we identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs. In the No-Go decay mRNA surveillance pathway, mRNAs containing stalled ribosomes are cleaved by an endoribonuclease. Here the authors show the endonucleolytic cleavage on the artificial No-Go decay target mRNAs, revealing downstream decay process by Trl1 kinase and the 5′ to 3′ exonuclease Dxo1.