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.

No abstract

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 unambiguously demonstrated. We used mRNAs expressing a 3'-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allowed 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. A similar analysis of mRNAs containing rare codons showed that at least three stacked ribosomes are also necessary for endonucleolytic cleavage of these mRNAs. However, we observed that NGD RNA fragments can also be trimmed by the 5'-3' exoribonuclease activity of Dxo1, creating new extremities in the region theoretically covered by disomes. Finally, we show that endonucleolytic cleavage produces 5'-hydroxylated RNA fragments requiring 5'phosphorylation prior to digestion by 5'-3' exoribonucleases. We identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs.