TY - JOUR T1 - A unique No-Go Decay cleavage in mRNA exit-tunnel of ribosome produces 5’-OH ends phosphorylated by Rlg1 JF - bioRxiv DO - 10.1101/465633 SP - 465633 AU - Albertas Navickas AU - Sébastien Chamois AU - Rénette Saint-Fort AU - Julien Henri AU - Claire Torchet AU - Lionel Benard Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/07/18/465633.abstract N2 - The No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. An endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence. However, 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 and produce more precise ribosome stalling events and cleavages than mRNAs containing contiguous rare codons. This technique allowed us to analyse ribosome associated NGD cleavage products at single-nucleotide resolution. We show that (i) the 5’-3’ exoribonuclease Xrn1 is the principal contributor to NGD fragment production and (ii) we can detect endonucleolytic cleavage events starting at the third collided ribosome. This cleavage, which we show to be Hel2-dependent, maps 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 3 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 NGD endonucleolytic cleavage produces 5’-hydroxylated RNA fragments requiring 5’-phosphorylation prior to digestion by 5’-3’ exoribonucleases. We identify the RNA kinase Rlg1/Trl1 as a new essential player in the degradation of NGD RNAs. ER -