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

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 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.

Ribosome states signal RNA quality control

“…As described by Glover et al (2020), these homologs contain a polynucleotide kinase (PNK) domain thought to be involved in phosphorylating cleaved mRNAs for further processing by exonucleases. Cue2 lacks such a domain, but another protein in yeast, Tlr1, contains a PNK domain that is required to phosphorylate the 5 0 OH of cleaved mRNAs from NGD substrates for further processing (Navickas et al, 2020). Although N4BP2 has not yet been implicated in mRNA turnover in mammalian cells, mass spectrometry studies provide evidence that N4BP2 physically associates with colliding ribosomes (Sinha et al, 2020).…”

Oxidized RNA Bodies compartmentalize translation quality control inSaccharomyces cerevisiae

“…However, this nascent chain does accumulate in dom34Δ background (Fig 2C), due to NGD deficiency (3). Moreover, it was shown previously that this truncated URA3 mRNA in dom34Δ background is bound by an array of colliding ribosomes (34,35). Therefore, the HA-Ura3 nascent polypeptide in this strain can serve as a marker for the arrested RNC in situ.…”

“…If ORBs compartmentalize TQC, they should be enriched in a translationally arrested RNC because this is a TQC-specific substrate (33,34). An arrested RNC was generated on a URA3…”

“…Yeast plasmids were constructed using standard molecular biology procedures starting from p415ADH1 (59) and were described previously (Navickas et al, 2020). pLB138 expresses a truncated mRNA (Rz) from a 2HA-tagged-URA3-RZ (Ribozyme) construct.…”

Translational control of coronaviruses

“…The fact that SARS-CoV nsp1 does not show any intrinsic endonuclease activity means that nsp1 does not act directly on its substrate but rather activates one, or several, unknown cellular endonucleases. As nsp1 blocks ribosomes essentially during the initiation step, these stalled 40S ribosomes on the mRNA may trigger a quality control mechanism that could be reminiscent to the no-go decay which targets mRNAs on which elongation is stalled ( 161 , 162 ). Further studies have to be carried out to understand the molecular mechanism by which nsp1 can induce RNA cleavage.…”