Yeast elongation factor homolog New1 protects a subset of mRNAs from degradation by no-go decay.

New1 is a homologue of the essential yeast translation elongation factor eEF3. Lack of New1 has been shown to induce ribosome queuing upstream of the stop codon on messenger RNAs (mRNAs) with specific C-terminal lysine and arginine codons. Here, we used ultraviolet crosslinking and analysis of complementary DNA (cDNA), long-read nanopore sequencing, and proteomics to address the consequences such queues have for the yeast cell. We show that these queues represent collisions, recognized by collision sensor Hel2, triggering mRNA degradation via canonical no-go decay (NGD). We identified 139 target mRNAs, on which decay is initiated by Cue2-mediated cleavage upstream of the stop codon. Compared to other collision-prone mRNAs, ending on the same C-terminal codons, these targets are characterized by stronger secondary structures upstream of the stop codon, longer queues, and stronger queuing signatures. Nanopore sequencing enabled characterization of NGD cleavage fragments across targets. Ultimately, NGD in the absence of New1 leads to downregulation of encoded proteins, including highly abundant and essential metabolic enzymes like Pgk1 and Gpm1, as well as translation elongation factors such as eEF1-alpha and eEF1-beta. We show that New1 protects such mRNAs from degradation by NGD and that NGD is a major determinant of the cold sensitive growth phenotype observed in NEW1 deletants.