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

New1 is a homolog of fungal translation elongation factor 3 (eEF3) inSaccharomyces cerevisiae. Its absence has previously been described to lead to cold sensitivity, ribosome biogenesis defects, and ribosomal queues upstream of Stop codons on selected mRNAs. Here, we find that, especially in cold conditions, lack of New1 induces no-go decay (NGD) on specificmRNAs, leading, as a consequence, to decreased mRNA and protein levels of such genes.The effect correlates with the presence of specific Lysine and Arginine codons just upstream of the Stop codon. In total, more than 10% of yeast coding sequences end with the affected codons. We have confirmed NGD and subsequent downregulation at the protein level for the metabolic enzymes Pgk1, Gpm1, and Adh1, translation elongation factors EF-1 alpha and EF-1 beta (gamma-subunit), ribosomal protein Rps5, as well as ribosome biogenesis factor Nop1 and adenosine kinase Ado1. Overall design: We performed UV crosslinking and analysis of cDNA (CRAC) for the collision sensor and E3 ubiquitin ligase Hel2 and for New1, in presence or absence of the respective other player. To this end,in yeast Saccharomyces cerevisiae, BY4741, we genomically tagged Hel2 or New1 (only version in the genome, single copy) with a His-TEV-ProteinA tag in either a wildtype or new1-deleted (for Hel2) or hel2-deleted (for New1) strain background. Cells were cultured at 30°C or 20°C, as mentioned, and UV-crosslinked. The tagged protein was purified along with crosslinked RNA and sequencing libraries were prepared in a total of 4 replicates (2 at 30°C, 2 at 20°C). For each replicate, a control experiment was conducted in which no protein was tagged (BY), in the wildtype background, to assess background signals.