Endonucleolytic cleavage of eukaryotic mRNAs with stalls in translation elongation

Abstract

A fundamental aspect of the biogenesis and function of eukaryotic messenger RNA is the quality control systems that recognize and degrade non-functional mRNAs. Eukaryotic mRNAs where translation termination occurs too soon (nonsense-mediated decay) or fails to occur (non-stop decay) are rapidly degraded. We show that yeast mRNAs with stalls in translation elongation are recognized and targeted for endonucleolytic cleavage, referred to as 'no-go decay'. The cleavage triggered by no-go decay is dependent on translation and involves Dom34p and Hbs1p. Dom34p and Hbs1p are similar to the translation termination factors eRF1 and eRF3 (refs 3, 4), indicating that these proteins might function in recognizing the stalled ribosome and triggering endonucleolytic cleavage. No-go decay provides a mechanism for clearing the cell of stalled translation elongation complexes, which could occur as a result of damaged mRNAs or ribosomes, or as a mechanism of post-transcriptional control.

Figure 1. Stalling of elongating ribosomes leads to endonucleolytic cleavage of the PGK1-SL reporter transcript

Northern analysis of steady-state PGK1 and PGK1-SL reporter mRNA in wild-type strains and strains mutant either in 3′ → 5′ exosome activity (a) that include ski2Δ, ski3Δ, ski7Δ and ski8Δ, or a mutant in 5′ → 3′ exonucleolytic activity, xrn1Δ (b). The untransformed strain (Un) is shown in b. Northern analysis was performed with probes specific for mRNA regions 5′ (a) or 3′ (b) of the stall site, as illustrated at the bottom.

Figure 2. NGD is initiated by endonucleolytic cleavage of mRNA with various ribosomal stalls

Steady-state cultures of xrn1Δ (a) and dom34Δxrn1Δ (b) strains with PGK1, PGK1-SL, PGK1-PK (pseudoknot), PGK1-RC (rare codons), PGK1-stop (stop codon) and PGK1-proline-stop (proline-stop codon) reporters were grown at low temperature (16 8C). Northern analysis of steady-state reporter mRNAs was performed with a probe specific for mRNA regions 3′ of the stall sites.

Figure 3. NGD is dependent on translation by ribosomes

Northern analysis of steady-state PGK1-SL, SL-PGK1-SL (pRP1252) and PGK1-PTC-SL (pRP1253) reporter transcripts in wild-type, ski7Δ, upf1Δ ski7Δ (a) and xrn1Δ, upf1Δ xrn1Δ (b) mutant strains. Northern analysis was performed with probes specific for mRNA regions 5′ (a) and 3′ (b) of the stall site.

Figure 4. Dom34p and Hbs1p affect NGD

Northern analysis of steady-state PGK1-SL reporter mRNA in wild-type and mutant strains with probes specific for mRNA regions 5′ (a) and 3′ (b) of the stall site.