The Ccr4-Not complex monitors the translating ribosome for codon optimality.

INTRODUCTION The tightly controlled process of gene expression requires messenger RNAs (mRNAs), which represent DNA-derived blueprints for polypeptides, to be translated by the protein-producing machinery of the cell, the ribosomes. Therefore, protein levels depend largely on cellular mRNA levels, and the control of mRNA decay is one of the most critical processes for setting the overall level of gene expression. Half-lives of mRNAs vary greatly between different transcripts, and regulation of the mRNA decay rate is intimately connected to the elongation phase of mRNA translation. To that end, codon optimality has been established as a key parameter for determining mRNA half-life in multiple eukaryotic organisms. It has also been established that the timely decay of short-lived mRNAs enriched with nonoptimal codons requires the Ccr4-Not complex. Ccr4-Not is an essential protein complex, with its best understood role in mRNA degradation, where it serves as the major cytoplasmic 3′-poly(A)-tail deadenylase that initiates decay of most mRNAs. By deadenylation and subsequent activation of the mRNA decapping machinery, the Ccr4-Not complex renders mRNAs accessible to the major degrading exonucleases, such as Xrn1 on the 5′ end and the exosome on the 3′ end. The molecular mechanism underlying codon optimality monitoring and coordination with mRNA decay by the Ccr4-Not complex has remained elusive. RATIONALE Because nonoptimal codons affect decoding kinetics of the ribosome and mRNA degradation occurs largely cotranslationally, it is highly plausible that codon optimality is directly monitored on the ribosome. In addition, a direct physical link between the participating Ccr4-Not complex and the ribosome has been suggested previously, and the Not4 subunit of the complex, an E3 ligase, ubiquitinates the eS7 protein of the 40S ribosomal subunit in yeast. Therefore, we set out to gain insights into the connection between the Ccr4-Not complex and the translation machinery in the context of mRNA homeostasis by combining cryo–electron microscopy (cryo-EM), ribosome profiling, and biochemical analysis. RESULTS We used affinity-purified native Ccr4-Not–ribosome complexes from Saccharomyces cerevisiae for analysis by cryo-EM and found that recruitment of Ccr4-Not to the ribosome occurs via the Not5 subunit. The N terminus of Not5—in particular, a three α-helix bundle—interacted specifically with the ribosomal E-site, and deletion of the Not5 N-terminus resulted in the loss of stable ribosome association of the Ccr4-Not complex. However, ubiquitination of the small ribosomal subunit protein eS7 through the Not4 subunit still occurred. The Not5 interaction involved the ribosomal protein eS25 of the small subunit, in addition to transfer RNA (tRNA) and ribosomal RNAs (rRNAs). We found that Ccr4-Not interacts with both initiating and elongating ribosomes. In either case, Not5 engaged the E-site only when the ribosome adopted a distinct conformation lacking accommodated tRNA in the A-site, indicative of impaired decoding kinetics. Ribosome profiling revealed that low-optimality codons were enriched in the A-site in the Ccr4-Not–bound elongating ribosomes. This observation explained the low A-site tRNA occupancy observed with cryo-EM and suggested a link to codon optimality monitoring. Consistently, using mRNA stability assays, we found that loss of Not5 resulted in the inability of the mRNA degradation machinery to sense codon optimality. The observed dysregulation of mRNA half-life was detected upon Not5 deletion, Not5 N-terminal deletion, eS25 deletion, and loss of eS7 ubiquitination by Not4, which apparently serves as an upstream prerequisite for further Ccr4-Not activity on the ribosome. In addition, mRNA decapping was found to be impaired in these mutants, which confirmed that, in this pathway, Ccr4-Not triggers decapping downstream of optimality monitoring. CONCLUSION Our analysis elucidates a direct physical link between the mRNA decay–mediating Ccr4-Not complex and the ribosome. Dependent on preceding ubiquitination of eS7 by the Not4 subunit, the Ccr4-Not complex binds (via the Not5 subunit) specifically to the ribosomal E-site when the A-site lacks tRNA because of slow decoding kinetics. This state of the ribosome occurs in the presence of nonoptimal codons in the A-site, which explains the shorter half-lives of transcripts enriched in nonoptimal codons. Thus, our findings provide mechanistic insights into the coordination of translation efficiency with mRNA stability through the Ccr4-Not complex.