How Degrading! Trapped Translation Factors Get Trashed

Using distinct small molecules that trap elongation or termination factors within the A-site of translating ribosomes, Gurzeler et al.¹ and Oltion et al.² identify a new branch of the ribosome-associated quality control pathway. This new mode of ubiquitin-dependent translation regulation expands the growing number of mechanistically distinct ribosome-associated quality control pathways that surveil translation progression.

Translation is tightly regulated by an interconnected network of pathways to ensure protein synthesis is carried out efficiently and with minimal errors. Ribosome progression can be stalled during translation for a variety of reasons, including defective mRNAs and damaged or improperly assembled ribosomes. Persistent ribosome stalls lead to collisions that are sensed and acted upon by a growing list of factors, many conserved across eukaryotes, that regulate ribosome-associated quality control (RQC) pathways. Regulatory ribosomal ubiquitylation is triggered upon activation of RQC pathways and often necessary for successful ribosome collision resolution. These conserved ubiquitylation events occur in a site-specific manner on the ribosome and are catalyzed and acted upon by distinct cellular factors.³ Prior to the results described by Oltion et al. and Gurzeler et al., two distinct ubiquitin-dependent RQC branches have been characterized. The canonical RQC (eRQC) pathway targets ribosomes that collide during elongation and is characteristically stimulated by moderate doses of elongation inhibitors.³ Here, the ubiquitin ligase ZNF598 ubiquitylates ribosomal proteins eS10 and uS10, initiating a series of RQC steps that ultimately catalyze ribosome recycling and degradation of the nascent peptide chain.^4,5 A more recently described RQC pathway (iRQC) is triggered both by activation of the integrated stress response and by translation initiation inhibitors, resulting in uS3 and uS5 ubiquitylation by a distinct ubiquitin ligase, RNF10, and culminating in small ribosomal subunit (40S) protein degradation.^6,7 Additional observations that many other ribosomal proteins and translation factors are ubiquitylated in response to various stressors suggests that ribosome ubiquitylation may play a larger role in regulating translation beyond the currently identified RQC pathways.⁸

The Taunton, Mühlemann, and Reinhardt research teams identify a novel branch of the RQC pathway induced by small molecules that trap translation factors in the tRNA-binding A-site of the ribosome.^1,2 Oltion and colleagues demonstrate that ternatin-4, an FDA-approved drug for treating multiple myeloma previously shown to inhibit translation by trapping eEF1A on the ribosome and stalling elongation, induces eEF1A degradation.⁹ The observation that intermediate but not high concentrations of ternatin-4 lead to a sharp decline in eEF1A levels was reminiscent of previous RQC studies using classic elongation inhibitors, suggesting a model where eEF1A degradation requires ribosome collisions. Extending previous studies identifying a class of small molecules that induce readthrough of premature termination codons (PTCs) by degrading eRF1,¹⁰ the Mühlemann and Reinhardt groups identify and optimize small molecules (NVS1.1 and NVS2.1) that enhance PTC readthrough and demonstrate that these molecules also act by inducing eRF1 degradation. Despite the different agents used to induce the degradation of separate translation factors, independent CRISPR screens from both studies converged on the ribosomal collision sensor GCN1 and the ubiquitin ligases RNF14 and RNF25 as critical factors necessary for eEF1A and eRF1 degradation. Using proteomic approaches that enrich for ubiquitylated peptides, the groups discovered that these small molecules lead to ribosomal protein eS31 ubiquitylation, which serves as a primary step in a previously undiscovered RQC pathway. Oltion and colleagues further deconvolute the role of RNF14 and RNF25 using knockout cell lines and demonstrate that RNF25 ubiquitylates eS31, with K113 being the consequential ubiquitylation site. This ribosomal ubiquitylation event leads to subsequent RNF14-dependent ubiquitylation of either eEF1A or eRF1 trapped within the ribosomal A-site, targeting them for degradation and resolving the translational block that caused the collision. GCN1 has an established role as a ribosomal collision sensor by binding across both ribosomes involved in the collision event.³ Here, the authors present data that suggests GCN1 acts as a scaffold to recruit RNF14 to the ribosomal collision, positioning it near the occluded A site for ubiquitylation of either eEFA1 or eRF1. The proposed model represents an entirely novel RQC branch in which a distinct site-specific ribosomal ubiquitylation event is necessary for ribosome stall resolution.

Beyond the description of this novel RQC pathway, the NVS compounds generated by the Mühlemann and Reinhardt teams have particular clinical relevance. Several human diseases result from nonsense mutations that lead to premature translation termination and a subsequent decrease in disease-relevant protein abundance. As such, promoting PTC readthrough is a promising strategy to restore protein expression. However, current readthrough-promoting agents (aminoglycosides that reduce translation fidelity) have shown modest clinical performance and have toxicity when used long-term, making them unsuitable treatments for genetic disorders.¹⁰ The NVS compounds specifically leverage a distinct strategy for readthrough: the depletion of eRF1, delaying translation termination and increasing the likelihood of incorporation of near-cognate tRNA at nonsense mutations.¹ Gurzeler and colleagues test the ability of the NVS compounds to promote full length CFTR and IDUA expression in disease models for cystic fibrosis and Hurler syndrome, respectively. They first demonstrate that the NVS compounds induce robust full-length CFTR and IDUA expression and activity in cell-based reporter assays. To establish physiological relevance, they utilized Hurler syndrome patient-derived fibroblasts and a Hurler syndrome rat model. Hurler syndrome is characterized by a deficiency in IDUA activity due to various nonsense mutations, which impairs the patient’s ability to breakdown glycosaminoglycans (GAG). In both the patient-derived fibroblasts and rat models, Gurzeler and colleagues found that NVS1.1 and NVS2.1 substantially restore IDUA levels and reduce GAG accumulation. Intriguingly, the effect of the NVS compounds is PTC-specific and does not lead to enhanced readthrough of normal termination codons.¹⁰ Together, these results indicate the optimized NVS compounds have a strong potential for broad therapeutic value in the many human diseases caused by nonsense mutations.

The discovery and characterization of this novel RQC pathway expands our understanding of a complex ubiquitin code on the ribosome that produces distinct functional outputs. These pathways all share a common theme of regulatory ribosomal ubiquitylation on the 40S subunit. Here, the site-specific ubiquitylation event by RNF25 on eS31 promotes the activity RNF14. Future structural studies are needed to understand how eS31 ubiquitylation, as well as other regulatory ubiquitylation events, act to promote downstream pathway outcomes. Interestingly, Oltion and colleagues identify several additional ubiquitylation events on 60S subunit proteins in the vicinity of GCN1, RNF14, and eEF1A binding. Future research will determine if these ubiquitylation events are critical for subsequent steps in the pathway or represent bystander ubiquitylation events. Because both ternatin-4 and the NVS compounds lead to ribosome collisions, they result in ribosomal “traffic jams” that trigger the characteristic eRQC and iRQC ubiquitylation events as well. These previously characterized ribosomal ubiquitylation events and their associated ubiquitin ligases are not necessary for eEF1A or eRF1 degradation,^1,2 but this finding raises interesting questions regarding potential crosstalk between the RQC branches. Finally, while it is clear that various pharmacological agents can potently stimulate the distinct RQC branches, the physiologically relevant conditions that both trigger RQC pathways and require RQC activation for appropriate phenotypic outcomes remain unknown.

Figure 1.

Overview of RQC pathways Small molecule agents that trap translation factors within the A site of elongating 80S ribosomes activate a new RQC pathway which induces ubiquitylation of the ribosome and the trapped translation factor (center, right). This new RQC pathway also stimulates the canonical and initiation RQC pathways (left).