EDF1 coordinates cellular responses to ribosome collisions

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation

Borrelidin Induces the Unfolded Protein Response in Oral Cancer Cells and Chop-Dependent Apoptosis

Oral squamous cell carcinoma (OSCC) is the most common cancer affecting the oral cavity, and US clinics will register about 30,000 new patients in 2015. Current treatment modalities include chemotherapy, surgery, and radiotherapy, which often result in astonishing disfigurement. Cancers of the head and neck display enhanced levels of glucose-regulated proteins and translation initiation factors associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Previous work demonstrated that chemically enforced UPR could overwhelm these adaptive features and selectively kill malignant cells. The threonyl-tRNA synthetase (ThRS) inhibitor borrelidin and two congeners were discovered in a cell-based chemical genomic screen. Borrelidin increased XBP1 splicing and led to accumulation of phosphorylated eIF2α and UPR-associated genes, prior to death in panel of OSCC cells. Murine embryonic fibroblasts (MEFs) null for GCN2 and PERK were less able to accumulate UPR markers and were resistant to borrelidin. This study demonstrates that UPR induction is a feature of ThRS inhibition and adds to a growing body of literature suggesting ThRS inhibitors might selectively target cancer cells.National Institutes of Health/[DE019678]/NIH/Estados UnidosInternational Cooperative Biodiversity Groups/[U01 TW007404]/ICBG/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

Identification and Characterization of Key Regulatory Factors Mediating Ribosomal Ubiquitylation and Quality Control during Translation

Protein ubiquitylation plays a critical role in shaping proteome dynamics and responding to proteostasis dysfunction. Activation of the integrated stress response (ISR) or the ribosome-associated quality control (RQC) pathway stimulates regulatory ribosomal ubiquitylation (RRub) on distinct 40S ribosomal proteins, yet the cellular role for these ubiquitylation events remains unclear. We previously demonstrated that conserved monoubiquitylation events are required for downstream RQC events following the translation of poly(A) sequences. We identified the E3 ubiquitin ligase, ZNF598, which is responsible for initiating RQC by catalyzing the ubiquitylation of eS10 and uS10. An additional set of ubiquitylation events on uS5 and uS3 are triggered upon activation of the ISR and appear to function outside of the RQC pathway, however the critical regulators remained unknown. In this dissertation I establish that RRub events diminish over time following exposure to UV stress, implicating a role for deubiquitylating enzymes (Dubs) within the RQC pathway. I identified the Dubs OTUD3 and USP21 that, when overexpressed, result in read-through of poly(A)-mediated stalls, and directly antagonize ZNF598. USP21 or OTUD3 knockout cell lines revealed that loss of expression for either Dub results in enhanced stalling on poly(A) sequences and prolonged site-specific RRub following UV exposure. Additionally, I establish a hierarchical structure for the ribosome ubiquitin code by demonstrating that eS10 and uS3 ubiquitylation is necessary for subsequent uS10 and uS5 ubiquitylation, respectively, suggesting a specific order of ribosome ubiquitylation events occurs to ensure optimal resolution of RQC nucleating events. These results demonstrate that Dubs can constrain RQC activation and may serve to remove ubiquitin from 40S subunits to allow for subunit recycling. uS5 and uS3 ubiquitylation events operate outside of the canonical RQC pathway. Here I demonstrate that translation initiation inhibition, either through moderate ISR activation which produces low levels of eIF2a phosphorylation, or overt pharmacological inhibition of translation initiation trigger these site-specific modifications. I identify the E3 ubiquitin ligase RNF10 and the Dub USP10 as the regulators of uS3 and uS5 ubiquitylation. Additionally, I show that prolonged ubiquitylation results in 40S but not 60S ribosomal protein degradation in an autophagy-independent manner. This study identifies and characterizes a discrete ribosome-associated quality control pathway that surveys preinitiation complex status during mRNA translation initiation

iRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity

Orphan quality control shapes network dynamics and gene expression

AbstractAll eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb crucial interactions between network components often result in disease, but how the composition and dynamics of complex networks are established is unknown. Here, we identify the tumor suppressor E3 ligase UBR5 as a quality control enzyme that helps degrade unpaired subunits of multiple transcription factors that operate within a single network. By constantly turning over orphan subunits, UBR5 forces cells to continuously replenish network components through new protein synthesis. The resulting cycles of transcription factor synthesis and degradation allow cells to effectively execute the gene expression program, while remaining susceptible to environmental signals. We conclude that orphan quality control plays an essential role in establishing the dynamics of protein networks, which may explain the conserved need for protein degradation in transcription and offers unique opportunities to modulate gene expression in disease