Mechanisms of readthrough mitigation reveal principles of GCN1-mediated translational quality control.

Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.

RNF14-dependent atypical ubiquitylation promotes translation-coupled resolution of RNA-protein crosslinks.

Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.

Disome and Trisome Profiling Reveal Genome-wide Targets of Ribosome Quality Control.

The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3' UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2α, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.

The integrated stress response in metabolic adaptation.

The integrated stress response (ISR) refers to signaling pathways initiated by stress-activated eIF2α kinases. Distinct eIF2α kinases respond to different stress signals, including amino acid deprivation and mitochondrial stress. Such stress-induced eIF2α phosphorylation attenuates general mRNA translation and, at the same time, stimulates the preferential translation of specific downstream factors to orchestrate an adaptive gene expression program. In recent years, there have been significant new advances in our understanding of ISR during metabolic stress adaptation. Here, I discuss those advances, reviewing among others the ISR activation mechanisms in response to amino acid deprivation and mitochondrial stress. In addition, I review how ISR regulates the amino acid metabolic pathways and how changes in the ISR impact the physiology and pathology of various disease models.

Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation.

The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.

Structure of Gcn1 bound to stalled and colliding 80S ribosomes.

The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.

Emerging Role of GCN1 in Disease and Homeostasis.

GCN1 is recognized as a factor that is essential for the activation of GCN2, which is a sensor of amino acid starvation. This function is evolutionarily conserved from yeast to higher eukaryotes. However, recent studies have revealed non-canonical functions of GCN1 that are independent of GCN2, such as its participation in cell proliferation, apoptosis, and the immune response, beyond the borders of species. Although it is known that GCN1 and GCN2 interact with ribosomes to accomplish amino acid starvation sensing, recent studies have reported that GCN1 binds to disomes (i.e., ribosomes that collide each other), thereby regulating both the co-translational quality control and stress response. We propose that GCN1 regulates ribosome-mediated signaling by dynamically changing its partners among RWD domain-possessing proteins via unknown mechanisms. We recently demonstrated that GCN1 is essential for cell proliferation and whole-body energy regulation in mice. However, the manner in which ribosome-initiated signaling via GCN1 is related to various physiological functions warrants clarification. GCN1-mediated mechanisms and its interaction with other quality control and stress response signals should be important for proteostasis during aging and neurodegenerative diseases, and may be targeted for drug development.

The amino acid sensor GCN2 suppresses terminal oligopyrimidine (TOP) mRNA translation via La-related protein 1 (LARP1).

La-related protein 1 (LARP1) has been identified as a key translational inhibitor of terminal oligopyrimidine (TOP) mRNAs downstream of the nutrient sensing protein kinase complex, mTORC1. LARP1 exerts this inhibitory effect on TOP mRNA translation by binding to the mRNA cap and the adjacent 5'TOP motif, resulting in the displacement of the cap-binding protein eIF4E from TOP mRNAs. However, the involvement of additional signaling pathway in regulating LARP1-mediated inhibition of TOP mRNA translation is largely unexplored. In the present study, we identify a second nutrient sensing kinase GCN2 that converges on LARP1 to control TOP mRNA translation. Using chromatin-immunoprecipitation followed by massive parallel sequencing (ChIP-seq) analysis of activating transcription factor 4 (ATF4), an effector of GCN2 in nutrient stress conditions, in WT and GCN2 KO mouse embryonic fibroblasts, we determined that LARP1 is a GCN2-dependent transcriptional target of ATF4. Moreover, we identified GCN1, a GCN2 activator, participates in a complex with LARP1 on stalled ribosomes, suggesting a role for GCN1 in LARP1-mediated translation inhibition in response to ribosome stalling. Therefore, our data suggest that the GCN2 pathway controls LARP1 activity via two mechanisms: ATF4-dependent transcriptional induction of LARP1 mRNA and GCN1-mediated recruitment of LARP1 to stalled ribosomes.

Aminoacyl-tRNA synthetase inhibition activates a pathway that branches from the canonical amino acid response in mammalian cells.

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.

Inducible Systemic Gcn1 Deletion in Mice Leads to Transient Body Weight Loss upon Tamoxifen Treatment Associated with Decrease of Fat and Liver Glycogen Storage.

GCN1 is an evolutionarily-conserved ribosome-binding protein that mediates the amino acid starvation response as well as the ribotoxic stress response. We previously demonstrated that Gcn1 mutant mice lacking the GCN2-binding domain suffer from growth retardation and postnatal lethality via GCN2-independent mechanisms, while Gcn1-null mice die early in embryonic development. In this study, we explored the role of GCN1 in adult mice by generating tamoxifen-inducible conditional knockout (CKO) mice. Unexpectedly, the Gcn1 CKO mice showed body weight loss during tamoxifen treatment, which gradually recovered following its cessation. They also showed decreases in liver weight, hepatic glycogen and lipid contents, blood glucose and non-esterified fatty acids, and visceral white adipose tissue weight with no changes in food intake and viability. A decrease of serum VLDL suggested that hepatic lipid supply to the peripheral tissues was primarily impaired. Liver proteomic analysis revealed the downregulation of mitochondrial ß-oxidation that accompanied increases of peroxisomal ß-oxidation and aerobic glucose catabolism that maintain ATP levels. These findings show the involvement of GCN1 in hepatic lipid metabolism during tamoxifen treatment in adult mice.

Perturbations in actin dynamics reconfigure protein complexes that modulate GCN2 activity and promote an eIF2 response.

Genetic and pharmacological interventions in yeast and mammalian cells have suggested a cross-talk between the actin cytoskeleton and protein synthesis. Regulation of the activity of the translation initiation factor 2 (eIF2) is a paramount mechanism for cells to rapidly adjust the rate of protein synthesis and to trigger reprogramming of gene expression in response to internal and external cues. Here, we show that disruption of F-actin in mammalian cells inhibits translation in a GCN2-dependent manner, correlating with increased levels of uncharged tRNA. GCN2 activation increased phosphorylation of its substrate eIF2α and the induction of the integrated stress response master regulator, ATF4. GCN2 activation by latrunculin-B is dependent on GCN1 and inhibited by IMPACT. Our data suggest that GCN2 occurs in two different complexes, GCN2-eEF1A and GCN2-GCN1. Depolymerization of F-actin shifts GCN2 to favor the complex with GCN1, concomitant with GCN1 being released from its binding to IMPACT, which is sequestered by G-actin. These events might further contribute to GCN2 activation. Our findings indicate that GCN2 is an important sensor of the state of the actin cytoskeleton.

The inhibition of protein translation mediated by AtGCN1 is essential for cold tolerance in Arabidopsis thaliana.

In yeast, the interaction of General Control Non-derepressible 1 (GCN1) with GCN2 enables GCN2 to phosphorylate eIF2α (the alpha subunit of eukaryotic translation initiation factor 2) under a variety of stresses. Here, we cloned AtGCN1, an Arabidopsis homologue of GCN1. We show that AtGCN1 directly interacts with GCN2 and is essential for the phosphorylation of eIF2α under salicylic acid (SA), ultraviolet (UV), cold stress and amino acid deprivation conditions. Two mutant alleles, atgcn1-1 and atgcn1-2, which are defective in the phosphorylation of eIF2α, showed increased sensitivity to cold stress, compared with the wild type. Ribosome-bound RNA profiles showed that the translational state of mRNA was higher in atgcn1-1 than in the wild type. Our result also showed that cold treatment reduced the tendency of the tor mutant seedlings to produce purple hypocotyls. In addition, the kinase activity of TOR was transiently inhibited when plants were exposed to cold stress, suggesting that the inhibition of TOR is another pathway important for plants to respond to cold stress. In conclusion, our results indicate that the AtGCN1-mediated phosphorylation of eIF2α, which is required for inhibiting the initiation of protein translation, is essential for cold tolerance in Arabidopsis.

Keeping the eIF2 alpha kinase Gcn2 in check.

The protein kinase Gcn2 is present in virtually all eukaryotes and is of increasing interest due to its involvement in a large array of crucial biological processes. Some of these are universally conserved from yeast to humans, such as coping with nutrient starvation and oxidative stress. In mammals, Gcn2 is important for e.g. long-term memory formation, feeding behaviour and immune system regulation. Gcn2 has been also implicated in diseases such as cancer and Alzheimer's disease. Studies on Gcn2 have been conducted most extensively in Saccharomyces cerevisiae, where the mechanism of its activation by amino acid starvation has been revealed in most detail. Uncharged tRNAs stimulate Gcn2 which subsequently phosphorylates its substrate, eIF2α, leading to reduced global protein synthesis and simultaneously to increased translation of specific mRNAs, e.g. those coding for Gcn4 in yeast and ATF4 in mammals. Both proteins are transcription factors that regulate the expression of a myriad of genes, thereby enabling the cell to initiate a survival response to the initial activating cue. Given that Gcn2 participates in many diverse processes, Gcn2 itself must be tightly controlled. Indeed, Gcn2 is regulated by a vast network of proteins and RNAs, the list of which is still growing. Deciphering molecular mechanisms underlying Gcn2 regulation by effectors and inhibitors is fundamental for understanding how the cell keeps Gcn2 in check ensuring normal organismal function, and how Gcn2-associated diseases may develop or may be treated. This review provides a critical evaluation of the current knowledge on mechanisms controlling Gcn2 activation or activity.

Evolutionarily conserved IMPACT impairs various stress responses that require GCN1 for activating the eIF2 kinase GCN2.

In response to a range of environmental stresses, phosphorylation of the alpha subunit of the translation initiation factor 2 (eIF2α) represses general protein synthesis coincident with increased translation of specific mRNAs, such as those encoding the transcription activators GCN4 and ATF4. The eIF2α kinase GCN2 is activated by amino acid starvation by a mechanism involving GCN2 binding to an activator protein GCN1, along with association with uncharged tRNA that accumulates during nutrient deprivation. We previously showed that mammalian IMPACT and its yeast ortholog YIH1 bind to GCN1, thereby preventing GCN1 association with GCN2 and stimulation of this eIF2α kinase during amino acid depletion. GCN2 activity is also enhanced by other stresses, including proteasome inhibition, UV irradiation and lack of glucose. Here, we provide evidence that IMPACT affects directly and specifically the activation of GCN2 under these stress conditions in mammalian cells. We show that activation of mammalian GCN2 requires its interaction with GCN1 and that IMPACT promotes the dissolution of the GCN2-GCN1 complex. To a similar extent as the overexpression of YIH1, overexpression of IMPACT in yeast cells inhibited growth under all stress conditions that require GCN2 and GCN1 for cell survival, including exposure to acetic acid, high levels of NaCl, H2O2 or benomyl. This study extends our understanding of the roles played by GCN1 in GCN2 activation induced by a variety of stress arrangements and suggests that IMPACT and YIH1 use similar mechanisms for regulating this eIF2α kinase.

Exploring the multifaceted role of GCN1: Implications in cellular responses and disease pathogenesis.

GCN1 is a highly conserved protein present widely across eukaryotes. As an upstream activator of protein kinase GCN2, GCN1 plays a pivotal role in integrated stress responses, such as amino acid starvation and oxidative stress. Through interaction with GCN2, GCN1 facilitates the activation of GCN2, thus initiating downstream signaling cascades in response to cellular stressors. In these contexts, the activation of GCN2 necessitates the presence and action of GCN1. Notably, GCN1 also operates as a ribosome collision sensor, contributing significantly to the translation quality control pathway. These discoveries offer valuable insights into cellular responses to internal stresses, vital for maintaining cellular homeostasis. Additionally, GCN1 exhibits the ability to regulate the cell cycle and suppress inflammation, among other processes, independently of GCN2. Our review outlines the structural characteristics and biological functions of GCN1, shedding light on its significant involvement in the onset and progression of various cancer and non-cancer diseases. Our work underscores the role of GCN1 in the context of drug therapeutic effects, hinting at its potential as a promising drug target. Furthermore, our work delves deep into the functional mechanisms of GCN1, promising innovative avenues for the diagnosis and treatment of diseases in the future. The exploration of GCN1's multifaceted roles not only enhances our understanding of its mechanisms but also paves the way for novel therapeutic interventions. The ongoing quest to unveil additional functions of GCN1 holds the promise of further enriching our comprehension of its mode of action.

RNAi-Mediated Knockdown of Acidic Ribosomal Stalk Protein P1 Arrests Egg Development in Adult Female Yellow Fever Mosquitoes, Aedes aegypti.

After taking a blood meal, the fat body of the adult female yellow fever mosquito, Aedes aegypti, switches from a previtellogenic state of arrest to an active state of synthesizing large quantities of yolk protein precursors (YPPs) that are crucial for egg development. The synthesis of YPPs is regulated at both the transcriptional and translational levels. Previously, we identified the cytoplasmic protein general control nonderepressible 1 (GCN1) as a part of the translational regulatory pathway for YPP synthesis. In the current study, we used the C-terminal end of GCN1 to screen for protein-protein interactions and identified 60S acidic ribosomal protein P1 (P1). An expression analysis and RNAi-mediated knockdown of P1 was performed to further investigate the role of P1 in mosquito reproduction. We showed that in unfed (absence of a blood meal) adult A. aegypti mosquitoes, P1 was expressed ubiquitously in the mosquito organs and tissues tested. We also showed that the RNAi-mediated knockdown of P1 in unfed adult female mosquitoes resulted in a strong, transient knockdown with observable phenotypic changes in ovary length and egg deposition. Our results suggest that 60S acidic ribosomal protein P1 is necessary for mosquito reproduction and is a promising target for mosquito population control.

Boron stress signal is transmitted through the TOR pathway.

Although boron is an essential element for many organisms, an excess amount of it can cause toxicity, and the mechanism behind this toxicity is not yet fully understood. The Gcn4 transcription factor plays a crucial role in the boron stress response by directly activating the expression of the boron efflux pump Atr1. More than a dozen transcription factors and multiple cell signaling pathways have roles in regulating the Gcn4 transcription factor under various circumstances. However, it is unknown which pathways or factors mediate boron signaling to Gcn4. Using the yeast Saccharomyces cerevisiae as a model, we analyzed the factors that converge on the Gcn4 transcription factor to assess their possible roles in boron stress signaling. Our findings show that the GCN system is activated by uncharged tRNA stress in response to boron treatment and that GCN1, which plays a role in transferring uncharged tRNAs to Gcn2, is necessary for the kinase activity of Gcn2. The SNF and PKA pathways were not involved in mediating boron stress, even though they interact with Gcn4. Mutations in TOR pathway genes, such as GLN3 and TOR1, abolished Gcn4 and ATR1 activation in response to boric acid treatment. Therefore, our study suggests that the TOR pathway must be functional to form a proper response against boric acid stress.

Drug-induced eRF1 degradation promotes readthrough and reveals a new branch of ribosome quality control.

Suppression of premature termination codons (PTCs) by translational readthrough is a promising strategy to treat a wide variety of severe genetic diseases caused by nonsense mutations. Here, we present two potent readthrough promoters-NVS1.1 and NVS2.1-that restore substantial levels of functional full-length CFTR and IDUA proteins in disease models for cystic fibrosis and Hurler syndrome, respectively. In contrast to other readthrough promoters that affect stop codon decoding, the NVS compounds stimulate PTC suppression by triggering rapid proteasomal degradation of the translation termination factor eRF1. Our results show that this occurs by trapping eRF1 in the terminating ribosome, causing ribosome stalls and subsequent ribosome collisions, and activating a branch of the ribosome-associated quality control network, which involves the translational stress sensor GCN1 and the catalytic activity of the E3 ubiquitin ligases RNF14 and RNF25.

General control nonderepressible 1 interacts with cationic amino acid transporter 1 and affects Aedes aegypti fecundity.

BACKGROUND: The amino acid transporter protein cationic amino acid transporter 1 (CAT1) is part of the nutrient sensor in the fat body of mosquitoes. A member of the SLC7 family of cationic amino acid transporters, it is paramount for the detection of elevated amino acid levels in the mosquito hemolymph after a blood meal and the subsequent changes in gene expression in the fat body. METHODS: We performed a re-annotation of Aedes aegypti cationic amino acid transporters (CATs) and selected the C-terminal tail of CAT1 to perform a yeast two-hybrid screen to identify putative interactors of this protein. One interesting interacting protein we identified was general control nonderepressible 1 (GCN1). We determined the expression pattern of GCN1 in several adult organs and structures using qRT-PCR and western blots. Finally, we knocked down GCN1 using double-stranded RNA and identified changes in downstream signaling intermediates and the effects of knockdown on vitellogenesis and fecundity. RESULTS: In a screen for Ae. aegypti CAT1-interacting proteins we identified GCN1 as a putative interactor. GCN1 is highly expressed in the ovaries and fat body of the mosquito. We provide evidence that eukaryotic translation initiation factor 2 subunit alpha (eIF2α) phosphorylation changed during vitellogenesis and that RNA interference knockdown of GCN1 in whole mosquitoes reduced egg clutch sizes of treated mosquitoes relative to controls. CONCLUSIONS: Aedes aegypti CAT1 and GCN1 are likely interacting partners and GCN1 is likely necessary for proper egg development. Our data suggest that GCN1 is part of a nutrient sensor mechanism in various mosquito tissues involved in vitellogenesis.

Ribo-Seq and RNA-Seq of TMA46 ( DFRP1) and GIR2 ( DFRP2) knockout yeast strains.

In eukaryotes, stalled and collided ribosomes are recognized by several conserved multicomponent systems, which either block protein synthesis in situ and resolve the collision locally, or trigger a general stress response. Yeast ribosome-binding GTPases RBG1 (DRG1 in mammals) and RBG2 (DRG2) form two distinct heterodimers with TMA46 (DFRP1) and GIR2 (DFRP2), respectively, both involved in mRNA translation. Accumulated evidence suggests that the dimers play partially redundant roles in elongation processivity and resolution of ribosome stalling and collision events, as well as in the regulation of GCN1-mediated signaling involved in ribosome-associated quality control (RQC). They also genetically interact with SLH1 (ASCC3) helicase, a key component of RQC trigger (RQT) complex disassembling collided ribosomes. Here, we present RNA-Seq and ribosome profiling (Ribo-Seq) data from S. cerevisiae strains with individual deletions of the TMA46 and GIR2 genes. Raw RNA-Seq and Ribo-Seq data as well as gene-level read counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE185458 and GSE185286.