Biology subjects

Kadijk, E.

Publications and source records attributed to Kadijk, E..

4 recordsLinked to original sources

bioRxiv2026

PRRC2A, PRRC2B and PRRC2C are Stress Granule Proteins that Promote Translation Through Association with the eIF3 complex

Regulation of mRNA translation is essential for cellular homeostasis, and its dysregulation contributes to cancer, neurodegeneration, and developmental disorders. Stress granules are cytosolic condensates that form during stress-induced translation arrest and are enriched in mRNAs, translation factors, and RNA-binding proteins, but how stress granule proteins modulate translation remains poorly understood. Here, we identify the stress granule components Proline-Rich Coiled-Coil A, B, and C (PRRC2 proteins) as translation regulators. PRRC2 proteins are large, intrinsically disordered paralogs conserved across jawed vertebrates. Functional proteomics revealed that all PRRC2 proteins associate with the 48S translation initiation complex (PIC), whereas PRRC2B additionally interacts with nuclear proteins. Under stress, the proximal interaction network of PRRC2 proteins undergoes dynamic remodeling, including increased interactions with the stress granule scaffold G3BP1. Genetic perturbation shows that the PRRC2 proteins influence stress granule assembly in a context-specific manner, and are collectively required for cell growth in basal conditions due to their essential role in translation. Cells with reduced PRRC2 proteins exhibit a significant reduction in the abundance of more than half of the proteome, with a bias toward translational targets of eIF3d and eIF4G2. Interaction domain mapping and AlphaFold3 modeling revealed that an helix within the putative coiled-coil domain of PRRC2C mediates interactions with the eIF3 core complex. This modeling places the PRRC2C helix in a previously unassigned region of a published cryo-EM density map, validating the protein interaction and the mechanistic role of PRRC2C in translation control. Together, these findings establish PRRC2 proteins as components of the translation initiation machinery that regulate translation through their interactions with the eIF3 complex and other components of the 48S PIC factors, providing a direct mechanistic link between stress granule proteins and translational control.

cell biology↗

bioRxiv2025

Dynamic Proximal Interactomics and Chemical Genetic Screening Reveal CCR4-NOT Sequestration in Stress Granules as a Mechanism for Transcript Stabilization

Cells adapt to stress by rewiring their post-transcriptional gene regulation. Stress granules--biomolecular condensates composed of polyadenylated RNAs and RNA-binding proteins--are implicated in this process, yet their precise functional roles remain debated. To address this, we mapped the dynamic proteomic landscapes of stress granules formed under oxidative and hyperosmotic stress using multi-bait BioID proximity profiling coupled with quantitative mass spectrometry. This analysis revealed context-specific remodeling of proximal interaction networks and identified a conserved, stress-dependent shift in association with the CCR4-NOT deadenylase complex. A complementary genome-wide chemical genetic screen further implicated CCR4-NOT in stress granule biology, showing that reduced CCR4-NOT activity bypassed lipoamide-mediated inhibition of stress granule assembly. Microscopy showed sequestration of the CCR4-NOT complex into stress granules, and global transcriptomic analyses revealed that this relocalization promotes poly(A) tail lengthening and increased abundance of stress-induced survival transcripts. Together, integration of proteomics, chemical genetics, and transcriptomics uncovers a spatial mechanism by which stress granule assembly promotes cellular adaptation to stress through sequestration of CCR4-NOT from the cytosol and consequent remodeling of post-transcriptional regulation.

cell biology↗

bioRxiv2023

Influenza A virus NS1 effector domain is required for PA-X mediated host shutoff

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their own protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine if NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X effects on host gene expression are not simply additive and that these factors functionally interact in mediating host shutoff. IMPORTANCERespiratory viruses including influenza A virus continue to cause annual epidemics with high morbidity and mortality due to limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff - a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the non-structural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterised some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs development of improved live attenuated vaccines.

microbiology↗

bioRxiv2020

Thiopurines activate an antiviral unfolded protein response that blocks viral glycoprotein accumulation in cell culture infection model

Enveloped viruses, including influenza A viruses (IAVs) and coronaviruses (CoVs), utilize the host cell secretory pathway to synthesize viral glycoproteins and direct them to sites of assembly. Using an image-based high-content screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that selectively disrupted the processing and accumulation of IAV glycoproteins hemagglutinin (HA) and neuraminidase (NA). Selective disruption of IAV glycoprotein processing and accumulation by 6-TG and 6-TGo correlated with unfolded protein response (UPR) activation and HA accumulation could be partially restored by the chemical chaperone 4-phenylbutyrate (4PBA). Chemical inhibition of the integrated stress response (ISR) restored accumulation of NA monomers in the presence of 6-TG or 6-TGo, but did not restore NA glycosylation or oligomerization. Thiopurines inhibited replication of the human coronavirus OC43 (HCoV-OC43), which also correlated with UPR/ISR activation and diminished accumulation of ORF1ab and nucleocapsid (N) mRNAs and N protein, which suggests broader disruption of coronavirus gene expression in ER-derived cytoplasmic compartments. The chemically similar thiopurine 6-mercaptopurine (6-MP) had little effect on the UPR and did not affect IAV or HCoV-OC43 replication. Consistent with reports on other CoV Spike (S) proteins, ectopic expression of SARS-CoV-2 S protein caused UPR activation. 6-TG treatment inhibited accumulation of full length S0 or furin-cleaved S2 fusion proteins, but spared the S1 ectodomain. DBeQ, which inhibits the p97 AAA-ATPase required for retrotranslocation of ubiquitinated misfolded proteins during ER-associated degradation (ERAD) restored accumulation of S0 and S2 proteins in the presence of 6-TG, suggesting that 6-TG induced UPR accelerates ERAD-mediated turnover of membrane-anchored S0 and S2 glycoproteins. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and disrupt accumulation of viral glycoproteins. Importantly, our data demonstrate for the first time the efficacy of these thiopurines in limiting IAV and HCoV-OC43 replication in cell culture models. IMPORTANCESecreted and transmembrane proteins are synthesized in the endoplasmic reticulum (ER), where they are folded and modified prior to transport. During infection, many viruses burden the ER with the task of creating and processing viral glycoproteins that will ultimately be incorporated into viral envelopes. Some viruses refashion the ER into replication compartments where viral gene expression and genome replication take place. This viral burden on the ER can trigger the cellular unfolded protein response (UPR), which attempts to increase the protein folding and processing capacity of the ER to match the protein load. Much remains to be learned about how viruses co-opt the UPR to ensure efficient synthesis of viral glycoproteins. Here, we show that two FDA-approved thiopurine drugs, 6-TG and 6-TGo, induce the UPR in a manner that impedes viral glycoprotein accumulation for enveloped influenza viruses and coronaviruses. These drugs may impede the replication of viruses that require precise tuning of the UPR to support viral glycoprotein synthesis for the successful completion of a replication cycle.

microbiology↗