Biology subjects

Bekker-Jensen, S.

Publications and source records attributed to Bekker-Jensen, S..

8 recordsLinked to original sources

bioRxiv2026

RNF25 restrains GCN2 hyperactivation to sustain protein synthesis and cell proliferation in response to RNA damage

Regulation of protein synthesis is essential for maintaining cellular homeostasis during stress. The integrated stress response (ISR) is a conserved signaling pathway that modulates global mRNA translation through four eIF2 kinases--GCN2, PKR, PERK, and HRI. However, how these kinases are selectively activated and tuned to distinct stress signals to direct appropriate cell fate decisions remains poorly understood. Here, we employ ultra-deep mutagenesis screens to systematically map regulators of protein synthesis across diverse stress perturbations in human cells. This comparative approach identifies stress-specific translational control factors, including a previously unrecognized role for the E3 ubiquitin ligase RNF25 in selectively sustaining translation following UV irradiation and other RNA-damaging treatments. In this context, we demonstrate that RNF25 operates independently of its partner RNF14, and that its ubiquitin ligase activity, as well as its RWD-domain, is required to restrain excessive activation of the eIF2 kinase GCN2. Accordingly, loss of RNF25 results in hyperactivation of GCN2, exacerbated translation shutdown, and impaired cell proliferation following RNA damage--phenotypes that can be fully reversed by genetic or pharmacological inhibition of GCN2. Together, these findings uncover a previously unappreciated RNF25-GCN2 signaling axis and identify ISR-driven toxicity as a potential vulnerability in combination with RNA-damaging chemotherapeutics.

cell biology↗

bioRxiv2025

Resolution of collided ribosomes by ZNF598 is required for adipocyte energy dissipation

Cellular adaptation to stress evoked by high energy demand requires tight coordination between transcriptional programs, protein synthesis, and organelle function. In many physiological contexts, metabolic remodeling depends on increased mitochondrial capacity and activity, placing high demands on the translational machinery. Beige and brown adipocytes, which undergo rapid mitochondrial expansion and activation in response to physiological cues, provide a powerful model to study how cells adapt their translational output to meet increased metabolic demand. How translational fidelity is maintained, and stress-associated conflicts in protein synthesis are resolved during such adaptive processes, remains incompletely understood. Here, we identify ZNF598 as a key factor that resolves translation stress caused by ribosome collisions during metabolic adaptation of adipocytes. We show that diverse physiological stimuli, including hormonal signaling, environmental challenges, and dietary changes, induce ZNF598 and associated enzymes in metabolically active cells. ZNF598 is required for efficient mitochondrial biogenesis and function, supporting adaptive increases in respiration and energy expenditure. Loss of ZNF598 compromises these adaptive responses and leads to metabolic dysfunction, whereas enhancement of ZNF598 activity improves cellular and organismal metabolic flexibility. Together, these findings indicate that distal steps of the translational machinery are a rate-limiting factor for adaptation to increased energy demand.

cell biology↗

bioRxiv2025

ZAKα is a sensor of mRNA stasis at the ribosomal exit channel

Despite a growing interest in the ribotoxic stress response (RSR), it remains unknown how the upstream p38 and JNK-activating MAP3 kinase ZAK senses translational impairment. Combining Alphafold3 prediction and RNA crosslinking and immunoprecipitation (CLIP), we uncover that ZAK dynamically monitors the mRNA exit channel of elongating ribosomes for mRNA stasis. This is accomplished by ZAK via its direct interactions with the ribosomal proteins RACK1 and RPS27 as well as with the 18S rRNA helix-26. In this conformation, four mRNA-binding peptides in ZAK span across the path of ribosome-exiting mRNA. Progressive elongation effectively threads ZAK off the ribosome, while mRNA stasis stabilizes the interaction allowing for kinase activation. Prolonged binding of ZAK to slow-elongating, stalled and collided ribosomes is associated with sequestration of the inhibitory SAM domain on RACK1, allowing for transient ZAK dimerization, activation loop trans-autophosphorylation and RSR activation. We propose that compromised ribosome processivity constitutes a common ribotoxic stress signal and that ZAK is a ribosome collision-agnostic sensor of such perturbations.

molecular biology↗

bioRxiv2023

Initiation of a ZAKa-dependent Ribotoxic Stress Response by the Innate Immunity Endoribonuclease RNase L

RNase L is a regulated endoribonuclease in higher vertebrates that functions in antiviral innate immunity. Interferons induce OAS enzymes that sense double-stranded RNA of viral origin leading to synthesis of 2,5-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L inhibits viral infections. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A was directly introduced into cells. Here we report that RNase L activation by 2-5A causes a ribotoxic stress response that requires the ribosome-associated MAP3K, ZAK. Subsequently, the stress-activated protein kinases (SAPK) JNK and p38 are phosphorylated. RNase L activation profoundly altered the transcriptome by widespread depletion of mRNAs associated with different cellular functions, but also by SAPK-dependent induction of inflammatory genes. Our findings show that 2-5A is a ribotoxic stressor that causes RNA damage through RNase L triggering a ZAK kinase cascade leading to proinflammatory signaling and apoptosis. HighlightsO_LIRNase L signals antiviral innate immunity from the ribosome C_LIO_LIThe ribotoxic stress response requires the MAP3K ZAK C_LIO_LIRNA cleavage leads to transcription of proinflammatory genes C_LIO_LIZAK contributes to apoptosis downstream of RNase L activity C_LI

immunology↗

bioRxiv2023

Mechanistic basis for nigericin-induced NLRP1 inflammasome activation in human epithelial cells

Nigericin, an ionophore derived from Streptomyces hygroscopicus, is arguably the most commonly used tool compound to study the NLRP3 inflammasome. Recent findings, however, showed that nigericin also activates the NLRP1 inflammasome in human keratinocytes. In this study, we resolve the mechanistic basis of nigericin-driven NLRP1 inflammasome activation. In multiple non-hematopoietic cell types, nigericin rapidly and specifically inhibits the elongation stage of the ribosome cycle by depleting cytosolic potassium ions. This activates the ribotoxic stress response (RSR) sensor kinase ZAK[a], p38 and JNK, as well as the hyperphosphorylation of the NLRP1 linker domain. As a result, nigericin-induced pyroptosis in human keratinocytes is blocked by extracellular potassium supplementation, ZAK[a] knockout or pharmacologic inhibitors of ZAK[a] and p38 kinase activities. By surveying a diverse panel of ionophores, we show that the electroneutrality of potassium efflux is essential to activate ZAK[a]-driven RSR, likely because a greater extent of K+ depletion is necessary to activate ZAK[a]-NLRP1 than NLRP3. These findings resolve the mechanism by which nigericin activates NLRP1 in nonhematopoietic cell types and demonstrate an unexpected connection between RSR, perturbations of potassium ion flux and innate immunity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/546021v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1afe96aorg.highwire.dtl.DTLVardef@53c59org.highwire.dtl.DTLVardef@1bd14d1org.highwire.dtl.DTLVardef@a1bccc_HPS_FORMAT_FIGEXP M_FIG C_FIG SIGNIFICANCENigericin is familiar to the inflammasome field as the most robust and commonly used NLRP3 inducer. It has enabled numerous breakthroughs in the field linking NLRP3 activation to potassium efflux. In this manuscript, we report that nigericin activates an alternate inflammasome sensor, NLRP1 in primary human skin, nasal and corneal epithelial cells. NLRP1 activation by nigericin requires K+ efflux-driven ribosome stalling and the ribotoxic stress response (RSR) sensor MAP3K, ZAK[a]. We further identify the key biophysical principles that explain why only a subset of K+ ionophores, exemplified by nigericin, function as super inflammasome agonists that can activate either NLRP1 or NLRP3, depending on cell type. These results reveal an unexpected connection between RSR, potassium ion flux and innate immunity.

immunology↗

bioRxiv2022

PRMT5-mediated regulatory arginine methylation of RIPK3

The TNF receptor-interacting protein kinases (RIPK)-1 and 3 are regulators of extrinsic cell death response pathways, where RIPK1 makes the cell-survival or death decisions by associating with distinct complexes mediating survival signaling, caspase activation or RIPK3-dependent necroptotic cell death in a context dependent manner. Using a mass spectrometry-based screen to find new components of the ripoptosome/necrosome, we discovered the protein-arginine methyltransferase (PRMT)-5 as a direct interaction partner of RIPK1. Interestingly, RIPK3 but not RIPK1 was then found a target of PRMT5-mediated symmetric arginine dimethylation. A conserved arginine residue in RIPK3 (R486 in human, R415 in mouse) was identified as the evolutionarily conserved target for PRMT5-mediated symmetric dimethylation and the mutations R486A and R486K in human RIPK3 almost completely abrogated its methylation. Rescue experiments using these non-methylatable mutants of RIPK3 demonstrated PRMT5-mediated RIPK3 methylation to act as an efficient mechanism of RIPK3-mediated feedback control on RIPK1 activity and function. Therefore, this study reveals PRMT5-mediated RIPK3 methylation as a novel modulator of RIPK1-dependent signaling.

cell biology↗

bioRxiv2021

Spatial-proteomics reveal in-vivo phospho-signaling dynamics at subcellular resolution

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry (MS)-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmarked the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in-vitro in HeLa cells and in-vivo in mouse tissues. Finally, we investigated the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io.

systems biology↗

bioRxiv2018

Alternative translation initiation generates a functionally distinct isoform of the stress-activated kinase MK2

Shaping of the proteome by alternative translation is an important mechanism of post-transcriptional gene regulation. It can lead to the expression of multiple protein isoforms originating from the same mRNA. Here we show that a novel, abundant and long isoform of the stress/p38MAPK-activated kinase MK2, a key regulator of transcription, migration, death signaling and post-transcriptional gene regulation, is constitutively translated from an alternative CUG translation initiation start site located in the 5'UTR of its mRNA. GC-rich sequences and putative G-quadruplex structures influence the usage of that codon as a translation initiation start site and the RNA helicase eIF4A1 is needed to ensure alternative isoform translation. We recapitulated the usage of the alternative start codon and determined the molecular properties of the short and a long MK2 isoforms. Phenotypically, only the short isoform phosphorylated Hsp27, supported migration and stress-induced immediate early gene (IEG) expression. Interaction profiling by quantitative mass-spectrometry revealed short isoform-specific binding partners that were associated with migration. In contrast, the long isoform contains additional putative phosphorylation sites in its unique N-terminus. In sum, our data reveal a longer and previously non-described isoform of MK2 with distinct physiological properties originating from alternative translation.

molecular biology↗