ZAKα Recognizes Stalled Ribosomes through Partially Redundant Sensor Domains
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ABSTRACT:
PROVIDER: PRJNA542891 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9054403.fastq.gz | Fastqsanger.gz | |||
| SRR9054404.fastq.gz | Fastqsanger.gz |
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Project description:Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) re-mains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix14 of 18S ribosomal RNA (rRNA). An adjacent domainin ZAKa also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxinaction. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.
2020-04-15 | GSE131201 | GEO
Project description:Local protein synthesis is a crucial process that maintains synaptic proteostasis. Ribosomes stalled at elongation make up a sizable percentage of mRNAs translated in developing neurons. The protein lost in Fragile X syndrome (FMRP) is highly enriched in RNA granules containing stalled ribosomes, both of which play crucial roles in regulating forms of synaptic plasticity such as metabotropic glutamate receptor-dependent long-term depression (mGLUR-LTD). Previous examination of ribosome protected fragments (RPFs) from neuronal stalled ribosomes have identified motifs matching those found in mRNAs associated with FMRP, as recognized by FMRP cross-linking immunoprecipitation (CLIP) (Anadolu et al., 2023, Journal of Neuroscience doi: 10.1523/JNEUROSCI.1002-22.2023). To investigate if FMRP recognition of these sequences is important for determining where mRNAs are stalled, we examined stalled ribosomes RPFs isolated from P5 mice lacking the FMRP protein. We found that the loss of FMRP had no effect on the proteins associated with RNA granules, structure of the ribosomes and stalling sites, or where accumulations of RPFs occurred. However, there was a significant decrease in the amount of stalling on mRNAs previously shown to be associated with FMRP by cross-linking immunoprecipitation (CLIP). In addition, there was a decrease in the number of RNA granules containing stalled polysomes as assayed by ribopuromycylation in distal neurites and, unlike RNA granules in WT neurons, the remaining distal RNA granules were resistant to reactivation by stimuli that induce mGLuR-LTD. These results show important roles for FMRP in regulating RNA granules containing stalled ribosomes, but not in determining where ribosomes are stalled.
2026-06-18 | GSE291701 | GEO
Project description:FMRP Regulates Neuronal RNA Granules Containing Stalled Ribosomes, Not Where Ribosomes Stall
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Project description:Ribosome stalling at problematic sequences in mRNAs leads to collisions that trigger a collection of quality control events including ribosome rescue, targeting the nascent polypeptide for decay (Ribosome-mediated Quality Control or RQC), and targeting of the mRNA for decay (No Go Decay or NGD). Using a reverse genetic screen in yeast, we identify Cue2 as the endonuclease that is recruited to stalled ribosomes to promote NGD. Following Cue2-mediated cleavage, ribosomes upstream of the cleavage site translate to the end of the truncated mRNA and are rescued by the Dom34:Hbs1 complex. We also show that the putative helicase Slh1 (part of the RQC Trigger or RQT complex) removes collided ribosomes behind the lead stalled ribosome and thereby reduces endonucleolytic cleavage by Cue2. The synergistic activities of Cue2 and Slh1 define two parallel pathways that allow cells to recognize and respond to ribosomes trapped on problematic mRNAs.
2019-06-25 | GSE129128 | GEO
Project description:Despite a growing interest in the ribotoxic stress response (RSR), it remains unknown how the upstream p38 and JNK-activating MAP3 kinase ZAKɑ senses translational impairment. Combining Alphafold3 prediction and RNA crosslinking and immunoprecipitation (CLIP), we uncover that ZAKɑ dynamically monitors the mRNA exit channel of elongating ribosomes. This is accomplished by ZAKɑ via its direct interactions with the ribosomal proteins RACK1 and RPS27 as well as with the 18S rRNA helix-26. In this conformation, four RNA-binding peptides in ZAKɑ span across the path of ribosome-exiting mRNA. Progressive elongation effectively threads ZAKɑ off the ribosome, while mRNA stasis stabilizes the interaction allowing for kinase activation. Prolonged binding of ZAKɑ to slow-elongating, stalled and collided ribosomes is associated with sequestration of the SAM domain on RACK1, allowing for transient ZAKɑ dimerization, activation loop trans-autophosphorylation and RSR activation. Our findings highlight how ZAKɑ senses both stalled and collided ribosomes through overlapping mechanisms.
2026-06-01 | GSE292064 | GEO
Project description:Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on non-colliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S non-functional rRNA decay via poly-ubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting a mRNA stasis sensing activity, and Fap1 sterically hinders formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.
2022-09-15 | GSE159329 | GEO
Project description:Cue2 and Slh1 define parallel pathways to rescue stalled ribosomes
| PRJNA530200 | ENA
Project description:Impairment of translation can lead to stalling and collision of ribosomes which constitute an activation platform for several ribosomal stress-surveillance pathways. Among these is the Ribotoxic Stress Response (RSR), where ribosomal sensing by the MAP3K ZAKa leads to activation of p38 and JNK kinases. Despite these insights, the physiological ramifications of ribosomal impairment and downstream RSR signaling remain elusive. Here we show that stalling of ribosomes is sufficient to activate ZAKa. In response to amino acid deprivation and full nutrient starvation, RSR impacts on the ensuing metabolic responses in cells, nematodes and mice. The RSR-regulated responses in these model systems include regulation of AMPK and mTOR signaling, survival under starvation conditions, stress hormone production and regulation of blood sugar control. In addition, ZAK-/- mice present with a lean phenotype. Our work highlights stalled ribosomes as metabolic signals and demonstrates a role for RSR signaling in metabolic regulation.
2022-11-16 | GSE205191 | GEO
Project description:Mechanism of RACK1-dependent ZAKɑ activation at stalled and collided ribosomes
| PRJNA1236635 | ENA
Project description:Stalled ribosomes are signals for metabolic regulation by the ribotoxic stress response
| PRJNA844063 | ENA