Cycloheximide resistant ribosomes reveal adaptive translation dynamics in C. elegans [RNA-Seq]
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PROVIDER: PRJNA1281580 | ENA |
REPOSITORIES: ENA
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Project description:Cycloheximide resistant ribosomes reveal adaptive translation dynamics in C. elegans
| PRJNA1281666 | ENA
Project description:Cycloheximide resistant ribosomes reveal adaptive translation dynamics in C. elegans [Ribo-Seq]
| PRJNA1282121 | ENA
Project description:Protein translation regulation is critical for cellular responses and development, yet how disruptions during the elongation stage shape these processes remains incompletely understood. Here, we identify and validate a single amino acid substitution (P55Q) in the ribosomal protein RPL-36A of Caenorhabditis elegans that confers complete resistance to high concentrations of the elongation inhibitor cycloheximide (CHX). Heterozygous animals carrying both wild-type RPL-36A and RPL-36A(P55Q) exhibit normal development but intermediate CHX resistance, indicating a partial dominant effect. Leveraging RPL-36A(P55Q) as a single-copy positive selection marker for CRISPR-based genome editing, we introduced targeted modifications into multiple ribosomal protein genes, confirming its broad utility for altering essential loci. In L4-stage heterozygotes, where CHX-sensitive and CHX-resistant ribosomes coexist, ribosome profiling revealed increased start-codon occupancy, suggesting early stalling of CHX sensitive ribosomes. Chronic CHX reduced ribosome collisions, evidenced by fewer disomes and unchanged codon distributions in monosomes. Surprisingly, prolonged elongation inhibition did not activate well characterized stress pathways–including ribosome quality control (RQC), the ribotoxic stress response (RSR), or the integrated stress response (ISR)–as indicated by absence of changes in RPS-10 ubiquitination, eIF2α phosphorylation, PMK-1 phosphorylation, or the transcriptional upregulation of ATF-4 target genes. Instead, RNA-normalized ribosome footprints revealed gene-specific changes in translation efficiency, with nucleolar and P granule components significantly decreased while oocyte development genes were increased. Consistent with these observations, we detected premature oogenesis in L4 animals, suggesting that partial translation elongation inhibition reshapes translation efficiency, to fine-tune developmental timing.
2025-06-29 | GSE300687 | GEO
Project description:Protein translation regulation is critical for cellular responses and development, yet how disruptions during the elongation stage shape these processes remains incompletely understood. Here, we identify and validate a single amino acid substitution (P55Q) in the ribosomal protein RPL-36A of Caenorhabditis elegans that confers complete resistance to high concentrations of the elongation inhibitor cycloheximide (CHX). Heterozygous animals carrying both wild-type RPL-36A and RPL-36A(P55Q) exhibit normal development but intermediate CHX resistance, indicating a partial dominant effect. Leveraging RPL-36A(P55Q) as a single-copy positive selection marker for CRISPR-based genome editing, we introduced targeted modifications into multiple ribosomal protein genes, confirming its broad utility for altering essential loci. In L4-stage heterozygotes, where CHX-sensitive and CHX-resistant ribosomes coexist, ribosome profiling revealed increased start-codon occupancy, suggesting early stalling of CHX sensitive ribosomes. Chronic CHX reduced ribosome collisions, evidenced by fewer disomes and unchanged codon distributions in monosomes. Surprisingly, prolonged elongation inhibition did not activate well characterized stress pathways–including ribosome quality control (RQC), the ribotoxic stress response (RSR), or the integrated stress response (ISR)–as indicated by absence of changes in RPS-10 ubiquitination, eIF2α phosphorylation, PMK-1 phosphorylation, or the transcriptional upregulation of ATF-4 target genes. Instead, RNA-normalized ribosome footprints revealed gene-specific changes in translation efficiency, with nucleolar and P granule components significantly decreased while oocyte development genes were increased. Consistent with these observations, we detected premature oogenesis in L4 animals, suggesting that partial translation elongation inhibition reshapes translation efficiency, to fine-tune developmental timing.
2025-06-29 | GSE300790 | GEO
Project description:Protein translation regulation is critical for cellular responses and development, yet how disruptions during the elongation stage shape these processes remains incompletely understood. Here, we identify and validate a single amino acid substitution (P55Q) in the ribosomal protein RPL-36A of Caenorhabditis elegans that confers complete resistance to high concentrations of the elongation inhibitor cycloheximide (CHX). Heterozygous animals carrying both wild-type RPL-36A and RPL-36A(P55Q) exhibit normal development but intermediate CHX resistance, indicating a partial dominant effect. Leveraging RPL-36A(P55Q) as a single-copy positive selection marker for CRISPR-based genome editing, we introduced targeted modifications into multiple ribosomal protein genes, confirming its broad utility for altering essential loci. In L4-stage heterozygotes, where CHX-sensitive and CHX-resistant ribosomes coexist, ribosome profiling revealed increased start-codon occupancy, suggesting early stalling of CHX sensitive ribosomes. Chronic CHX reduced ribosome collisions, evidenced by fewer disomes and unchanged codon distributions in monosomes. Surprisingly, prolonged elongation inhibition did not activate well characterized stress pathways–including ribosome quality control (RQC), the ribotoxic stress response (RSR), or the integrated stress response (ISR)–as indicated by absence of changes in RPS-10 ubiquitination, eIF2α phosphorylation, PMK-1 phosphorylation, or the transcriptional upregulation of ATF-4 target genes. Instead, RNA-normalized ribosome footprints revealed gene-specific changes in translation efficiency, with nucleolar and P granule components significantly decreased while oocyte development genes were increased. Consistent with these observations, we detected premature oogenesis in L4 animals, suggesting that partial translation elongation inhibition reshapes translation efficiency, to fine-tune developmental timing.
2025-06-28 | GSE300626 | GEO
Project description:Regulation of the efficiency with which an mRNA is translated into proteins represents a key mechanism for controlling gene expression. Such regulation impacts the number of actively translating ribosomes per mRNA molecule, referred to as translation efficiency (TE), which can be monitored using ribosome profiling and RNA-seq, or by evaluating the position of an mRNA in a polysome gradient. Here we show that in budding yeast, under nutrient limiting conditions, the commonly used translation inhibitor cycloheximide induces rapid transcriptional upregulation of hundreds of genes involved in ribosome biogenesis. Cycloheximide also prevents translation of these newly transcribed messages, leading to an apparent drop in TE of these genes under conditions that include key transitions of the yeast metabolic cycle, meiosis, and amino acid starvation; an effect which is abolished when cycloheximide pretreatment is omitted. This response requires TORC1 signaling, and is modulated by the genetic background as well as the vehicle used to deliver the drug. The present work highlights an important caveat to the use of translation inhibitors when measuring TE, and will hopefully aid in future experimental design as well as interpretation of prior results
2019-03-08 | GSE125038 | GEO
Project description:Global restriction of protein synthesis is a hallmark of cellular stress. Using hydrogen peroxide, we monitor the transcript level and also the translation status for each RNA using cycloheximide to freeze elongating ribosomes. Polyribosome fractionation of cell extracts was used to separate highly translated and poorly translated mRNAs that were then separately analysed.
2006-06-01 | E-MEXP-526 | biostudies-arrayexpress
Project description:Selective translation by alternative bacterial ribosomes
| PRJNA525595 | ENA
Project description:Ribosome profiling measures genome-wide translation dynamics at sub-codon resolution. Cycloheximide (CHX), a widely used translation inhibitor to arrest ribosomes in these experiments, has been shown to induce biases in yeast, questioning its use. However, whether such biases are present in datasets of other organisms including humans is unknown. Here we compare different CHX-treatment conditions in human cells and yeast in parallel experiments using an optimized protocol. We find that human ribosomes are not susceptible to conformational restrictions by CHX, nor does it distort gene-level measurements of ribosome occupancy, measured decoding speed or the translational ramp. Furthermore, CHX-induced codon-specific biases on ribosome occupancy are not detectable in human cells or other model organisms. This shows that reported biases of CHX are species-specific and that CHX does not affect the outcome of ribosome profiling experiments in most settings. Our findings provide a solid framework to conduct and analyze ribosome profiling experiments.
2021-07-28 | GSE136940 | GEO
Project description:Translation is initiated by binding of the eIF4F complex to the 5' cap of the mRNA, which is followed by scanning of the initiation codon by scanning ribosomes. Here we demonstrate that the ASC-1 complex (ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with the scanning ribosomes to regulate translation initiation. Sel-TCP-seq analysis revealed that ASCC3, a subunit of ASCC with a helicase domain, localizes predominantly to the 5' untranslated region of mRNAs. Knockdown of ASCC3 resulted in reduced translation efficiency associated with reduced 43S preinitiation complex (PIC) loading and a reduced speed of scanning ribosomes. In addition, depletion of the ubiquitin ligase ZNF598, a sensor of collided 80S ribosomes, also reduces the PIC loading and speed of scanning ribosomes. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes, but also for efficient translation initiation by scanning ribosomes.
2023-03-31 | GSE186502 | GEO