Development of High-throughput 5Pseq and detection of ribosome stalls at termination level in Saccharomyces cerevisiae
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ABSTRACT:
PROVIDER: PRJNA639129 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12004671_1.fastq.gz | Fastqsanger.gz | |||
| SRR12004671_2.fastq.gz | Fastqsanger.gz | |||
| SRR12004672_1.fastq.gz | Fastqsanger.gz | |||
| SRR12004672_2.fastq.gz | Fastqsanger.gz | |||
| SRR12004673_1.fastq.gz | Fastqsanger.gz |
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Project description:We developed a high throughput 5’P sequencing approach (HT-5PSeq) to investigate 5'P mRNA degradome in relation with translation process. This approach is easy and scalable; and uses an affordable rRNA depletion based on duplex-specific nuclease treatment. We use it to investigate in vivo ribosome stalls in S. cerevisiae and S. pombe at single nucleotide resolution. We investigate the 5’P degradation profiles associated to ribosome pausing, its regulation in stress conditions and the relative poly(A) length of mRNA degradation intermediates.
2021-04-02 | GSE152375 | GEO
Project description:We investigated cryptic transcription start site usage, chromatin organization and post-transcriptional consequences in Saccharomyces cerevisiae. We used 5PSeq approach, which measures ribosome dynamics by sequencing the presence of co-translation mRNA degradation intermediates, to assess if cryptic transcripts are engaged in active translation. We show that chromatin-dependent cryptic transcripts can be recognized by ribosomes and have the potential to produce truncated polypeptides by using downs-stream, in-frame start codons. Our work suggests that a significant fraction of chromatin-dependent internal cryptic promoters are in fact alternative truncated mRNA isoforms.
2019-10-01 | GSE118758 | GEO
Project description:It is generally assumed that mRNAs undergoing translation are protected from decay. Here, we show that mRNAs are, in fact, co-translationally degraded. This is a widespread and conserved process affecting most genes, where 5′–3′ transcript degradation follows the last translating ribosome, producing an in vivo ribosomal footprint. By sequencing the ends of 5′ phosphorylated mRNA degradation intermediates, we obtain a genome-wide drug-free measurement of ribosome dynamics. We identify general translation termination pauses in both normal and stress conditions. In addition, we describe novel codon-specific ribosomal pausing sites in response to oxidative stress that are dependent on the RNase Rny1. Our approach is simple and straightforward and does not require the use of translational inhibitors or in vitro RNA footprinting that can alter ribosome protection patterns. RNA samples were quantified according to their 5’modification. Samples were quantified using 2 alternative methods. 5PSeq (measures 5’P RNAs) and 5PSeq-fragmented (measures the sequencing bias present in any sample by randomly fragmenting RNA, re-phosphorilating the 5’ends and subjecting the sample to 5PSeq; it is used as a negative control). Different strains of S. cerevisiae and S. pombe samples were analyzed. Cells were grown both in rich media (YPD or YES) and synthetic defined media (SD). Cells were subjected to different treatments such as inhibition of translation elongation with cyclohexamide (CHX), oxidative stress (0.2 mM H2O2) or inhibition of histidine biosyntesys (3-AT; 100mM 3-amino-1,2,4-triazole). S. cerevisiae samples grown in rich media were also analyzed after CHX treatment and sucrose fractionation into monosome and polyribosome fractionation. All samples were analyzed in biological duplicates.
2015-06-04 | E-GEOD-63120 | biostudies-arrayexpress
Project description:In this study, we examine the impact of poor nutrient conditions on -1 ribosome frameshifting and its effect on mRNA degradation.To explore this process, we utilized HT-5PSeq (Zhang & Pelechano, 2021, PMID:35474692) to measure the ribosome footprint of co-translationally degraded mRNAs in S.cerevisiae under different stresses in YPD and CSM (poor nutrients). To show the mechanism is conserved in bacteria, we performed HT-5PSeq in B.subtilis upon starvation .Our goal with HT-5PSeq is to identify gene- and codon-specific features that contribute to genome-wide frameshifting events.
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Project description:We report the effect of siRNA-mediated ABCE1 KD in HeLa cells, using a modified ribosome profiling protocol adapted from Ingolia et al., 2011 in order to specifically investigate ABCE1 role on translation termination
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Project description:Antimicrobial resistance (AMR) poses a significant threat to public health. Rapid and accurate antimicrobial sensitivity testing is essential to guide effective treatment. Here, we present “simplified 5PSeq” (s5PSeq), a streamlined protocol for profiling 5’ monophosphorylated (5’P) mRNA degradation intermediates that reflect ribosome dynamics in vivo. By capturing antibiotic-induced, context-specific ribosome stalling events, s5PSeq provides a molecular proxy for bacterial growth inhibition—offering a molecular phenotypic readout without the need for culturing. s5PSeq reduces library preparation time to under four hours and incorporates a novel rRNA blocking strategy. We demonstrated its clinical utility by identifying erythromycin-resistant and sensitive Clostridioides difficile clinical isolates. Combining s5PSeq with real-time nanopore sequencing enables fast AMR diagnosis with as few as 3000 reads. In addition to simplifying the study of 5’P co-translational mRNA decay, our work suggests that utilizing information-rich phenotypic molecular readouts can significantly improve AMR diagnostics.
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Project description:Ribosome composition can vary depending on cell type, stress or developmental state. Here we describe circadian clock-dependent changes in ribosome composition in Neurospora crassa. Mass spectrometry of ribosomes isolated at different circadian times identified six ribosomal proteins and one ribosome-associated protein with clock-controlled abundance. We confirmed clock control of eL31-HA abundance in purified ribosomes and found that deletion of eL31 altered translation of many mRNAs and inhibited translation rhythms for 42% of rhythmically translated mRNAs. Because eL31 homologs promote translation termination fidelity, we examined ribosome protected footprint (RPF)-seq reads mapping past stop codons over circadian time to reveal clock-dependent and eL31-enhanced rhythms in translation termination fidelity. We further discovered that eL31 promotes proper ion homeostasis and translation elongation fidelity. Taken together these studies demonstrated that the circadian clock governs daily changes in ribosome composition that control rhythms in translation and impact translation fidelity, likely through changes in intracellular Mg2+ levels.
2025-10-26 | GSE282317 | GEO
Project description:Ribosome composition can vary depending on cell type, stress or developmental state. Here we describe circadian clock-dependent changes in ribosome composition in Neurospora crassa. Mass spectrometry of ribosomes isolated at different circadian times identified six ribosomal proteins and one ribosome-associated protein with clock-controlled abundance. We confirmed clock control of eL31-HA abundance in purified ribosomes and found that deletion of eL31 altered translation of many mRNAs and inhibited translation rhythms for 42% of rhythmically translated mRNAs. Because eL31 homologs promote translation termination fidelity, we examined ribosome protected footprint (RPF)-seq reads mapping past stop codons over circadian time to reveal clock-dependent and eL31-enhanced rhythms in translation termination fidelity. We further discovered that eL31 promotes proper ion homeostasis and translation elongation fidelity. Taken together these studies demonstrated that the circadian clock governs daily changes in ribosome composition that control rhythms in translation and impact translation fidelity, likely through changes in intracellular Mg2+ levels.
2025-10-26 | GSE282318 | GEO
Project description:Candida albicans is a leading cause of fungal infections in immunocompromised patients. Management of candidemia relies on a few antifungal agents, with fluconazole being first line therapy. The emergence of fluconazole-resistant strains highlights the pressing need to improve our molecular understanding of the drug response mechanisms. By sequencing the 5’P mRNA degradation intermediates, we show that co-translational mRNA decay is common in C. albicans and characterize how in vivo 5´-3´ exonuclease degradation trails the last translating ribosome. Thus, the study of the 5'P mRNA degradome (5PSeq) offers a simple and affordable way to measure ribosome dynamics and identify codon specific ribosome stalls in response to drugs and amino acid deprivation. Building upon this, we combine RNA-Seq and 5PSeq to study the early response of sensitive and resistant C. albicans isolates to fluconazole. Our results highlight that transcriptional responses, rather than changes in ribosome dynamics, are the main driver of Candida resistance to fluconazole.
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Project description:Candida albicans is a leading cause of fungal infections in immunocompromised patients. Management of candidemia relies on a few antifungal agents, with fluconazole being first line therapy. The emergence of fluconazole-resistant strains highlights the pressing need to improve our molecular understanding of the drug response mechanisms. By sequencing the 5’P mRNA degradation intermediates, we show that co-translational mRNA decay is common in C. albicans and characterize how in vivo 5´-3´ exonuclease degradation trails the last translating ribosome. Thus, the study of the 5'P mRNA degradome (5PSeq) offers a simple and affordable way to measure ribosome dynamics and identify codon specific ribosome stalls in response to drugs and amino acid deprivation. Building upon this, we combine RNA-Seq and 5PSeq to study the early response of sensitive and resistant C. albicans isolates to fluconazole. Our results highlight that transcriptional responses, rather than changes in ribosome dynamics, are the main driver of Candida resistance to fluconazole.
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