RNA-sequencing of RNAs found on eRpL22 paralogue-specific ribosomes in Drosophila melanogaster testis
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE124371 | GEO | 2019/06/14
REPOSITORIES: GEO
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Project description:RNA-sequencing of RNAs found on eRpL22 paralogue-specific ribosomes in Drosophila melanogaster testis
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Project description:Ribosomes execute the transcriptional program in every cell. Critical to sustain nearly all cellular activities, ribosome biogenesis requires the translation of ~200 factors of which 80 are ribosomal proteins (RPs). As ribosome synthesis depends on RP mRNAs translation, a priority within the translatome architecture should exist to ensure the preservation of ribosome biogenesis capacity, particularly under adverse growth conditions. Here we show that under critical metabolic constraints characterized by mTOR inhibition, LARP1 complexed with the 40S subunit protects from ribophagy the mRNAs regulon for ribosome biogenesis and protein synthesis, acutely preparing the translatome to promptly resume ribosomes production after growth conditions return permissive. Characterizing the LARP1-protected translatome revealed a set of 5’TOP transcript isoforms other than RPs involved in energy production and in mitochondrial function, among other processes, indicating that the mTOR-LARP1-5’TOP axis acts at the translational level as a primary guardian of the cellular anabolic capacity
2021-10-30 | GSE186779 | GEO
Project description:In this study, we systematically identified ribosome associated RNAs. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a, expressed under control of its native promoter, was affinity purified from whole cell extracts of cultures grown to mid-log phase. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. To analyze how changes in steady-state mRNA levels (= transcriptome) are related to changes of respective messages in the translatome, total RNA and ribosome associated RNA from stress-treated and untreated cells were analyzed with yeast cDNA microarrays. To achieve this, fluorescently labeled (Cy5) cDNA was prepared from total RNA isolated from yeast cell extracts and from affinity-purified ribosomes, and each sample was mixed with differentially labeled (Cy3) cDNA prepared from a common reference RNA pool, and competitively hybridized on yeast cDNA microarrays. We performed five independent experiments with untreated cells grown in minimal medium (t=0 reference) and three or more independent biological replicates of treated cells. Altogether, we sampled the cell's response to five different "stress treatments" that were applied for relatively short periods of time (10 or 20 minutes) to avoid secondary effects triggered by transcriptional adaption. A stimulus or stress experiment design type is where that tests response of an organism(s) to stress/stimulus. e.g. osmotic stress, behavioral treatment Time: Duration of the treatment Growth Condition: Type of treatment stimulus_or_stress_design
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Project description:Translational regulation in in vivo tissue environments during viral pathogenesis has hardly been scrutinized due to the lack of tissue translatomes upon viral infection despite a number of the translatome studies on virus-infected cells cultured in vitro. We constructed the first temporal profile of lung translatomes during SARS-CoV-2 pathogenesis by applying ribosome profiling (Ribo-seq) to a severe COVID-19 mouse model. Unexpectedly, we observed gradual accumulations of non-canonical Ribo-seq reads representing hitherto-unidentified ribonucleoproteins (RNPs) that are likely involved in impeded translational elongation in the infected tissues. Contemporarily developing ribosome heterogeneity with prominently deviated 5S rRNP association supported the malfunction of elongating ribosomes. The analyses of canonical Riboseq reads (ribosome footprints) highlighted two obstructive characteristics to host gene expression: attenuated translation for transcriptionally up-regulated genes including immune response genes and ribosome stalling on codons within transmembrane domain-coding regions. Our study elucidates hidden molecular features of gene regulation in vivo underlying SARS-CoV-2 pathogenesis.
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