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VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

ABSTRACT: VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

PROVIDER: PRJNA593241 | ENA |

REPOSITORIES: ENA

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VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide

Project description:Roles for ribosomal RNA (rRNA) in gene regulation remain largely unexplored. With hundreds of rDNA units positioned across multiple loci, it is not possible to genetically modify rRNA in mammalian cells, hindering understanding of ribosome function. It remains elusive whether expansion segments (ESs), tentacle-like rRNA extensions that vary in sequence and size across eukaryotic evolution, may have functional roles in translation control. Here, we develop VELCRO-IP RNA-seq: a versatile methodology to generate species-adapted ESs and to map specific mRNA regions across the transcriptome that preferentially associate with ESs. By applying VELCRO-IP RNA-seq to a mammalian ES, ES9S, a large array of transcripts was identified that are selectively recruited to ribosomes via an ES. We further characterize a set of 5’ UTRs that facilitate cap-independent translation through ES9S-mediated ribosome binding. Thus, we present a technology for studying the enigmatic ESs of the ribosome, revealing their function in gene-specific translation.

2021-01-29 | GSE141382 | GEO

VELCRO-IP RNA-seq reveals ribosome expansion segment function in translation genome-wide.

Project description: Not available

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tRNA synthetases are recruited to yeast ribosomes by rRNA expansion segment 7L but do not require association for func-tionality

Project description:Ribosomes expanded during evolution on the protein and rRNA level. The expanded regions do not change the active sites of ribosomes and thus do not alter the peptide bond formation reaction catalysed by the ribosome. To what purpose these expansions were selected is not fully understood. Here, we deleted one rRNA expansion segment (ES7La) and addressed physiological consequences. We found a mild ribosome biogenesis defect accompanied by slightly reduced translation. On a molecular level, ribosomes displayed reduced association to three aminoacyl-tRNA synthetases. Loss of ribosome binding however did not alter synthetase activity suggesting, that tRNAs are not channelled for enhanced local translation in the case of these synthetases.

2022-02-16 | PXD029751 | Pride

Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells

Project description:Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells. Ribosome profiling or stranded RNAseq (ribominus) with murine embryonic stem cells treated with either DMD-pateamineA, Puromycin, Harringtonine or vehicle (no drug control). Two replicates per condition.

2016-01-12 | E-GEOD-67741 | biostudies-arrayexpress

A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis [aramis mRNA IP]

Project description:Ribosomal defects perturb stem cell differentiation, causing diseases called ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discovered three RNA helicases are required for ribosome biogenesis and for Drosophila oogenesis. Loss of these helicases, which we named Aramis, Athos and Porthos, lead to aberrant stabilization of p53, cell cycle arrest and stalled GSC differentiation. Unexpectedly, Aramis is required for efficient translation of a cohort of mRNAs containing a 5’-Terminal-Oligo-Pyrimidine (TOP)-motif, including mRNAs that encode ribosomal proteins and a conserved p53 inhibitor, Novel Nucleolar protein 1 (Non1). The TOP-motif co-regulates the translation of growth-related mRNAs in mammals. As in mammals, the La-related protein co-regulates the translation of TOP-motif containing RNAs during Drosophila oogenesis. Thus, a previously unappreciated TOP-motif in Drosophila responds to reduced ribosome biogenesis to co-regulate the translation of ribosomal proteins and a p53 repressor, thus coupling ribosome biogenesis to GSC differentiation.

2022-03-24 | GSE190969 | GEO

Translation Inhibitors Cause Abnormalities in Ribosome Profiling Experiments

Project description:Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in wide-spread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods. Ranging concentrations of cycloheximide and various stress contitions were tested with Ribo-seq

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Ribosome profiling on polysomes extracted via sucrose gradient and IP

Project description:Technical feasibility study to determine data quality and reproducibilty. Ribosome profiling was performed by either sucrose gradient (SG) or immunoprecipitation (IP). 1 sucrose gradient sample, 2 immunoprecipitation samples in two concentrations of beads

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Genome-Wide Translation Profiling by Ribosome-Bound tRNA Capture

Project description:In the ribosome complex, tRNA is a critical element of mRNA translation. We reported a new technology for profiling ribosome-embedded tRNAs and their modifications. With the method, we generated a comprehensive survey of the quanity and quality of intra-ribosomal tRNAs (Ribo-tRNA-seq). Ribo-tRNA-seq can provide new insights on translation control mechanism in diverse biological contexts.

2018-03-19 | E-MTAB-6576 | biostudies-arrayexpress

Ribosome profiling on polysomes extracted via sucrose gradient and IP

Project description:Technical feasibility study to determine data quality and reproducibilty. Ribosome profiling was performed by either sucrose gradient (SG) or immunoprecipitation (IP).

2016-02-17 | GSE74511 | GEO

Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells

Project description:Ribosome profiling with translation inhibitors reveals pervasive translation in murine ES cells.

2016-01-12 | GSE67741 | GEO