Ontology highlight
ABSTRACT:
PROVIDER: PRJNA512900 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Ribosome profiling has emerged as a powerful method to assess global gene translation, but methodological and analytical challenges often lead to inconsistencies across labs and model organisms. A critical issue in ribosome profiling is nuclease treatment of ribosome-mRNA complexes, as it is important to ensure both stability of ribosomal particles and complete conversion of polysomes to monosomes. We performed comparative ribosome profiling in yeast and mice with various ribonucleases including I, A, S7 and T1, characterized their cutting preferences, trinucleotide periodicity patterns, and coverage similarities across coding sequences, and showed that they yield comparable estimations of gene expression when ribosome integrity is not compromised. However, ribosome coverage patterns of individual transcripts had little in common between the ribonucleases. We further examined their potency at converting polysomes to monosomes across other commonly used model organisms, including bacteria, nematodes and fruit flies. In some cases, ribonuclease treatment completely degraded ribosome populations. Ribonuclease T1 was the only enzyme that preserved ribosomal integrity while thoroughly converting polysomes to monosomes in all examined species. This study provides a guide for ribonuclease selection in ribosome profiling experiments across most common model systems
2016-09-20 | GSE82220 | GEO
Project description:Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosomeprofiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5â and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3â end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation. Ribosome profiling and mRNA-seq libraries from wt yeast comparing different library preparation approaches using different combinations of randomized and non-randomized linkers and RT primers.
2016-08-01 | E-GEOD-84746 | biostudies-arrayexpress
Project description:Ribosome profiling of whi3 mutant yeast
| PRJNA221377 | ENA
Project description:Techniques for systematically monitoring protein translation have lagged far behind methods for measuring mRNA levels. Here we present a ribosome profiling strategy, based on deep sequencing of ribosome protected mRNA fragments, that enables genome-wide investigation of translation with sub-codon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control both for determining absolute protein abundance and for responding to environmental stress. We also observed distinct phases during translation involving a large decrease in ribosome density going from early to late peptide elongation as well as wide-spread, regulated initiation at non-AUG codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible. Examine replicates of ribosome footprints and mRNA abundance in biological replicates of log-phase growth and acute amino acid starvation
2009-02-12 | E-GEOD-13750 | biostudies-arrayexpress
Project description:Techniques for systematically monitoring protein translation have lagged far behind methods for measuring mRNA levels. Here we present a ribosome profiling strategy, based on deep sequencing of ribosome protected mRNA fragments, that enables genome-wide investigation of translation with sub-codon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control both for determining absolute protein abundance and for responding to environmental stress. We also observed distinct phases during translation involving a large decrease in ribosome density going from early to late peptide elongation as well as wide-spread, regulated initiation at non-AUG codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible.
2009-02-12 | GSE13750 | GEO
Project description:The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here
we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and
genome-wide ribosome profiling analyses we show that this mechanism, initially derived from studies
of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in
membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common
features. First, they contain long and highly structured CDSs, including a region located around 42 to
120 nucleotides after the translation initiation site, second, they are directly bound by Dhh1 with a
specific binding distribution and third, complementary experimental approaches suggest that they are
activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a conserved layer of translational control that involve
RNA helicases and RNA folding within CDS providing novel opportunities for regulation of
membrane and secretome proteins.
2016-11-04 | GSE87892 | GEO
Project description:The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site, second, they are directly bound by Dhh1 with a specific binding distribution and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins.
2016-11-04 | GSE87888 | GEO