Nuclease-mediated depletion biases in ribosome footprint profiling libraries
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ABSTRACT:
PROVIDER: PRJNA613736 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11362448.fastq.gz | Fastqsanger.gz | |||
| SRR11362449.fastq.gz | Fastqsanger.gz | |||
| SRR11362450.fastq.gz | Fastqsanger.gz | |||
| SRR11362451.fastq.gz | Fastqsanger.gz | |||
| SRR11362452.fastq.gz | Fastqsanger.gz |
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Project description:Ribosome footprint profiling is a high throughput sequencing based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally abundant, ribosomal RNA sequences in order to save on sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.
2020-03-27 | GSE147324 | GEO
Project description:Ribosome footprint profiling is a high-throughput sequencing-based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally very abundant, ribosomal RNA sequences that dominate libraries and increase sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued as a standalone product and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them generally unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.
| S-EPMC7491325 | biostudies-literature
Project description:Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5'-untranslated regions. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast. Ribosome-footprint profiling and RNA-seq (total RNA, poly(A) selected, RiboMinus treated, or Ribo-Zero treated) from log-phase S. cerevisiae. The study includes a reanalysis of the two Samples from GSE53313. The reanalyzed data is linked to the Series record.
2016-02-10 | E-GEOD-75897 | biostudies-arrayexpress
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:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library. Ribosome profiling in cultured mammalian cells under three different footprinting conditions
2012-07-26 | E-GEOD-37744 | biostudies-arrayexpress
Project description:Nucleases, i.e. enzymes that catalyze the hydrolysis of phosphodiester bonds in nucleic acids, are essential tools in molecular biology and biotechnology. Staphylococcus aureus nuclease (MNase) is particularly interesting due to its thermostability and Ca2+-dependence, making it the prime choice for applications where nuclease modulation is critical, such as ribosome profiling in bacteria and halophilic archaea. The latter poses a technical and economical challenge, as high salt reaction conditions negatively impact MNase activity, necessitating large amounts of nuclease to be used for achieving efficient cleavage. Here, we set out to generate an optimized production protocol for two forms of MNase — fully processed MNaseA and the 19 aa propeptide containing MNaseB — and to biochemically benchmark them against a commercial nuclease. Our results show that both MNases are highly active in normal reaction conditions, but MNaseA maintains higher enzymatic activity in high salt concentrations than MNaseB. MNaseA also retains >90% of its activity after multiple freeze-thaw cycles when stored at -80°C in a buffer containing 5% glycerol. Importantly, ribosome profiling experiments in Haloferax volcanii demonstrated that MNaseA produces ribosome footprints highly comparable to those obtained with the commercial nuclease, making it a suitable alternative for high-salt ribosome profiling applications. In conclusion, our method can be easily implemented for efficient MNaseA production, thereby providing access to an effective, robust, and cost-efficient alternative to commercial nucleases, as well as facilitating future translation studies into halophilic organisms.
2024-06-22 | GSE269836 | GEO
Project description:Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5'-untranslated regions. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast.
2016-02-10 | GSE75897 | GEO
Project description:Accurate annotations of protein coding regions are essential for understanding how genetic information is translated into biological functions. The recent development of ribosome footprint profiling provides an important new tool for measuring translation. Here we describe riboHMM, a new method that uses ribosome footprint data along with gene expression and sequence information to accurately infer translated sequences. We applied our method to human lymphoblastoid cell lines and identified 7,863 previously unannotated coding sequences, including 445 translated sequences in pseudogenes and 2,442 translated upstream open reading frames. We observed an enrichment of harringtonine-treated ribosome footprints at the inferred initiation sites, validating many of the novel coding sequences. In aggregate, the novel sequences exhibit significant signatures of purifying selection indicative of protein-coding function, suggesting that many of the novel sequences are functional. We observed that nearly 40% of bicistronic transcripts showed significant negative correlation in the levels of translation of their two coding sequences, suggesting a key regulatory role for these novel translated sequences. Despite evidence for their functional importance, the novel peptide sequences were detected by mass spectrometry at a lower rate than predicted based on data from annotated proteins, thus suggesting that many of the novel peptide products may be relatively short-lived. Our work illustrates the value of ribosome profiling for improving coding annotations, and significantly expands the set of known coding regions.
2015-12-25 | GSE75290 | GEO
Project description:Ribosome profiling — RNase footprinting of ribosome-bound mRNA — has been a unique and powerful method, applied to widespread organisms to survey ribosome traversal along mRNAs. In contrast to eukaryotes, bacterial ribosome footprints show a broad range of sizes, reflecting the differential states of ribosomes. However, the origin remains unclear. Here, we show that rotated state of ribosomes and intramolecular RNA duplexes each extend bacterial ribosome footprints at the 5′ end. Combining elongation inhibitors, cryo-electron microscopy, and ribosome profiling, we demonstrated that the rotated state of ribosomes results in long footprints. Along the subunit rotation, ribosomal protein S1 — a 30S-subunit RNA-binding protein — sterically protects mRNA at the 5′ end of the ribosome from RNase digestion and facilitates elongation of the ribosome. Moreover, we found that ribosomes stalled on ycbZ mRNA generate prolonged footprints because of their unique RNA secondary structure proximal to ribosomes. Through the studies of footprint extension, our results revealed S1-mediated stabilization of translation elongation and provide ribosome profiling approach to probe the conformational diversity of ribosomes in bacteria.
2023-07-28 | GSE155243 | GEO
Project description:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library.
2012-07-26 | GSE37744 | GEO