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The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments in HuH-7 cells

ABSTRACT: To globally analyse the lncRNAs with potential coding ability of HCC cells, we performed Ribo-seq using HuH-7 cells. The cycloheximide (CHX) was added to inhibit the translational elongation of ribosomes of the HuH-7 cells.Furthermore,we identified the candidate ORFs with the selected RPF reads using the RiboCode software.

ORGANISM(S): Homo sapiens

PROVIDER: GSE128320 | GEO | 2020/04/30

REPOSITORIES: GEO

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p53-inducible long non-coding RNAs encode functional peptides in hepatocellular carcinoma cells [Ribo-seq]

Project description:To globally analyse the lncRNAs with potential coding ability upon DNA damage, we performed Ribo-seq using ADR-treated HepG2 cells. The HepG2 cells were treated with 500 ng/mL ADR for 24 h, and cycloheximide (CHX) was added to inhibit the translational elongation of ribosomes.Furthermore,we identified the candidate ORFs with the selected RPF reads using the RiboCode software.

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Ribosome profiling of HEK293 cells.

Project description:The development of the ribosome profiling (ribo-seq) technique has enabled the measurement of translation at a genome-wide level. There are several variants of the ribosome profiling technique that use different translation inhibitors. The regular ribo-seq utilizes Cycloheximide (CHX), a translation elongation inhibitor to freeze all translating ribosomes. In contrast to CHX, the translation inhibitor lactimidomycin (LTM) and harringtonine (Harr) have a much stronger effect on initiating ribosomes. The use of these inhibitors allows for the global mapping of translating initiating sites (TISs) when they are coupled with ribosome profiling (TI-seq). We have developed a computational tool to detect and/or quantitatively compare translation initiation from TI-seq data, and predict novel ORFs from regular CHX-based ribo-seq data. Two replicates of CHX-based ribo-seq experiments and one Harr based ribo-seq were performed in HEK293 cells for confirming the ORFs predicted using public available data.

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REPARATION: Ribosome Profiling Assisted (Re-) Annotation of Bacterial genomes

Project description:The delineation of genes in bacteria has remained an important challenge because prokaryotic genomes are often tightly packed frequently resulting in overlapping genes. We hereby present a de novo approach called REPARATION (RibosomeE Profiling Assisted (Re-)AnnotaTION) to delineate translated open reading frames (ORFs) in bacteria independent of (available) genome annotation. By deep sequencing of ribosome protected mRNA fragments (RPF) to map translating ribosomes across the entire genome, REPARATION takes advantage of the recently developed ribosome profiling (Ribo-seq) technique. REPARATION starts by traversing the entire genome to generate all possible ORFs and then collects their corresponding RPF signal information. Based on a growth curve model to estimate minimum ORF read density and Ribo-seq RPF coverage, thresholds indicative of translation is estimated. Finally, our algorithm applies a random forest model to build a classifier to classify putative protein coding ORFs. We evaluated the performance of REPARATION on 3 annotated bacterial species using in-house generated Ribo-seq data and matching N-terminal and shotgun proteomics data next to publically available Ribo-seq data. In all cases, about 80% of the ORFs predicted by REPARATION were previously annotated as protein coding. While 13-20% were variants of previously annotated ORFs and about 3-4% point to novel translated ORFs within intergenic or other regions previously annotated as non-coding. Without stringent length restrictions REPARATION was able to identify several small ORFs (sORFs). Multiple supportive evidence from matching MS data and sequence conservation analysis was obtained to validate predicted ORFs.

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The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments

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

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jaw-D;pTCP4::mTCP4:GR_Cycloheximide + Dexamethasone microarray

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The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments.

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. In addition, 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 pretreating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5-7 days to generate a completed ribosome profiling sequencing library. Sequencing and data analysis require a further 4-5 days.

| S-EPMC3535016 | biostudies-literature

The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments

2012-07-26 | GSE37744 | GEO

The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments in HuH-7 cells

Project description:The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments in HuH-7 cells

| PRJNA527113 | ENA

Humans and other commonly used model organisms are resistant to cycloheximide-mediated biases in ribosome profiling experiments

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

Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments

Project description:During translation elongation, the ribosome ratchets along its mRNA template, incorporating each new amino acid and translocating from one codon to the next. The elongation cycle requires dramatic structural rearrangements of the ribosome. We show here that deep sequencing of ribosome-protected mRNA fragments reveals not only the position of each ribosome but also, unexpectedly, its particular stage of the elongation cycle. Sequencing reveals two distinct populations of ribosome footprints, 28-30 nucleotides and 20-22 nucleotides long, representing translating ribosomes in distinct states, differentially stabilized by specific elongation inhibitors. We find that the balance of small and large footprints varies by codon and is correlated with translation speed. The ability to visualize conformational changes in the ribosome during elongation, at single-codon resolution, provides a new way to study the detailed kinetics of translation and a new probe with which to identify the factors that affect each step in the elongation cycle. Ribosome profiling, or sequencing of ribosome-protected mRNA fragments, in yeast. We assay ribosome footprint sizes and positions in three conditions: untreated yeast (3 replicates) and yeast treated with translation inhibitors cycloheximide (2 replicates) and anisomycin (2 biological replicates, one technical replicate). We also treat yeast with 3-aminotriazole to measure the effect of limited histidine tRNAs on ribosome footprint size and distribution (two treatment durations).

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