Effects of Triad1-knockdown on ribosome/polysome profiles are reversed by Gcn1-knockdown
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ABSTRACT:
PROVIDER: PRJNA1196904 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31682719.fastq.gz | Fastqsanger.gz | |||
| SRR31682720.fastq.gz | Fastqsanger.gz | |||
| SRR31682721.fastq.gz | Fastqsanger.gz | |||
| SRR31682722.fastq.gz | Fastqsanger.gz | |||
| SRR31682723.fastq.gz | Fastqsanger.gz |
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Project description:Previously, we identified ARIH2, the gene encoding Triad1, as a HoxA10 target gene. We found that HoxA10-induced expression of Triad1 was necessary for termination of emergency granulopoiesis, and antagonized leukemogenesis in a murine model of KMT2A-rearranged AML. In this study, we found that knockdown Triad1 increased polysome-specific mRNAs regulating the cellular response to stress, the DNA-damage response, ribosomal biogenesis, or protein catabolism, but decreased mRNAs regulating mitosis, DNA-repair, DNA and RNA metabolism, or Tp53 activity in U937 cells. Polysome-specific mRNAs for Solute Carrier Proteins (SLCs) were also increased by Triad1-knockdown, consistent with correction of metabolic defects during the ISR. This included SLCs involved in import of amino acids, long chain fatty acids and glucose, and lactate export. In comparison of polysome-specific mRNA profiles with Triad1-knockdown versus knockdown of Triad1 plus Gcn1, we identified the same gene and pathway differences as in comparison of Triad1-knockdown to control cells. There were only minor differences in polysome specific mRNAs in cells with Triad1- plus Gcn1-knockdown versus control cells, encompassing fewer differences than with Triad1-knockdown alone. This suggested that Triad1-knockdown on ribosome/polysome profiles are reversed by Gcn1-knockdown in U937 cells.
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Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs.
2014-06-12 | GSE52799 | GEO
Project description:We use mRNA-seq in combination with polysome profiling to determine translational status for all mRNAs in Drosophila mature oocytes and activated eggs. Puromycin-treated lysates are used as a negative control in polysome profiling experiments. Additionally, we use ribosome footprinting to globally measure translational efficiency of mRNAs in wild type mature oocytes as well as wild type and png mutant activated eggs. Lysates of hand-dissected Drosophila mature oocytes (containing ~540 M-NM-<g of total RNA) were subjected to separation by velocity sedimentation through sucrose gradients. In this way, free mRNAs (present in RNPs fraction) or those comigrating with ribosomal subunits (40S or 60S+80S fractions) or with varying numbers of bound ribosomes (low polysomes (2-4 ribosomes), medium polysomes (5-9 ribosomes), and heavy polysomes (more than 10 ribosomes) can be separated based on their size and collected as sucrose gradient fractions. To compare quantitatively the levels of every mRNA across the polysome gradient fractions, we added 5ng of S. cerevisiae mRNA as an exogenous spike-in to each of the six fractions of interest: RNPs, 40S, 60S+80S, low polysomes, medium polysomes and heavy polysomes. RNA was extraced from these fractions, follwing proteinase K treatment, by hot acid phenol method. In case of unfractionated lysates, RNA was extracted using TRIzol (Invitrogen) according to manufacturerM-bM-^@M-^Ys instructions. mRNA-seq samples were prepared from 1 M-NM-<g of total RNA (in case of sucrose gradient fractions and unfractionated lysates) and subject to Illumina based sequencing. Puromycin-treated lysates of mature oocytes or 0-2h Drosophila activated eggs (containing ~540 M-NM-<g of total RNA) were also subjected to separation by velocity sedimentation through sucrose gradients. Puromycin causes premature termination of elongating ribosomes and thus it can be used to determine whether the mRNAs co-sedimenting with the polysomal peaks (defined here as M-bM-^IM-%5 ribosomes) were actively engaged in translation. As an independent approach to assess translation and obtain information on the position of ribosomes on mRNAs, we employed ribosome footprinting. In addition to analyzing the same samples, as by polysome profiling, we also analyzed png mutant activated eggs by ribosome footprinting. Ribosome footprint profiling measures the number of ribosome-protected fragments (RPFs) derived from the mRNAs of each gene, resulting in a singular value of translational efficiency (TE) for each gene (TE=RPF/RNA).
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Project description:The assessment of transcriptome-wide ribosome binding to mRNAs is useful for studying the dynamic regulation of protein synthesis. Two methods frequently applied in eukaryotic cells that operate at different levels of resolution are polysome profiling, which reveals the distribution of ribosome loads across the transcriptome, and ribosome footprinting (also termed ribosome profiling or Ribo-Seq), which when combined with appropriate data on mRNA expression can reveal ribosome densities on individual transcripts. In this study we develop methods for relating the information content of these two methods to one another, by reconstructing theoretical polysome profiles from ribosome footprinting data. Our results validate both approaches as experimental tools. Although we show that both methods can yield highly consistent data, some published ribosome footprinting datasets give rise to reconstructed polysome profiles with non-physiological features. We trace these aberrant features to inconsistencies in RNA and Ribo-Seq data when compared to datasets yielding physiological polysome profiles, thereby demonstrating that modelled polysomes are useful for assessing global dataset properties such as its quality in a simple, visual approach. Aside from using polysome profile reconstructions on published datasets, we propose that this also provides a useful tool for validating new ribosome footprinting datasets in early stages of analyses.
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Project description:We performed ribosome profiling of polysomes (Poly-RIBOseq) to monitor translation of RNAs associated with polysomes in brain extracts from a mouse model Keywords: ribosome profiling, translation, polysome profiling, Poly-RIBOseq
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Project description:Raw mass spectrometry data associated with the following paper: EDF1 coordinates cellular responses to ribosome collisions.
Related to Figure 6B-6C, Figure 6-figure supplement 1 and Figure 6-source data 1.
Files detail: Light-Polysome (12 BPRP fractions - labeled AO3310-3321); Heavy-Polysome (12 BPRP fractions - labeled AO3322-3333)
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Project description:In this study, we identify leucyl-tRNA synthetase (LARS) as a breast tumor suppressor. To identify the mechanism underlying LARS-mediated breast tumor suppression, we first conducted charged tRNA profiling to assess charged and total tRNA-Leu expression in cell lines with high and low LARS expression. Expression of select charged and total tRNA-Leu isoacceptors were reduced in cell lines with lower LARS expression. We then conducted RNA-Seq and translation profiling including ribosome profiling (Ribo-Seq), polysome profiling in LARS-depleted cell lines. To test our hypothesis in vivo, we also performed RNA-Seq of ribosome-associated RNAs in Lars depleted PyMT tumors within RiboTag mice. The analyses implicate LARS as a regulator of leucine-rich translation.
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