Translational profiling of RBMX-overexpressed HEK293 cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE68990 | GEO | 2019/12/01
REPOSITORIES: GEO
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Project description:Wild-type and RBMX-knockdown HEK293 cells where profiled at the transcriptome and translatome levels for the expression of 50 homeotic genes. Targeted RNA-seq libraries were built and sequenced on an Illumina MiSeq machine to allow for accurate gene expression quantification of this specific set of genes. To assess the binding of RBMX to the same set of mRNAs, RNA immunoprecipitation was also performed on HIS-HA tagged RBMX expressing HEK293 cells.
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Project description:This experiment investigates the effect of mild cold-shock (32 degrees C, 24 hours) on the translatome of human HEK293 cells. After cold-shock, cells were treated with cycloheximide to block elongation, then sucrose density gradients were used to separate post-nuclear extracts into polysome and subpolysome fractions. The pooled polysomal fraction was labelled with Cy5, the pooled subpolysomal fraction was labelled with Cy3, and both were hybridised to the same array.
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Project description:To characterize the binding sites of RBMX and RPA on chromatin, 293T cells were treated with CPT and binding profiles were obtained by ChIP-seq assay. RBMX ChIP-seq profile was highly correlated with RPA ChIP-seq profile, both showing strong peaks at centromeres. To characterize the occupancy sites bound by both RBMX and RPA, we annotated their shared peaks. The results showed that these sequences are mostly enriched on repetitive DNAs including rRNA, simple repeats and satellite DNAs.
2019-07-27 | GSE134980 | GEO
Project description:In this study, we used CAGE followed by deep sequencing to globally profile the transcript 5’ isoforms in the translatome (i.e., polysome-associated RNA) and transcriptome (i.e., total RNA) of human HEK293 cells at single-nucleotide resolution. After removing low-quality sequencing reads, we obtained approximately 18 million and 14 million CAGE tags respectively for the translatome and transcriptome of HEK293 cells. These tags were then mapped to the human genome (assembly GRCh37) using bowtie with two mismatches allowed. Tags mapped to rRNA were less than 17.9% for translatome and 7.5% for transcriptome, indicating high quality of the two CAGE libraries. By comparing CAGE tags between HEK293's translatome and transcriptome, we revealed selective usage of the TSS-derived 5’ ends by polysome.
2019-06-13 | E-MTAB-7382 | biostudies-arrayexpress
Project description:RNA binding proteins (RBPs) are key arbiters of post-transcriptional regulation that have been found to be dysregulated in hematological malignancies. Here, we identify the RBP RBMX and its retrogene RBMXL1 to be required for murine and human myeloid leukemogenesis. RBMX/L1 are overexpressed in acute myeloid leukemia (AML) primary patients compared to healthy individuals, and RBMX/L1 loss delayed leukemia development. Complex chromosomal karyotyping analysis and assay for transposase-accessible chromatin sequencing demonstrated that RBMX/L1 loss led to significant changes in chromatin accessibility and compaction. We found that RBMX/L1 directly binds to mRNAs and affects transcription of multiple loci, including the heterochromatin protein 1 alpha (HP1a) mRNA. Using single molecule resolution RNA fluorescence in situ hybridization, we uncovered that RBMX/L1 controls the nascent transcription of the HP1a locus. Forced HP1a expression rescued the RBMX/L1 depletion effects on cell growth and apoptosis. Overall, we determine that RBMX/L1 control leukemia cell survival by regulating chromatin state through its downstream target HP1a. These findings illuminate a novel mechanism for RBPs directly promoting transcription and suggest RBMX/L1 as well as HP1a as potential novel therapeutic targets in myeloid malignancies.
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Project description:Bladder cancer (BCa) is one of the most common malignant tumors of urinary system and has high incidence rate and mortality but there is a lack of effective treatment. Therefore, an in-depth study of the molecular mechanisms involved in BCa is of great significance for improving the survival of patients with advanced BCa. We found that the expression of RBMX was significantly declined in BCa, especially in muscle-invasive BCa. BCa patients with low levels of RBMX had poor prognoses. By in vitro and in vivo experiments, RBMX was shown to inhibit BCa cells growth and metastasis. Co-IP coupled with MS and GO analysis identified hnRNP A1 as a RBMX-binding protein. Mechanistically, RBMX competitively bond to the RGG box of hnRNP A1 and antagonized the hnRNP A1-mediated regulation of PKM splicing by blocking the binding of the RGG motif of hnRNP A1 to the sequences flanking PKM exon 9, resulting in downregulation of PKM2 and upregulation of PKM1. By decreasing the PKM2/PKM1 ratio, RBMX suppressed BCa cells aerobic glycolysis, which further inhibited tumorigenicity and progression of BCa.
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Project description:We subjected MCF7 cells to starvation with 0.5% charcoal treated serum for 48h and then we added 17-beta estradiol (E2) at final concentration of 10 nM, profiling before and after 60 minutes of treatment the transcriptome and the translatome, coming from the polysomal pool of mRNAs after sucrose gradient separation. Comparison of translatome profile changes with corresponding transcriptome profile changes represents a way of studying translational control networks and the degree of concordance between cellular controls affecting mRNA abundance and cellular controls affecting mRNA availability to translation. It is well known that E2 is a strong transcriptional regulator, while its translational control activity is less characterized. To provide a direct experimental evaluation of E2 induced translational regulation, we compared translatome and transcriptome profiles of E2 treated cells. Keywords: polysomal profiling, translatome profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, estradiol stimulation, estrogen receptor. The comparison between transcriptional and polysomal profiling was used for the discovery of general and mRNA-specific changes in the translation state of the serum starved MCF7 cells transcriptome in response to E2 stimulus. To identify translationally regulated mRNA molecules, gene expression signals derived from the polysomal populations were compared by microarrays analysis to those obtained from unfractionated total RNAs. Polysomal RNA and total RNA were isolated from MCF7 cells serum starved and treated with E2. Cells lysates were collected before (t = 0 min) and after (t = 60 min) E2 treatment. All experiments were run in quadruplicates.
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Project description:Translational profiling of RBMX-overexpressed HEK293 cells
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Project description:Genome-wide analysis of translation has the potential to provide major contributions in understanding the pathophysiology of infection processes, given the complex interplay between pathogens and host cells. This study uncovers the reshaping undergoing in the translational control system of the host in response to staphylococcal α-hemolysin oligomers (rAHL). Keywords: translatome profiling, polysomal profiling, polysomal RNA, translational control, translational profiling, polysome profiling, post-transcriptional regulation, staphylococcal α-hemolysin, pore forming toxins, PTF. The comparison between translatome and transcriptome profiling was used to discover mRNA-specific changes of the SH-SY5Y cells transcriptome and translatome in response to staphylococcal α-hemolysin oligomers (rAHL). To identify translationally regulated mRNAs, gene expression signals derived from the polysomal mRNA populations were compared by microarrays analysis to those obtained from total RNAs. Polysomal mRNA and total mRNA were isolated from SH-SY5Y cells treated with 3nM of extracted oligomers (rAHL) for 2 hours. Cells lysates were collected from untreated cells (control) and from treated cells. All experiments were run in biological triplicates.
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