Initiation Pausing of 80S Ribosome on mRNA Is Governed by 5’UTR and Responsive to mTORC1 Signaling
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Shu-Bing Qian
PROVIDER: E-GEOD-25627 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5M-bM-^@M-^Y untranslated region (5M-bM-^@M-^Y UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway. Monitor the translational status of transcriptome in mammalian cells under different conditions
2011-11-12 | E-GEOD-25626 | biostudies-arrayexpress
Project description:Recent studies have revealed that the mRNA translation is punctuated by ribosomal pauses through the body of transcripts. However, little is known about its physiological significance and regulatory aspects. Here we present a multi-dimensional ribosome profiling approach to quantify the dynamics of initiation and elongation of 80S ribosomes across the entire transcriptome in mammalian cells. We show that a subset of transcripts have a significant pausing of 80S ribosome around the start codon, creating a major barrier to the commitment of translation elongation. Intriguingly, genes encoding ribosome proteins themselves exhibit an exceptionally high initiation pausing on their transcripts. Our studies also reveal that the initiation pausing is dependent on the 5’ untranslated region (5’ UTR) of mRNAs and subject to the regulation of mammalian target of rapamycin complex 1 (mTORC1). Thus, the initiation pausing of 80S ribosome represents a novel regulatory step in translational control mediated by nutrient signaling pathway. Untreated TSC2 WT MEFs, TSC2 KO MEFs and TSC2 WT MEFs, TSC2 KO MEFs treated with 20nM rapamycin for 30 minutes or 3hours were harvested for ribosme profiling. The fraction samples were pooled into three groups based on velocity sedimentation: single ribosome fraction (Small group), fractions with 2 ~ 4 ribosomes (Medium group), and the one with ≥5 ribosomes (Large group). RNA were extracted from the whole cell lysis and each fraction group.
2011-11-12 | E-GEOD-25331 | biostudies-arrayexpress
Project description:Mitochondrial oxidative function is tightly controlled to maintain energy homeostasis in response to nutrient and hormonal signals. An important cellular component in the energy sensing response is the target of rapamycin (TOR) kinase pathway; however whether and how mTOR controls mitochondrial oxidative activity is unknown. Here, we show that mTOR kinase activity stimulates mitochondrial gene expression and oxidative function. In skeletal muscle cells and TSC2-/- MEFs, the mTOR inhibitor rapamycin largely decreased gene expression of mitochondrial transcriptional regulators such as PGC-1alpha and the transcription factors ERRalpha and NRFs. As a consequence, mitochondrial gene expression and oxygen consumption were reduced upon mTOR inhibition. Using computational genomics, we identified the transcription factor YY1 as a common target of mTOR and PGC-1alpha that controls mitochondrial gene expression. Inhibition of mTOR resulted in a failure of YY1 to interact and be coactivated by PGC-1alpha. Notably, knock-down of YY1 in skeletal muscle cells caused a significant decrease in mRNAs of mitochondrial regulators and mitochondrial genes that resulted in a decrease in respiration. Moreover, YY1 was required for rapamycin-dependent repression of mitochondrial genes. Thus, we have identified a novel mechanism in which a nutrient sensor (mTOR) balances energy metabolism via transcriptional control of mitochondrial oxidative function. These results have important implications for our understanding of how these pathways might be altered in metabolic diseases and cancer. Experiment Overall Design: Using Affymetrix MOE430 v2 gene chips, biological triplicates of each condition were analyzed: vehicle-treated, rapamycin-treated, gfp-infected, and pgc-1alpha-infected resulting in a total of 12 samples. Experiment Overall Design: Data were analyzed by RMA (with default settings) in BioConductor 1.2 -- one batch for the Rapamycin vs. Vehicle, and another batch for the PGC vs GFP.
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2021-03-16 | PXD020537 | Pride
Project description:The mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation. Inhibitors of mTOR are being evaluated as anti-tumor agents. Given the emerging role of microRNAs (miRNAs) in tumorgenesis we hypothesized that miRNAs could play important roles in the response of tumors to mTOR inhibitors. Rapamycin resistant myogenic cells developed by long-term rapamycin treatment showed extensive reprogramming of miRNAs expression, characterized by up-regulation of the mir-17~92 and related clusters and down-regulation of tumor-suppressor miRNAs. Antagonists of oncogenic miRNA families and mimics of tumor suppressor miRNAs (let-7) restored rapamycin sensitivity in resistant tumor cells. This study identified miRNAs as new downstream components of the mTOR-signaling pathway, which may determine the response of tumors to mTOR inhibitors. Total RNA extraction and hybridization on Affymetrix microarrays of rapamycin sensitive (RS) cells (BC3H1, mouse brain tumor cell line with myogenic properties, ATCC) cultured in Dulbecco’s modified essential medium (DMEM) media supplemented with 20% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 mg/ml). Rapamycin resistant cells (RR1) were developed by culturing BC3H1 cells in the presence of 1 uM rapamycin for 6 months. Three samples in triplicates: 1) Rapamycin sensitive cells treated with DMSO for 24 h(BC3H1, reference), 2) Rapamycin sensitive cells treated for 24 h with 100 nM rapamycin (BC3H1+R), 3) Rapamycin resistant cells constantly treated with 1uM Rapamycion (RR1+R).
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Project description:Aberrant activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a common molecular event in a large variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell intrinsic consequences of mTORC1 activation remain poorly defined. Here, we identify global trancriptional changes in TSC1 and TSC2 null MEFs, which exhibit constitutive activation of mTORC1, compared to wild-type littermate control lines. A rapamycin time course is included to determine those changes that are dependent on mTORC1 signaling, revealing mTORC1 induced and repressed transcripts. In order to identify mTORC1-dependent transcriptional changes, we compared wild-type MEFs to both Tsc1-/- and Tsc2-/- MEFs following serum starvation, where mTORC1 signaling is off in wild-type cells and fully active in TSC-deficient cells. All cell lines were serum-starved for 24 h, and the Tsc1-/- and Tsc2-/- cells were treated with a time course of rapamycin prior to the isolation of mRNA for microarray analysis. Immortalized wild-type (Tsc2+/+ p53-/-), Tsc1-/- (p53+/+, 3T3-immortalized), and Tsc2-/- (p53-/-, derived from a littermate of the wild-type cell line) MEFs are the three cell lines used in this study and were derived in the laboratory of David J. Kwiatkowski (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). Wild-type and null MEFs were grown to 70% confluence in 10 cm plates and were serum starved for 24 h in the presence of vehicle (DMSO) for 24 h or rapamycin (20 nM) for 2, 6, 12, or 24 h. All vehicle-treated samples (0 h time points) were plated in triplicate and all rapamycin time course samples were plated in duplicate. For each replicate, expression analysis was performed by hybridization to an Affymetrix Mouse 430_2 oligonucleotide microarray chip.
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