Unknown,Transcriptomics,Genomics,Proteomics

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Comparative analysis of transcripts from mia40-4int mutant and wild-type Saccharomyces cerevisiae strains quantified in RNA-seq experiment

ABSTRACT: The vast majority of the mitochondrial proteome originates from nuclear genes and is transported into the organelle after synthesis in the cytosol. Complex machineries, which maintain the specificity of protein import and sorting exist. Dysfunctions of mitochondrial protein sorting pathways result in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death. Cellular responses that are caused by slowdown in the transport of mitochondrial proteins are unknown. Here we have used RNA-seq transcription profiling of Saccharomyces cerevisiae to uncover the changes in the cellular transcriptome in the response to mitochondrial protein import dysfunction caused by mutation in the MIA40 gene. Mia40 is a key component of the mitochondrial intermembrane import and assembly (MIA) pathway. Mia40-4int mutant used in this study is not viable at high temperatures, while in the low, permissive temperature it expresses growth rate comparable to the wild-type strain. Even during the permissive temperature culture mutant cells are characterized by decreased accumulation of MIA substrate proteins. Thus permissive growth conditions were used in the present experiment to emphasise primary responses to mitochondrial protein import defect.

INSTRUMENT(S): MagNA Lyser, Ion Torrent Proton

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Piotr Bragoszewski

PROVIDER: E-MTAB-3588 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Long non-coding RNAs (lncRNAs) are recently characterized players that are involved in the regulatory circuitry of self-renewal in human embryonic stem cells (hESCs). However, the specific roles of lncRNAs in this circuitry are poorly understood. Here, we determined that growth-arrest-specific transcript 5 (GAS5), which is a known tumor suppressor and growth arrest gene, is abundantly expressed in the cytoplasm of hESCs and essential for hESC self-renewal. GAS5 depletion in hESCs significantly impaired their pluripotency and self-renewal ability, whereas GAS5 overexpression in hESCs accelerated the cell cycle, enhanced their colony formation ability and increased pluripotency marker expression. By RNA sequencing and bioinformatics analysis, we determined that GAS5 activates NODAL-SMAD2/3 signaling by sustaining the expression of NODAL, which plays a key role in hESC self-renewal but not in somatic cell growth. Further studies indicated that GAS5 functions as a competing endogenous RNA (ceRNA) to protect NODAL mRNA against degradation and that GAS5 transcription is directly controlled by the core pluripotency transcriptional factors (TFs). Taken together, we suggest that the core TFs, GAS5 and NODAL-SMAD2/3 form a feed-forward loop to maintain the hESC self-renewal process. These findings are specific to ESCs and did not occur in the somatic cell lines we tested; therefore, our findings also provide evidence that the functions of lncRNAs vary in different biological contexts. We analyzed long non-coding RNAs in two hESC cell lines (X-01 and H1), and found GAS5 is highly expressed and functional in maintaining hESC self-renewal. We generate stable overexpressed or knockdown hESC cell lines using lentiviral approach. We transfected cells initialy after passage, and lentiviruses are added with daily medium change for three days (at a final concentration of 10^5 IU/ml). Puromycin is added for selection and supplied with daily medium change. Stable cell lines are established after two passages and verified under fluorescence scope. Total RNAs and miRNAs are extracted separately of all three cell lines (LV-NC, LV-GAS5 and LV-shGAS5) and put to sequencing.

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Comparative analysis of transcripts from mia40-4int mutant and wild-type Saccharomyces cerevisiae strains quantified in RNA-seq experiment

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