Project description:The human mitochondrial degradosome is a complex of the ribonuclease PNPase (Q8TCS8, encoded by PNPT1 gene) and RNA helicase SUV3 (Q8IYB8, encoded by SUPV3L1 gene). The aim of the project was to identify proteins which co-purify with both subunits of the degradosome. To this end we used human 293 cells stably expressing hSuv3 or PNPase with a C-terminal TAP tag or TAP tag fused to a mitochondria targeting sequence (control bait) (cell lines are described in details in (PMID: 19864255). Mitochondria were isolated from the cells, lysed and the protein extracts were subjected to affinity chromatography. Co-purified proteins were identified by mass spectrometry and their amounts were quantified by a label-free approach (MaxQuant). These experiments are part of the project “Dedicated surveillance mechanism controls G-quadruplex forming non-coding RNAs in human mitochondria”.
Project description:The Hedgehog signaling pathway is essential for the maintenance and response of several types of stem cells. To study the transcriptional response of stem cells to HH signaling, we searched for proteins binding to GLI proteins, the transcriptional effectors of the HH pathway in mouse embryonic stem (ES) cells. We purified GLI protein complex from an ES cell line that contained a tamoxifen-inducible 3XFLAG-tagged GLI3 repressor allele by anti-FLAG immunoprecipitation and several novel GLI co-factors were identified in the complex by subsequent mass spectrometry analysis.
Project description:BRCA1-associated protein 1 (BAP1) is a multidomain deubiquitinase (DUB) with a ubiquitin carboxyl-terminal hydrolase (UCH) domain at its N-terminus. BAP1 mainly localizes in the nucleus and primarily functions as a transcriptional regulator in diverse cellular pathways, including cell proliferation, cell cycle progression, cell death and DNA repair. However, a comprehensive understanding of the mechanism by which the nucleocytoplasmic trafficking of BAP1 is regulated remains lacking. To identify protein factors that may be responsible for the nuclear import of BAP1, we used transiently expressed a N-terminally FLAG-tagged BAP1 in HEK293 freestyle (HEK293F) cells as a bait to pull down BAP1-interacting proteins for mass spectrometry-based proteomic analysis.
Project description:We investigated the peripheral mitochondrial localization of nuclear-encoded mRNAs (MLR) in various conditions in which translation was inhibited or the mRNA binding protein context altered (Delta puf3). We used cell fractionation protocols together with microarray to assess the distribution of mRNAs between free and mitochondrion-bound polysomes. Keywords: Mitochondrial-associated RNA localization in cells GSM239122-GSM239127: mitochondrial associated RNA (three biological replicates each in dye swap) GSM239128-GSM239131: mitochondrial associated RNA in presence of 200µg/ml cycloheximide (two biological replicates each in dye swap) GSM239132-GSM239135: mitochondrial associated RNA in presence of 2.1mM puromycin (two biological replicates each in dye swap) GSM239136-GSM239139: mitochondrial associated RNA in Delta Puf3 strain (two biological replicates each in dye swap)
Project description:Identification of a putative network of actin-associated cytoskeletal proteins in glomerular podocytes defined by co-purified mRNAs. An experimental purification in which a presumed RNA binding protein was used for an RNA immunoprecipitation and microarray analysis (RIP-CHIP). Total cell extracts were harvested from the conditionally immortalized mouse MPC-5 podocyte cell line 14 days after transfer from 33 to 37 degrees Celsius. The MPC-5 cell lines were stably expressing the Wilm's Tumor protein (+KTS) isoform carrying a modified TAP tag. A fraction of each extract (Input samples) was saved and the remaining extract was applied to a sepharose IgG column, washed, and eluates were harvested after addition of TEV protease and elution in microbiospin columns. Four input and four eluate samples were then used to harvest RNA using Trizol.
Project description:Mitochondrial biogenesis is regulated by signaling pathways sensitive to extracellular conditions and to the internal environment of the cell. We found that deletion of protein phosphatase 2A (PP2A) or of protein phosphatase 6 (PP6) diminishes the nuclear transcriptional response associated with mtDNA damage. Six samples were analyzed to determine message RNA levels.
Project description:The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP-protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP-TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP-mtRNAP fusion, pulled down associated proteins, and identified them by LC-MS-MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity.
Project description:Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.