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:To uncover novel components of the SOX2-associated protein complex, we established E14 mouse embryonic stem (ES) cells stably expressing Flag-HA-tagged SOX2. Whole-cell extracts were prepared and subjected to tandem affinity purification, sequentially using anti-Flag M2 and anti-HA agarose resins . The purified SOX2 complex was separated by SDS-PAGE and visualized by silver staining . Distinct protein bands specifically enriched in the SOX2 immunoprecipitate were excised and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) .
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:Chromatin Immunoprecipitation (ChIP) and Co-Immunoprecipitation (CoIP) assays are the most common approaches to characterize the genomic localization and protein interactors, respectively for a protein of interest. However, these approaches require the use of specific antibodies, which are costly reagents that often face sensitivity and specificity issues. Based on TurboID, we developed PLAMseq (Proximity Labelled Affinity-purified Mass spectrometry plus sequencing), after a short biotin pulse, DNA-protein crosslinks are induced by formaldehyde and the interactors of a protein of interest together with their associated DNA sequences are purified simultaneously and identified by mass spectrometry-based proteomics and Next Generation Sequencing, respectively. To validate PLAMseq, we performed the proteo-genomic characterization of two proteins which genomic loci are very well characterized, namely, RNA polymerase II and CTCF with excellent robustness and reproducibility. Next, we applied PLAMseq to characterize Histone H1 SUMOylation, a histone post-translational modification which study has remained elusive due to the lack of specific reagents. SETDB1 binds to SUMOylated histone H1 and, accordingly, SUMOylated histone H1 colocalize with H3K9me3 at repetitive regions of the genome.
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.