ABSTRACT: mTRAN proteins are components of the plant mitochondrial small subunits, thought to bind the mRNA 5' regions to initiate translation. This experiment was designed to identify the mTRAN1 binding regions on the plant mitochondrial mRNAs
Project description:The mitochondrial NADH:ubiquinone oxidoreductase, or complex I, is composed of a hydrophobic arm comprising the P module and a hydrophilic arm comprising the N and Q modules. The assembly of complex I is well characterized in humans and catalyzed by a series of assembly factors that join the Q, P, and N modules sequentially. The complex I of protists and plants contains additional ancestral features, like the ferredoxin bridge that connects the matrix and the membrane arms and the carbonic anhydrase domain, which raises the question of their assembly. In this work, a strain where the assembly factor NDUFAF3 has been tagged with a 3xFLAG at the C-terminal extremity has been investigated in the green microalga Chlamydomonas reinhardtii. Like its human homolog, NDUFAF3 interacts strongly with the classical subunits of the Q and P modules, but also with the carbonic anhydrase domain and C1-FDX, one of the subunits of the ferredoxin bridge. The predicted structural positioning of NDUFAF3 within the Q module suggests a role in the formation of the ferredoxin bridge. In contrast, subunits of the N module are only loosely associated with NDUFAF3. We further demonstrate that the N module is attached at a later stage of assembly, suggesting that Chlamydomonas complex I assembles in a human-like pathway. This contrasts with the situation in Angiosperms where the N and the Q modules are attached together before anchoring to the P module. Altogether, these results highlight a conserved and ancestral role of NDUFAF3 in complex I manufacture.
Project description:To study the possibility that the tissue-specific gene targets and regulation by FUL are due to a different composition in the FUL transcription factor (TF) protein complex/es in both tissues, we determined the protein-protein interactions of FUL in planta. Inflorescence meristems (IMs) of 35S:AP1-GR ap1 cal pFUL:FUL-GFP plants were collected to identify meristem-specific protein complexes of FUL, and stage 12 - 16 pistils of pFUL:FUL-GFP ful-1 plants were collected for the pistil-specific FUL protein complexes. Protein complexes were isolated by immunoprecipitation (IP) using anti-GFP antibodies and protein identification was performed using LC-MS/MS, followed by label-free protein quantification.
Project description:Different human mTEC subsets (MUC1, CEACAM5 and SGLT1) were purified by sequential enzymatic digestion (collagenase/dispase, trypsin) followed by enrichment using magnetic beads (CD45 beads, Miltenyi Biotech) and FACS sorting. Cells of the surface phenotype CD45-, CDR2-, EpCAM+ were further subdivided into MUC1+/MUC1-, CEACAM5+/CEACAM5- and SGLT1+/SGLT1- fractions. RNA was isolated using μMACS™ SuperAmp™ protocol (Miltenyi Biotec) and hybridized to Illumina Whole-Genome Expression Beadchips. Gene expression of Antigen-positive and Antigen-negative mTEC subsets was compared. Total RNA was isolated from ex-vivo isolated human mTEC subsets using μMACS™ SuperAmp™ protocol (Miltenyi Biotec)
Project description:We performed immunoprecipitation experiments in young worms using an antibody against Lys48-linked polyUb followed by single shot label-free proteomics.
Project description:We performed immunoprecipitation experiments in young worms using an antibody against Lys63-linked polyUb followed by single shot label-free proteomics.
Project description:In many plant species, roots maintain specific growth angles relative to the direction of gravity, known as gravitropic set point angles (GSA). These contribute to the efficient acquisition of water and nutrients. AtLAZY1/LAZY1-LIKE (LZY) genes are involved in GSA control by regulating auxin flow toward the direction of gravity in Arabidopsis. LZYs are plant-specific unknown proteins with no domain for which the function is inferable. To clarify the molecular function of LZYs, we identified the proteins that interact with them by immunoprecipitation coupled with mass spectrometry.
Project description:With no therapeutics available, there is an urgent need to better understand the pathogenesis of (ortho)flaviviruses which constitute a threat to public health worldwide. During infection, dengue virus (DENV) and Zika virus (ZIKV), two flavivirusesinduce alterations of mitochondria morphology to favor viral replication, suggesting a viral co-opting of mitochondria functions. Here, we performed an extensive transmission electron microscopy-based quantitative analysis to demonstrate that both DENV and ZIKV alter endoplasmic reticulum-mitochondria contact sites(ERMC). This correlated at the molecular level with an impairment of ERMC tethering protein complexes located at the surface of both organelles. Furthermore, virus infection expression modulated the mitochondrial oxygen consumption rate. Consistently, metabolomic and mitoproteomic analyses revealed a decrease in the abundance of several metabolites of the Krebs cycle and changes in the stoichiometry of the electron transport chain. Most importantly, ERMC destabilization by protein knockdown increased virus replication while dampening ZIKV-induced apoptosis. Overall, our results support the notion that flaviviruses hijack ERMCs to generate a cytoplasmic environment beneficial for sustained and efficient replication.
Project description:To identify mutations that occurred in the nuclear and mitochondrial DNA of the yeast subjected to mtDNA base editing or Mito-BE screen, we performed whole-genome sequencing of cultured yeast cells after isolation of mitochondrial DNA.
Project description:Long non-coding (lnc)RNAs play key roles in many biological processes. Elucidating the function of lncRNAs in cell type specification during organ development requires knowledge about their expression in individual progenitor types rather than in whole tissues. To achieve this during cortical development, we used a dual-reporter mouse line to isolate coexisting proliferating neural stem cells, differentiating neurogenic progenitors and newborn neurons and assessed the expression of lncRNAs by paired-end, high-throughput sequencing. We identified 379 genomic loci encoding novel lncRNAs and performed a comprehensive assessment of cell-specific expression patterns for all, annotated and novel, lncRNAs described to date. Our study provides a powerful new resource for studying these elusive transcripts during stem cell commitment and neurogenesis. mRNA profiles of Proliferating Progenitors, Differentiating Progenitors and Neurons from lateral cortex of E14.5 mouse embryos. Each cell type in three biological replicates.
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)