Project description:Dysfunction of the motor subunit of the TIM23 translocase, the PAM complex located on the matrix side of the mitochondrial inner membrane in Saccharomyces cerevisiae, was shown to cause a decrease in mitochondrial protein import and precursor accumulation in the cytosol. We used an analogous model to study the non-mitochondrial response to defective mitochondrial import machinery in Caenorhabditis elegans in which we depleted DNJ-21 as the functional homolog of yeast Pam18. To gain a broader insight in potential changes in Caenorhabditis elegans proteome upon DNJ-21 depletion we performed a quantitative, label-free proteomics analysis. We compared protein levels upon knockdown of dnj-21 (dnj-21 RNAi) with control conditions (Empty vector RNAi). Synchronized N2 wild type worms were grown on NGM plates seeded with E. coli HT115(DE3) transformed with a construct targeting dnj-21 gene or with empty vector L4440 as a control.
Project description:Comprehensive list of SUMO targets from the nematode Caenorhabditis elegans. SUMO conjugates isolated from transgenic worms carrying 8His and GFP tagged SUMO. The constructs rescues the lethal knock-out of a single SUMO gene, smo-1. SUMO conjugates where isolated from heat shock, arsenite exposure, and UV treated SUMO-GFP worms as well as from control non treated animals. In parallel identical purification procedure was performed with non-transgenic worms and proteins identified with this control where excluded.
Project description:One of the most abundant RNA modifications is N6-methyladenosine (m6A). RNA from all forms of life, including viruses, contain m6A. This modification has been detected in many types of RNAs, such as mRNA, ribosomal RNA, long non-coding RNAs, small nuclear RNAs and microRNAs. Diverse set of proteins have been characterized to methylate, demethylate and specifically bind to this modification in different organisms. C. elegans is a unique model organism with abundant m6A modification, although its genome does not code for orthologs of the well characterized m6A methyltransferase METTL3/METTL14 complex or the demethylases FTO or ALKBH5. Furthermore, orthologs of the YTH family m6A reader proteins seem to be absent from the worm genome as well. To gain insights into how this modification is installed in this organism, we set out to identify enzymes that contribute to the abundant level of m6A in C. elegans. We designed a targeted RNAi screen by which the expression of 22 candidate putative RNA methyltransferase genes are knocked down. We measured global RNA methylation level by HPLC-MS/MS analysis after two generations of RNAi-mediated knock down. The knock down of two candidate methyltransferases resulted in a decrease in global m6A level in total RNA. The first methyltransferase, F33A8.4, is an ortholog of the human ZCCHC4 gene. The second methyltransferase, C38D4.9, is an ortholog of the human METTL5 gene. In order to determine if ZCCHC4 or METTL5 are involved in the deposition of m6A at the mRNA level, m6A-RIP-seq experiments were performed in mRNA derived from WT (N2), ZCCHC4 KO, METTL5 KO and ZCCHC4/METTL5 dKO C. elegans embryos.
Project description:We have discovered a novel gene, mekk-3/idr-1, which when knocked down by RNAi dramatically increases life span. In order to understand how this gene works, we performed microarray analysis to compare the gene expression profiles of control and mekk-3/idr-1 RNAi-grown worms.
Project description:Comparison of gene expression profiles from C. elegans mutant strain CF1038 treated with L4440 and K02A4.1 RNAi and C. elegans mutant strain TU3311 treated with L4440 and B0412.2 RNAi for 5 days after L4 larvae stage. Jena Centre for Systems Biology of Ageing - JenAge (www.jenage.de)