Project description:Ribosome biogenesis is a highly energy-demanding process in eukaryotes which requires the concerted action of all three RNA polymerases. In RNA polymerase II transcription, the general transcription factor TFIIH is recruited by TFIIE to the initiation site of protein-coding genes. Distinct mutations in TFIIH and TFIIE give rise to the degenerative disorder trichothiodystrophy (TTD). Here we uncovered an unexpected role of TFIIE in ribosomal RNA synthesis by RNA polymerase I. With high resolution microscopy we detected TFIIE in the nucleolus where TFIIE binds to actively transcribed rDNA. Mutations in TFIIE affects gene-occupancy of RNA polymerase I, rRNA maturation, ribosomal assembly and performance. In consequence, the elevated translational error rate with imbalanced protein synthesis and turnover results in an increase in heat-sensitive proteins. Collectively, mutations in TFIIE – due to impaired ribosomal biogenesis and translational accuracy – lead to a loss of protein homeostasis (proteostasis) which can partly explain the clinical phenotype in TTD.
Project description:The ability to adapt quickly to changing environmental conditions is crucial for growth and survival of bacteria in their natural environment. The common strategy that bacteria utilize to increase their survival under stressful conditions, including antibiotic treatment, is the entry into a non-actively growing state (quiescence). In the wide host-range pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and other Gram negative bacteria, this temporary arrest of proliferation induces the expression of the alternative sigma subunit σS/RpoS of RNA polymerase (RNAP) which remodels global gene expression to reshape the cell physiology and ensure survival in various stress conditions and nutritional deficiencies (i.e. general stress resistance) and long-term cell persistence. σS is also required for virulence and biofilm formation. Our work aims at understanding key molecular and physiological determinants responsible for persistence of non-growing bacteria, using the ubiquitous pathogen S. Typhimurium as a working system. We expect to identify novel functional proteins that are both physiologically important and of wide biological significance. One approach to characterize those proteins is to compare mutant proteome to that of the wild-type strain to unravel potential effects of the mutations on the expression and/or stability of proteins.
Project description:Deficiency of proteostasis and RNA metabolism are increasingly recognized as critical drivers of neurodegenerative diseases. In this study, we focused on RNA polymerase II (Pol2) as the converging point of these two elements to understand how transcriptional machinery is affected by ALS-associated VCP mutations. Pol2 highly depends on its accessory factors to initiate, elongate, and terminate the transcription properly with accurate RNA splicing and processing. We, therefore, studied proteins co-localised with Pol2 in neural precursors obtained from ALS-patient iPS cells. To do that, we used the SPACE method as a highly sensitive proteomic tool to eliminate the artificial interactions during purification.
Project description:High Throughput bulk-sample ChIP-seq data targeted to RNA-polymerase II was collected for duplicate samples of two distinct mutations of Pcf11 and a matched wildtype w303 samples under standard growth conditions. The overall goal was quantification of changes in transcriptional activity in response to these mutations.
Project description:Mitochondrial oxidative phosphorylation (OXPHOS) is important for cancer cell growth and the persistence of therapy-resistant cancer stem cells. Here, we have developed novel first-in-class inhibitors of mitochondrial transcription (IMTs) targeting mtDNA gene expression. We show that IMTs specifically target mitochondrial RNA polymerase (POLRMT) and we defined the IMT binding site using exome sequencing of cells rendered resistant to IMTs by chemical mutagenesis and cryo-EM. IMTs act as allosteric inhibitors of POLRMT, impairing binding of the DNA-RNA hybrid and translocation of the nascent RNA. IMT treatment impairs OXPHOS in a dose-dependent way and decreases cell viability in various tumour cells. Surprisingly, several weeks of per oral IMT treatment is well tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in human cancer xenografts. We thus show that the IMTs represent a novel promising class of drugs for tumour treatment acting by inhibition of mtDNA gene expression.