Mitochondrial RNA processing and stability

Hertha Firnberg Position T 105 Mitochondrial RNA processing and stability Gerlinde WIESENBERGER 27.06.2000 Gene expression in organelles is mainly regulated at the post-transcriptional level. RNA processing and degradation play a prominent role, nevertheless only limited information is available about nuclearly encoded genes controlling these processes. In this project the yeast Saccharomyces cerevisiae will be used as a model system to elucidate molecular mechanisms of RNA metabolism in mitochondria. We have previously identified a nuclear gene, PET127, which affects stability and processing of multiple mitochondrial transcripts in S. cerevisiae. PET127 encodes a mitochondrial membrane protein, which is most likely involved in 5` end processing of several mitochondrial transcripts and/or might play a role in RNA surveillance. In the proposed project it is planned to elucidate the in vivo function of PET127 by investigating suppressors of a pet127 deletion and characterization of mutations causing synthetic phenotypes in combination with a pet127 deletion. Furthermore, factors alleviating the mtDNA instability phenotype of strains overexpressing the PET127 gene will be cloned and characterized. The functions of genes identified in these screens will be studied, particularly in connection with pet127, and molecular consequences of deletion and overexpression of the genes will be characterized biochemically. A conserved dodecamer sequence present at the 3` ends of all major mitochondrial mRNAs seems to be involved in 3` end processing and stabilization of mitochondrial mRNAs. Mutational, analysis of the dodecamer sequence by mitochondrial transformation will be performed to test the proposed rolo,in vivo. The genes encoding proteins binding to the dodecamer sequence and supposedly preventing 3`-5` degradation are unknown. A combination of biochemical and genetic approaches should allow to identify these components and to analyze their role by gene disruption and overexpression experiments. Informations obtained on the yeast model system should permit valuable insights into similar processes of RNA processing and degradation occurring in mitochondria of plants and fungal species not amenable to genetic analysis.

Mitochondria, the power plants of the eukaryotic cell, contain their own genome, which encodes mainly proteins required for respiration. Contrary to the nuclear genome, where transcription of individual genes can be turned on and off, mitochondrial genes are transcribed in large pieces and resulting precursers have to be processed extensively to form the mature transcripts. RNAs deriving from a single precurser can be individually stabilized or degraded. The processing events and the mechanisms leading to stable or unstable RNAs are performed by proteins, which are nuclearly encoded, synthesized in the cytoplasm and imported into mitochondria. Little is known about the biochemical nature of these proteins. In the presented project we used baker`s yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe as model systems to study maturation and turnover of mitochondrial RNAs. To investigate the function of the Pet127 protein the PET127 gene was deleted and the consequences of this manipulation were studied with respect to growth phenotype and mitochondrial transcript patterns. this experiment was performed in baker`s yeast as well as in fission yeast. Furthermore, pet127 from fission yeast was expressed in baker`s yeast and its function on transcript processing and turnover was investigated. We could show that the fission yeast protein can partially take over the function of its S. cerevisiae counterpart. Database searches revealed that many genomes of lower eukaryotes (including several pathogenic fungi) could code for proteins homologous to the Pet127p from S. cerevisiae. However, we found no evidence for the occurrence of Pet127 related proteins in higher eukaryotes (plants and animals), suggesting that Pet127p is only required for RNA metabolism in lower eukaryotes and the animals and plants use different mechanisms. Knowledge about specific differences in the mitochondrial gene expression between yeast (a fungus) and plants/mammals could pave the ground for the development of antifungal therapeutics. Another aspect of mitochondrial biogenesis was investigated during this project: mitochondrial metal ion homeostasis. The maintenance of physiological ion concentrations is of fundamental importance for all living organisms. Disturbances in the mitochondrial metal ion homeostasis can be the cause for severe human diseases (e.g. Friedreich`s ataxia, Menkes syndrome, Wilson disease). Friedreich`s ataxia is caused by reduction of the steady state levels of a mitochondrial protein (frataxin) connected with dramatically increased mitochondrial iron concentrations. In this project I investigated the function of two yeast proteins, Mrs3p and Mrs4p. These proteins belong to a family of conserved proteins (mitochondrial carrier family), which are located in the inner mitochondrial membrane and are required for transport of many metabolites into mitochondria. It was shown that (1) simultaneous deletion or overexpression of the MRS3 and MRS4 genes causes alterations in the cellular iron homeostasis and that (2) the Mrs3/4 proteins are required under certain conditions for transport of iron into mitochondria.