Regulation of post-transcriptional gene expression in human mitochondria

Abstract

Mitochondria are cellular organelles that have evolved from the eubacterial ancestor into highly specialized compartment of the eukaryotic cell. They are unique among animal cells in that they retain a level of autonomy through the genetic information in their genome. Human mtDNA is built of ~16.5 kbp encoding 13 polypeptides, which are synthesised by mitoribosomes. The latter consist of two RNA species also transcribed from mtDNA and approximately 80 proteins originating from the nucleus. All 13 products of intramitochondrial translation are incorporated into the inner mitochondrial membrane where they co-build the oxidative phosphorylation (OXPHOS) system. OXPHOS is a multicomplex machinery, the final product of which is adenosine triphosphate, ATP, a carrier of energy that is necessary to sustain cell homeostasis and growth. The malfunctions of mitochondria have a severe impact on the ‘host’ organism and are the causative factor in many human diseases. Pathological changes of mitochondrial function can be triggered by mutations in the mitochondrial genome and/or defects in nuclear genes involved in mitochondrial activity. The mitochondrial gene expression pathway has been increasingly investigated during last twenty years and combines both types of factors, those translated in the cytosol and those synthesised in the mitochondrial matrix. A functional mitochondrion requires over 1500 proteins to be imported from the cytosol, a significant subset of these are devoted to the maintenance, replication, transcription and subsequently for translation of the minimal mitochondrial genome fostered within. In the course of my PhD study three of these nuclear encoded but mitochondrially destined proteins were investigated. The first of these proteins that I contributed to investigating was SLIRP. As the specificity of this RNA binding protein had not been established I performed CLIP (cross-linking immunoprecipitation) assay in order to assess the ability of SLIRP to bind RNA. The data generated from this analysis directly showed that SLIRP can interact with all mt-mRNAs apart from MTND6. This work confirmed that SLIRP participates in the stability of mt-mRNA species, as has now been subsequently published by other research groups. A main part of my PhD studies centred on characterisation of MRPL12. This protein belongs to the pool of conserved mitochondrial proteins having the bacterial orthologue 3 called L7/L12. One of the unique features of these proteins is their dynamic character and ability to exchange location between ribosomal LSU and the free pool. This has been postulated to be a regulatory mechanism of translation process in response to fluctuations in cell metabolism. To test this hypothesis I characterised immortalised fibroblasts obtained from a patient with a homozygous mutation in MRPL12 caused by c.542C to T transition in exon 5. This cell line allowed me to study the consequence of this defect on the regulation of translation in human mitochondria. I could conclude that a reduced number of MRPL12 molecules per mt-LSU in subject fibroblasts did not affect overall mitoribosome assembly, but a visible decline in mitochondrial translation was detected although the reduction in translational efficiency for different mitochondrially encoded subunits varied. The third protein that I characterised was mitochondrial RBFA. This protein was identified in my host laboratory and preliminary characterisation performed prior to my involvement. My studies included the CLIP assay that showed direct interaction of this protein with a 3’ terminal stem loop of helix 45 of the 12S mt-rRNA. The methylation status of two conserved neighbouring adenines located in helix 45 was altered by changes in steady state level of RBFA. Moreover, the CLIP data identified a second rRNA species associated with RBFA. This was an unexpected RNA species in the form of 5S rRNA. The data regarding the mitochondrial localisation and specifically any submitochondrial location has been controversial. Intriguingly my data identified a number of chimeric CLIP sequences containing both 5S and 12S rRNA fragments, strongly suggesting that within the mitochondrial matrix RBFA interacts simultaneously with both RNA species. Similarity between the 5S rRNA secondary structure and snoRNA, which guides modifications on cytosolic rRNA, led to the hypothesis proposing a novel function for 5S rRNA guiding methylation at helix 45 of the 12S mtrRNA. My data therefore assign RBFA as a new member of the group of maturation factors of the mammalian mt-SSU.