|Ph.D Student||Vardi-Oknin Dikla|
|Subject||Localized Translation Near Mitochondria in Mammalian Cells|
|Department||Department of Nanoscience and Nanotechnology||Supervisor||Professor Yoav Arava|
|Full Thesis text|
Mitochondria exert their many functions through a repertoire of hundreds of proteins.
Although mitochondria contain their own DNA, only few mitochondrial proteins are encoded by the mitochondrial genome and most of them are encoded by the nuclear genome. Nuclear-encoded mitochondrial proteins are translated by cytosolic ribosomes and are imported into the mitochondria. The import of mitochondrial protein can occur post-translationally or concurrently with their translation, i.e. co-translationally. While the post-translation import mechanism is well-studied, co-translation import mechanism, and its molecular components are less known. Currently, most evidences for co-translation import were documented by studies in the yeast model, while in mammalian cells it is poorly investigated. Here we applied proximity-specific ribosome labeling strategy to address co-translation import of mitochondrial proteins in mammalian cells. This method is based on ribosome labeling in a specific subcellular location, the mitochondria in our case. Ribosome labeling is performed through biotinylation by the biotin ligase (BirA) which is spatially restricted to the mitochondria. Following a biotin pulse, BirA biotinylates ribosomes that are tagged by the biotin acceptor Avitag and are in close proximity to the mitochondria.
In this work, we demonstrated a clear biotinylation of ribosomes by mitochondrial
Tom20-BirA fusion protein, indicating the existent of mitochondria-associated active ribosomes. Isolation of mitochondria-associated ribosomes revealed their preferred association with mRNAs encoding mitochondrial proteins. We next knock down of the mitochondrial protein receptor Tom70 to explore its involvement in localized translation. siRNA treatment resulted in a decrease in biotinylation, concomitant with a decrease in mRNAs encoding Tom-70 target proteins. Intriguingly, association of mRNAs encoding Tom20-targets was increased. Together, this implies for the involvement of TOM complex in mitochondria localized translation. We deduce that an interaction between Tom20 and Tom70 protein receptors and their target proteins occurs while they are being translated, and this leads to anchoring of translating ribosomes to the mitochondria outer surface. 2 Additionally, we examined the role of the RNA binding protein CLUH in mitochondrial localized translation. CLUH is known to bind a subset of mRNAs encoding mitochondrial proteins and promote their translation. We hypothesized that CLUH has a role in the translocation of its target transcripts to the vicinity of the mitochondria for localized translation. Indeed, we found that CLUH depletion led to a decrease in mitochondrial biotinylation (i.e. localized translation). Surprisingly, however, CLUH knock down also led to an increased association of its target mRNAs with mitochondria, while non-target control was unaffected. We propose that CLUH depletion led to reduced protein levels of its target genes, by a reduction in the ribosome number that translate its target mRNAs, and that triggered the recruitment of mRNAs to the mitochondria for localized translation. Overall, these data shed light on mitochondrial localized translation mechanism in human cells, and the protein factors that contribute to the association of translating ribosomes with the mitochondria. Moreover, establishing the proximity-specific ribosome labeling method in mammalian's mitochondria, pave the way to investigate the importance of this mechanism in different physiological condition.