The mammalian mitochondria contain their own genome which encodes thirteen polypeptide components of the oxidative phosphorylation (OxPhos) system, and the mitochondrial (mt-) rRNAs and tRNAs required for their translation. The maturation of the mitochondrial ribosome requires both mt-rRNAs to undergo post-transcriptional chemical modifications, folding of the rRNA and assembly of the protein components assisted by numerous biogenesis factors. The post-transcriptional modifications of the mt-rRNAs include base methylations, 2’-O-ribose methylations and pseudouridylation. However, the exact function of these modifications is unknown. Many mitoribosome biogenesis factors still remain to be identified and characterised. This work aims to broaden our understanding of two proteins involved in mitoribosome biogenesis through the study of the function of an rRNA methyltransferase and a novel biogenesis factor. Firstly, we characterised MRM1 (mitochondrial rRNA methyltransferase 1), a highly conserved 2’-O-ribose methyltransferase. We confirmed that MRM1 modifies a guanine in the peptidyl (P) transferase region of the 16S mt-rRNA that specifically interacts with the 3’ end of the tRNA at the ribosomal P-site. In bacteria, the modification is dispensable for ribosomal biogenesis and cell viability under standard conditions. However, in yeast mitochondria, Mrm1p is vital for ribosomal assembly and function. We generated knockout cells lines using programmable nuclease technology, and characterised the possible effects of MRM1 depletion on mitochondrial translation and mitoribosome biogenesis. We demonstrated that neither the enzyme nor the modification is required for human mitoribosomal assembly and translation in our experimental setup. Secondly, we identified a novel mitochondrially-targeted putative RNA endonuclease, YbeY. Using YbeY knockout cell lines, we showed that depletion of YbeY leads to loss of cell viability and OxPhos function as a consequence of a severe decrease in mitochondrial translation. Northern blotting and transcriptomic analysis using next generation RNA-Seq revealed transcript-specific changes to steady state levels. This analysis identified mt-tRNASer as a potential target of YbeY. We investigated the effect of YbeY deficiency on mitoribosomal assembly by quantitative sucrose gradient fractionation and mass spectrometry. This analysis showed that the mt-SSU is depleted in YbeY knockout cells. Further, immunoaffinity purification identified MRPS11 as a key interactor of YbeY. We propose that YbeY is a multifunctional protein that performs endonucleolytic functions in the mitochondria and also acts as a mitochondrial ribosome biogenesis factor, assisting small subunit assembly through its interaction with MRPS11.
Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:744586 |
Date | January 2018 |
Creators | D'Souza, Aaron Raynold |
Contributors | Minczuk, Michal |
Publisher | University of Cambridge |
Source Sets | Ethos UK |
Detected Language | English |
Type | Electronic Thesis or Dissertation |
Source | https://www.repository.cam.ac.uk/handle/1810/273764 |
Page generated in 0.0018 seconds