Abstract
dc:description.abstractMitochondrial gene expression entails multiple steps, one of which is translation, performed by the mitochondrial ribosome. Post-transcriptional RNA nucleotide modifications have been identified in mitochondrial (mt)-rRNAs. However, little is known about their role in terms of mitochondrial translation and/or ribosome assembly. The mitochondrial methyltransferase MRM2 modifies the human 16S mt-rRNA by introducing a 2’-O-methyl moiety in the ribose of residue U1369 (Um1369). This nucleotide is located in the aminoacyl site (A-loop of 16S mt-rRNA) of the mitoribosome large subunit (mtLSU), contacting the acceptor stem of incoming tRNAs, which indicates a potentially relevant role for the Um1369 modification in the translation mechanism. To understand the molecular role of MRM2, a genetic ablation cell model was generated using zinc finger nuclease technology. The produced cells exhibit slow growth under regular conditions, and are unable to grow when the function of the mitochondrial respiratory chain is required for survival. Validation of the absence of modification in the target nucleotide was obtained by RNA mass spectrometry. Furthermore, mitochondrial function is affected as measured by oxygen consumption and extracellular acidification rates. When mitochondrial translation was investigated by labelling of nascent peptides, a severe disruption of this process was observed, providing biological basis for the previous findings. Moreover, mitochondrial ribosome profiling (mtRibo-Seq) revealed a marked and generalised decrease in the levels of ribosomes translating mitochondrial transcripts. Investigation of the composition and assembly state of the mtLSU was performed by sucrose density gradient ultracentrifugation and quantitative mass spectroscopy, revealing an accumulation of mtLSU, with decreased amounts of bL36m and an enrichment in mtLSU assembly factors. In order to conjugate a functionally defective, yet soundly structured mitochondrial ribosome, the atomic-level details of its structure were investigated by cryoEM, revealing the persistence of a late stage assembly containing the anti-association MALSU1:SMCR7L:mtACP complex and unfolded interfacial rRNA. Pseudouridine (Ψ), the most common RNA modification found in cells, is also present in mtRNAs. Investigation of pseudouridylation in mitochondria yielded the identification of four novel pseudouridine synthases: PUSL1, RPUSD3, RPUSD4 and TRUB2. Using targeted (RT-PEx) and transcriptome-wide (Psi-Seq) techniques PUSL1 was identified as the enzyme responsible for the modification of U39 of mt-tRNAs in the context of a consensus sequence. Overall, this work presents evidence that small chemical modifications may have a greater effect on the cellular homeostasis by affecting the correct function of basic mitochondrial processes.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2020
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Rebelo Guiomar, Pedro
- Advisor dc:contributor.advisor
-
- Minczuk, Michal
Subjects
dc:subject × 7Rights
dc:rightsIdentifiers
dc:identifier.*- DOI dc:identifier.doi
- https://doi.org/10.17863/CAM.56033
- OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/308942