Insights into the biogenesis of the human mitochondrial ribosomal large subunit – Characterisation of mL44 and mL45

by Elisa Hanitsch
Doctoral thesis
Date of Examination:2020-11-04
Date of issue:2020-11-16
Advisor:Dr. Ricarda Richter-Dennerlein
Referee:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Ralph Kehlenbach
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Michael Meinecke
Persistent Address: http://dx.doi.org/10.53846/goediss-8315

 

 

Files in this item

Name:Thesis_Elisa Hanitsch_Publication.pdf
Size:52.9Mb
Format:PDF

The following license files are associated with this item:

Abstract

English

Due to the presence of high-resolution cryo-EM structures of the human mitoribosome, the understanding about the function and assembly of the intriguing mitochondrial ribonucleoprotein complex increased tremendously during the last years. Even if the human mitoribosome descended from a bacterial ancestor, its structure and composition differs remarkably. During evolution, the RNA content decreased to approximately 50 % of the original bacterial. In contrast, many mitochondrial ribosome specific proteins were recruited and the existing proteins were extended leading to an inverted RNA to protein ratio. Many in vitro and in vivo studies about the assembly of the 70S bacterial ribosome have been conducted in the past. However, the assembly of the 55S human mitoribosome is not completely solved even if analyses about the biogenesis of the 54S subunit of Saccharomyces cerevisiae and the assembly of the mtLSU of Trypanosoma brucei contributed to our understanding. The number of mutations in genes encoding for proteins required for the mitochondrial translational apparatus which are implicated in severe early-onset diseases with various clinical phenotypes is growing. Hence, a deep understanding about the complex mechanisms of mitoribosomal assembly is required to shed light into the molecular basis leading to the manifestation of these severe human mitochondrial diseases. Within this thesis, a new purification protocol was established to obtain 55S human mitoribosomes from HEK293T cells. Therefore, the separation of mitoribosomal complexes on sucrose gradients was optimised in regard of sucrose concentration, buffer conditions and purity of starting material. This was done to ensure a reasonable separation of 28S mtSSU, 39S mtLSU and 55S monosome from each other as well as to improve the stability of the before mentioned complexes. By using the established protocol, 55S human mitoribosomes were successfully isolated for future analyses of the assembly pathway. In addition, to analyse the biogenesis of the 39S subunit, two proteins of the mtLSU were further characterised. The disease associated protein mL44 was shown to be crucial for de novo synthesis of mtDNA-encoded proteins as well as mtLSU assembly. Investigations using a FLAG-tagged variant of mL44 revealed that this protein is probably part of an early assembly intermediate together with bL20m and bL21m. Furthermore, by characterising mL45 it was observed that the mitoribosomal membrane anchor is required for functional mitochondrial translation and hierarchical assembly of the 39S mtLSU.
Keywords: mitochondrial ribosome; mitoribosome; 55S; 39S; mitochondrial ribosomal large subunit; mL44; mL45; human mitoribosome purification
 
Statistik