Characterisation of the RNA modifying enzyme ABH1
von Benedikt Hübner
Datum der mündl. Prüfung:2022-05-03
Betreuer:Prof. Dr. Markus Bohnsack
Gutachter:Prof. Dr. Michael Meinecke
Gutachter:Prof. Dr. Ralf Dressel
EnglischRibonucleic acid (RNA) modifications have been found in all coding and non-coding RNAs and represent a key mechanism in the homeostatic control of cell metabolism including gene expression and regulation. Therefore, the wobble base cytosine on position 34 (C34) of mitochondrial initiator and elongator transfer RNA methionine (mt-tRNAMet), that allows non-conventional codon-anticodon base pairing, may be of superior importance. Literature suggested a 5-formylated residue on C34 of mt-tRNAMet and the Bohnsack lab could identify methyltransferase NSUN3 to interact with and install m5C of mt-tRNAMet. However, the conversion to f5C34 and the enzyme involved remained unclear. The dioxygenase ABH1, that has been shown to localise to mitochondria, represents a potential candidate and its activity in the oxidation of m5C34 was my subject of investigation. RNA immunoprecipitation (RNA-IP) experiments after UV crosslinking followed by northern blotting and ethidium bromide staining of mt-tRNAMet and ABH1 were performed. Recombinant expression of ABH1 and catalytically inactive mutants, created by site-directed mutagenesis, was established in E. coli. The purified proteins were used in in vitro methylation and oxidation assays involving radioactive 3H (tritium). Sufficient depletion of ABH1 in human embryonic kidney 293 (HEK 293) cells was carried out by RNA interference (RNAi) and the modification status of C34 of mt-tRNAMet was monitored after bisulfite and reduced bisulfite treatment followed by Sanger sequencing. Taken together, I could identify ABH1 to specifically interact with mt-tRNAMet. Furthermore, I was able to monitor oxidation of m5C34 of mt-tRNAMet by recombinant ABH1 in vitro. ABH1 could be efficiently depleted by RNAi in Henrietta Lacks CCL2 (HeLa CCL2) human cervical carcinoma cells and a sequential modification pattern of unmodified mt-tRNAMet C34 to m5C34 by NSUN3, followed by ABH1 mediated oxidation to f5C34, could be described in vivo. ABH1 is an active enzyme capable of oxidising m5C into f5C and this activity is responsible for generating the f5C34 present in mt-tRNAMet in cells. Its target spectrum, the role of mt-tRNAMet C34 as epitranscriptomic hotspot and their involvement in pathologic metabolic processes in human cells are questions that need to be addressed in further research.
Keywords: ABH1; mitochondria; rna modification; translation; NSUN3