| dc.contributor.advisor | Bohnsack, Markus Prof. Dr. rer. nat. | |
| dc.contributor.author | Feußner, Konstantin | |
| dc.date.accessioned | 2024-04-18T13:11:07Z | |
| dc.date.available | 2024-05-17T00:50:12Z | |
| dc.date.issued | 2024-04-18 | |
| dc.identifier.uri | http://resolver.sub.uni-goettingen.de/purl?ediss-11858/15213 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-10456 | |
| dc.format.extent | 77 | de |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | |
| dc.subject.ddc | 610 | de |
| dc.title | Target recognition of an RNA methyltransferase | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Bohnsack, Markus Prof. Dr. rer. nat. | |
| dc.date.examination | 2024-04-24 | de |
| dc.description.abstracteng | To date more than 150 different RNA modifications have been detected in cellular RNAs and are collectively termed the epitranscriptome. RNA modifications occur at different positions within each of the four bases or the ribose moiety and enlarge the chemical and topological properties of the RNA building blocks. Due to its dynamic nature, the epitranscriptome adds an important layer of gene expression regulation as it influences the structure and function of many RNA molecules. tRNAs are the most extensively modified class of RNA. The anticodon loop is the hotspot of modifications in tRNAs and these modifications often have a direct impact on the function of tRNAs in translation. This can be through structural adjustments of the anticodon loop, interactions with the ribosome or an altered codon-anticodon base pairing.
Methyltransferase-like 6 (METTL6) has been shown to install the modification 3- methylcytidine at the cytosine at position 32 (m3C32), which is located at the 5’ end of the anticodon loop, in all mammalian cytoplasmic tRNASer. In the yeast S. cerevisiae, the homologue of METTL6 is Trm140 and its substrate recognition has been thoroughly examined. Important factors for its recognition of seryl-tRNAs are the presence of i6A37 or t6A37, the distinct and long variable loop of tRNASer and interaction with the cofactor Ses1. Mammalian seryl-tRNAs contain the same modifications at A37 nucleotides, and preliminary evidence suggests an interaction of METTL6 with SARS1, the mammalian homologue of Ses1. However, little was known about the requirement of i6A37 or t6A37 and SARS1 for substrate recognition by METTL6. Recently, a pathological relevance of METTL6 in neoplastic diseases like hepatocellular cancer and an entity of breast cancer has been suggested, which underlines the importance of a detailed understanding of its function.
This project established protocols for the recombinant expression and purification of METTL6 and SARS1 proteins and for in vitro transcription of full length tRNA substrates. A crystallization of recombinant METTL6 was attempted because the knowledge of its structure could also contribute to the understanding of its enzyme-substrate interaction. In vitro methylation assays yielded valuable insights regarding the importance of SARS1 as an METTL6 cofactor and highlighted interesting differences between the ability of unmodified versions of normally i6A37- and t6A37-containing seryl-tRNA isoacceptors to be methylated by METTL6. Moreover, METTL6 and SARS1 were transiently depleted in HEK293 cell lines via siRNA transfection. Two different RNA detection methods were tested for analyzing the effect of the preceded KDs on m3C32 levels in different cytoplasmatic seryl- tRNAs.
These insights present a valuable basis for further in vivo studies regarding the role of SARS1 as a METTL6 cofactor and A37 modifications as prerequisites for methylation by METTL6 as well as the biological role of m3C32 in cellular processes, especially translation. | de |
| dc.contributor.coReferee | Hillen, Hauke Prof. Dr. | |
| dc.subject.eng | RNA | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-ediss-15213-5 | |
| dc.affiliation.institute | Medizinische Fakultät | de |
| dc.subject.gokfull | Biologie (PPN619875151) | de |
| dc.description.embargoed | 2024-05-17 | de |
| dc.identifier.ppn | 1886408203 | |
| dc.notes.confirmationsent | Confirmation sent 2024-04-18T13:15:01 | de |