- Titel
- Mitochondrial tRNAs And Where To Find Them
- Zitierfähige Url:
- https://nbn-resolving.org/urn:nbn:de:bsz:15-qucosa2-1006593
- Datum der Einreichung
- 02.07.2025
- Datum der Verteidigung
- 18.11.2025
- Abstract (EN)
- Transfer RNAs (tRNAs), ancient and irreplaceable molecules, have well-defined features including sequence, secondary, and tertiary structure. As part of translation process, they play the role of an adapter and decode the mRNA into a protein. Translation is highly conserved across all domains of life, and changes to this process can be detrimental to organisms. An important part of eukaryotic cells is mitochondrion. That is the powerplant of the cell, generating the energy in ATP form. Mitochondria preserve the ancestral bacterial genome organization features, while part of the mitochondrial machinery is encoded in the nucleus. As bacterial and nuclear metazoan tRNAs demonstrate stable tRNA features, mitochondrial tRNAs show a surprisingly diverse portfolio of aberrant structures throughout metazoan evolution. This work focuses on the aberrant tRNAs in mitochondria across Metazoa clade. Reannotation of available mitogenomes in RefSeq was performed using mitos2 workflow. Based on individual cases of mitochondrial tRNA variants reported in the literature, this work identifies the respective hotspots of change, specifically in Acanthocephala (Lophotrochozoa), Nematoda, Acariformes, and Araneae (Arthropoda). Less dramatic deviations from the norm of mitochondrial tRNAs are observed throughout many other clades. Loss of arms in animal mitochondrial tRNA is a phenomenon that has clearly occurred independently many times, not limited to a small number of specific clades. It was shown that the annotation of aberrant structures is a bioinformatic challenge, and an approach with competitive mt-tRNA annotation with a set of artificial armless covariance models (CMs) increases the number of detected mt-tRNA genes in Romanomermis genus, but still cannot be better than clade-specific CMs. tRNA sequencing data of Romanomermis culicivorax, obtained with LOTTE-seq protocol, support gene annotation results obtained with the clade-specific CMs. Another example of challenge is Steganacarus magnus mt-tRNA annotation, although multiple attempts have been made to annotate and define them accurately, there is no accepted consensus. With LOTTE-seq data analysis, the annotation of mt-tRNAs was refined and can serve as a basis for clade-specific CMs. The question arises: what can be a driver for such significant changes? As a part of the translation machinery, mt-tRNAs must interact with its parts - rRNA and proteins. As a first step, mt-tRNAs should be transported to the ribosomal complex with Elongation Factor Tu (EF-Tu). Analysis of selective binding of mitochondrial EF-Tu with a randomized tRNA pool revealed no preferences in sequence or structure for selected tRNA. In addition to the main role in translation mt-tRNAs, can be involved in other processes. For example, they can be found in the nuclear genome as a part of nuclear mitochondrial DNA (NUMT) insertions. As they have been found to be expressed in experiments, the question is, how can they be expressed? Data from open databases on RNA polymerases binding with DNA, as well as chromatin accessibility, didn’t provide a clear answer. Analysis of tRNA-seq data, on the other hand, revealed enrichment for some of numt-tRNA candidates. The next question is what potential functions these molecules might have? To answer this question, data from protein pulldown were analysed, using numt-tRNA candidate as a bait. Analysis reveals that nuclear-encoded mttRNA can bind with proteins that interact with both nuclear and mitochondrial tRNAs, and potentially serves as a source for tRNA-derived fragments. Another part of the work involved studying the features of tRNA in bacteria. Analysis of the secondary structure as a part of testing the RNA probing protocol Led-Seq showed high stability of the cloverleaf structure of tRNA in Escherichia coli regardless of the temperature and probing conditions. Another layer of interaction with modification machinery was tested after the inactivation of the HD-domain in the CCA-adding enzyme in E.coli, connected with 2’,3’-cyclic phosphate cleavage. It was shown that major changes in the pool of molecules carrying 2’,3’-cyclic phosphate can be detected between normal and stress conditions, while domain inactivation-induced changes are less apparent regardless of the condition. This work connects together different sides of tRNA life cycle, highlighting the complexity of their interaction. It also provides a substantial basis for future research in this field.
- Freie Schlagwörter (EN)
- tRNA secondary structure, mitochondrial tRNAs, truncated tRNAs, armless tRNAs, tRNA loss, tRNA annotation, inter-nmtRNA
- Klassifikation (DDC)
- 500
- Den akademischen Grad verleihende / prüfende Institution
- Universität Leipzig, Leipzig
- Version / Begutachtungsstatus
- publizierte Version / Verlagsversion
- URN Qucosa
- urn:nbn:de:bsz:15-qucosa2-1006593
- Veröffentlichungsdatum Qucosa
- 28.11.2025
- Dokumenttyp
- Dissertation
- Sprache des Dokumentes
- Englisch
- Lizenz / Rechtehinweis
CC BY-NC-ND 4.0- Inhaltsverzeichnis
I Biological background 1 Glimpse into tRNA world and translation 1.1 tRNA role in translation 1.2 The tRNA 1.3 Prokaryotic, mitochondrial, and eukaryotic tRNAs 2 Mitochondrial tRNAs 2.1 Mitochondrion: power-plant with prokaryotic organization 2.2 Changes in mt-tRNA standards 2.3 Mitochondrial DNA traveling II Bioinformatical background 3 Sequencing data and data processing 3.1 Sequencing data types 3.2 Data quality control 3.3 Biological databases 3.4 Alignment and peak calling 4 Secondary structure analysis 4.1 RNA folding 4.2 Stochastic grammars in RNA structure prediction 5 Protein analysis 5.1 Differential interactomics 5.2 Enrichment analysis 5.3 Protein-protein interactions III Methods 6 Structure probing and tRNA processing 6.1 Structure probing 6.2 Analysis of RNA processing by modified CCA-adding enzyme 7 Mitochondrial tRNA annotation 7.1 Annotation with mitos2 7.2 Annotation with custom CM set 8 Aberrant mt-tRNA detection and insights in their processing 8.1 LOTTE-seq 8.2 Recognition of aberrant structures by Ef-TU 9 Search for inter-nmtRNA interaction 9.1 ChIP-seq, ATAC-seq, Ribo-seq, tRNA-seq data analysis 9.2 RNA-protein pulldown data analysis IV Results 10 What makes the molecules The tRNA 10.1 Led-Seq: structure on the transcriptomic level 10.2 tRNA repair by modified CCA-adding enzyme 11 Wide mt-tRNA annotation 11.1 Search for global patterns 11.2 Local patterns in mt-tRNA annotation 12 Aberrant mt-tRNA in real life 12.1 Aberrant mt-tRNA sequencing 12.2 mt-tRNA recognition by EF-Tu in R. culicivorax 13 Inter-nmtRNAs and their functions 13.1 If inter-nmtRNA are processed? 13.2 Inter-nmtRNAs candidates as tRNAs 13.3 Inter-nmtRNA protein interactions V Discussion and conclusions 14 Summary 14.1 Bacterial tRNAs 14.2 Mitochondial tRNAs 14.3 tRNA lookalikes with mitochondrial origin 14.4 Finale 15 Outlook 15.1 Open questions Appendices A Supplementary Data for Chapter 10 B Supplementary Data for Chapter 11 C Supplementary Data for Chapter 12 D Supplementary Data for Chapter 13 List of Abbreviations List of Figures List of Tables Bibliography