Surface-Driven RNA Refolding by the OB-Fold Proteins of the Trypanosoma brucei Editosome

• Main Author: Voigt, Christin
• Format: Others
• Language: en
• Published: 2017
• Online Access: http://tuprints.ulb.tu-darmstadt.de/6185/1/Voigt_Dissertation.pdf; Voigt, Christin <http://tuprints.ulb.tu-darmstadt.de/view/person/Voigt=3AChristin=3A=3A.html> : Surface-Driven RNA Refolding by the OB-Fold Proteins of the Trypanosoma brucei Editosome. Technische Universität Darmstadt, Darmstadt [Ph.D. Thesis], (2017)

RNA editing in African trypanosomes is an essential mitochondrial RNA processing reaction. It is required to decode otherwise untranslatable primary transcripts. The reaction is characterized by the insertion and/or deletion of, in several transcripts, hundreds of uridylyl residues. It relies on a specific class of small, non-coding RNA molecules, known as guide (g)RNAs. Guide RNAs act as templates in the process. They base-pair to the highly structured pre- and partially edited mitochondrial mRNAs before a multi-protein complex, the 20S editosome, catalyzes the individual steps of the RNA editing cycle. The 20S editosome executes a chaperone-like RNA remodeling activity. The activity acts on the pre-edited transcripts by enhancing the structural dynamics of primarily U-nucleotides in order to "loosen" the structure of the pre-mRNA substrate molecules. In chapter II, I present an in vitro assay, to experimentally monitor the consequences of the RNA chaperone activity for the entry of gRNAs. The assay uses short, gRNA-mimicking DNA oligonucleotides (gDNA) and probes the formation of gDNA-pre-mRNA hybrid molecules by ribonuclease H (RNaseH) digestion. The data demonstrate that the RNA chaperone activity stimulates the formation of gDNA/pre-mRNA hybrids thereby acting as an early facilitator of the editing reaction. The molecular nature of the editosome-inherent RNA chaperone activity is unknown. In chapter III, I test the hypothesis, that the oligonucleotide/oligosaccharide binding (OB)-fold proteins of the editosome are responsible for the activity. Importantly, these proteins are predicted to be in large parts intrinsically disordered. Furthermore, experimental evidence suggests that the proteins form a, structurally important, clustered subdomain within the editosome. To examine their potential chaperone activity I expressed the different proteins as recombinant polypeptides, both, as full-length and as OB-fold-only constructs. I verified the disorder prediction by circular dichroism (CD)-spectroscopy and analyzed their homo- and hetero-oligomerization behavior by size exclusion chromatography. Importantly, all OB-fold protein constructs show RNA chaperone activity with half maximal activities in the nM concentration range. The measured activities directly correlate to the surface areas of the different protein complexes suggesting that the RNA remodeling process is mainly surface-driven. The analysis of the induced RNA structure changes was performed with nucleotide resolution using selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE). The experiments demonstrate that the effect of the homo-oligomeric OB-fold protein complexes is qualitatively and quantitatively similar to the effect of 20S editosomes. The data further suggest an involvement of the intrinsically disordered protein regions. A coarse grained structural model of the 20S editosome is presented, which supports a preferential positioning of disordered protein domains on the surface of the complex and of ordered protein regions in the core of the editosome. An analysis, to address possible RNA substrate and nucleotide specificities of the OB-fold protein-mediated RNA chaperone activity, is presented in chapter IV. For that RNaseH-based gDNA annealing assays were performed comparing the OB-fold of the editosome protein TbMP24 with 20S editosomes. Two different mito-chondrial transcripts were used in the analysis: the pre-mRNA encoding ribosomal protein S12 (RPS12) and the pre-mRNA for apocytochrome b (CYb). I analyzed the RPS12 transcript at ten different regions of different structure and sequence contexts. The experiments demonstrate that both, TbMP24-OB and 20S editosomes, enhance the accessibility of a pre-mRNA for gDNA entry at every position in the RNA. The TbMP24-OB activity shows no preference for distinct RNA structures, but a preference for U-rich sequence regions. This U-preference was confirmed by SHAPE-experiments of the RPS12-RNA with TbMP24-OB. Both TbMP24-OB and 20S editosomes act on both transcripts, with a slightly higher activity on the larger CYb-RNA. TbMP24-OB thereby shows the same overall substrate preference as 20S editosomes, which enables the protein to function as a model protein for studying the RNA chaperone activity of the 20S editosome. RNA editing is differentially regulated throughout the lifecycle of Trypanosoma brucei, but the cause of that differential editing is unknown. The intracellular location of RNA editing, the mitochondrion, differs in the lifecycle stages, ultimately generating two different redox environments. In chapter V, I test whether the edito-somal OB-fold proteins can serve as "redox switches". I uncovered that some of the OB-fold proteins of the editosome are sensitive towards changes in the redox environment, leading to altered thermal stabilities and altered oligomerization characteristics. To investigate the effects of oxidation on protein oligomerization, I mutated the cysteine codons in the TbMP24-OB-coding gene. The exchange of all cysteines by alanines resulted in a mutated protein with an altered oligomerization behavior. The mutations cause an inability to switch the oligomerization state, resulting in a protein variant that stays dimeric, comparable to the wild-type protein in its reduced form. The interaction with the binding partner TbMP18 is not hindered by the cysteine mutations, but the ratio of interacting proteins is altered. These findings have implications for the assembly of the editing complex in vivo, suggesting that cysteine thiol redox switching could be a mechanism to regulate editing in a lifecycle-dependent manner.