Monitoring the Structural Changes of pre-edited mRNAs upon Editosome Binding -Evidence for the Evolutionary Origin of RNA-Editing

Chapter I: Mitochondrial transcript maturation in African trypanosomes requires an RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. The reaction is catalyzed by editosomes and requires guide (g)RNAs as templates. Recent data demonstrate that the binding of pre-edited mRNAs to editosomes is followed by a chaperone-type RNA remodeling reaction. Here we map the changes in RNA folding using selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that pre-mRNAs in their free state adopt intricately folded, highly stable 2D-structures. Editosome binding renders the pre-mRNAs to adopt 2D-conformations of reduced stabilities. On average about 30% of the nucleotides in every pre-mRNA are affected with a prevalence for U-nucleotides. The data demonstrate that the chaperone activity acts by increasing the flexibility of U-residues to lower their base-pairing probability. This results in a simplified RNA folding landscape with a reduced energy barrier to facilitate the binding of gRNAs. The data provide a first rational for the enigmatic U−specificity of the editing reaction. Chapter II: Mitochondrial transcript maturation in African trypanosomes requires a U-nucleotide specific RNA editing reaction. In its most extreme form hundreds of U’s are inserted into and deleted from primary transcripts to generate functional mRNAs. Unfortunately, the evolutionary origin and the biological necessity of the process have remained enigmatic. Here we report a so far unrecognized structural feature of pre-edited mRNAs, which suggests a defined evolutionary origin. We demonstrate that the cryptic pre-mRNAs contain numerous clustered G-nt, which fold into G-quadruplex (GQ) structures. We identified 27 GQ’s in the different pre-mRNAs and demonstrate a positive correlation between the steady state abundance of guide (g)RNAs and the sequence position of GQ elements. We postulate that the driving force for selecting G-rich sequences lies in the formation of DNA/RNA hybrid G-quadruplex (HQ) structures between the pre-edited transcripts and the non-template strands of mitochondrial DNA. HQ’s are transcription termination and replication initiation sites and thus guarantee an unperturbed replication of the mt-genome. This is essential for maintaining the life cycle of the parasite. In the transcription-on state, the identified GQ’s require editing as a GQ-resolving activity suggesting that the two processes coevolved and identifying the cell- and life-cycle of the parasite as evolutionary forces. Chapter III: Mitochondrial transcript maturation in African trypanosomes requires an RNA editing reaction in which non-functional pre-mRNAs are converted into translatable mRNAs. While the reaction is characterized by the site-specific insertion and deletion of exclusively U-nucleotides, some transcripts are edited through the insertion of hundreds of U’s, showcasing the cryptic nature of the various pre-mRNAs. In addition to the lack of nucleotide information, the different RNAs are further characterized by an unusual high G-content and as a consequence the formation of thermodynamically highly stable 2D-structures in the majority of cases even involving multiple G-quadruplex (GQ)-folds. Unfortunately, it is not clear whether these structures resemble the in vivo folds of the different RNAs given the extreme volume exclusion i.e. “crowding” conditions within the trypanosome mitochondrion. Here we analyze the effect(s) of volume exclusion on the structure of the mitochondrial RPS12 pre-mRNA. We use polyethylene glycol (PEG)4000 as a neutral macromolecular cosolute to mimick intra-mitochondrial solvent conditions and we monitor the structure of the RNA on a global scale and with nucleotide resolution. We demonstrate that macromolecular crowding has no impact on the 2D-fold of the RPS12 pre-mRNA and we conclude that the determined minimal free energy (MFE)-structure in dilute solvent conditions represent a good proxy for the folding of the RPS12 pre-mRNA within its mitochondrial solvent context. Chapter IV: African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds.