Molecular Crowding Inhibits U-Insertion/Deletion RNA Editing In Vitro: Consequences for the In Vivo Reaction

Abstract

Mitochondrial pre-mRNAs in African trypanosomes are edited to generate functional transcripts. The reaction is typified by the insertion and deletion of U nucleotides and is catalyzed by a macromolecular complex, the editosome. Editosomes bind pre-edited mRNA/gRNA pairs and the reaction can be recapitulated in vitro by using pre-mRNA- and gRNA-mimicking oligoribonucleotides together with enriched editosome preparations. Although the in vitro assay has been instrumental in unraveling the basic steps of the editing cycle it is performed at dilute solvent conditions. This ignores the fact that editing takes place inside the highly crowded mitochondria. Here we investigate the effects of molecular crowding on RNA editing. By using neutral, macromolecular cosolutes we generate defined dilute, semidilute and crowded solvent properties and we demonstrate different thermodynamic stabilities of the pre-mRNA/gRNA hybrid RNAs at these conditions. Crowded conditions stabilize the RNAs by -30 kJ/mol. Furthermore, we show that the rate constants for the association and dissociation (kass/kdiss) of substrate RNAs to editosomes decrease, ultimately inhibiting the in vitro reaction. The data demonstrate that the current RNA editing in vitro system is sensitive to molecular crowding, which suggests that the in vivo reaction cannot rely on a diffusion-controlled, collision-based mechanism. Possible non-diffusional reaction pathways are discussed.

Analyzing editosome function in high-throughput

“…3 shows a representative result. As demonstrated before (Katari et al, 2013), non-modified versions of the insertion and deletion pre-mRNA/gRNA hybrids melt with two well separated transitions with Tm-values of 68±1.5°C and 47±1.5°C for the U-insertion complex and 64±1.5°C and 55±1.5°C for the U-deletion hybrid. Importantly, all fluorophore-substituted versions of the two trimolecular RNA-complexes show qualitatively and quantitatively identical profiles.…”

“…Half-maximal melting temperatures (Tm) were calculated from 1 st -derivative plots of absorbance versus temperature dA260/dT=f(T) (Katari et al, 2013).…”

“…Third, the reaction cycle involves multiple enzyme reactions, all representing potential drug-interference points, and fourth, the high molecular mass protein complex (0.8MDa) offers a large drug-binding landscape (Golas et al, 2009). Despite these favorable features, only very few RNA-editing inhibitors have been identified (Liang and Connell, 2010;Moshiri and Salavati, 2010;Katari et al, 2013, Leeder et al, 2015Zimmermann et al, 2016). Moreover, only three studies have systematically searched for RNA-editing inhibiting compounds, which primarily is due to the lack of a robust, high-throughput compatible assay system.…”

Surface-driven RNA-refolding by the OB-fold proteins of theTrypanosoma bruceieditosome

“…It accounts for the fact that structurally flexible protein sequences can enlarge their effective "reach", which is of special importance for membrane-bound macromolecular complexes especially in crowded solvent conditions. RNA-editing takes place in the highly crowded mitochondria of African trypanosomes and preliminary evidence for an attachment of the editosome to mitochondrial ribosomes and/or the mitochondrial inner-membrane have been discussed 43,44 . We propose that the energy for the RNA-refolding reaction carried out by the editosome follows an "entropy transfer"-mechanism 40 .…”

The 2D-structure of theT. bruceipre-edited RPS12 mRNA is not affected by macromolecular crowding

“…properties. 24 Here we analyze the effect(s) of macromolecular crowding on the structure of the mitochondrial RPS12 pre-mRNA as an archetypical example of a mitochondrial transcript in African trypanosomes. We use high molecular mass polyethylene glycol (PEG) as a neutral macromolecular cosolute to mimic intra-mitochondrial solvent conditions and we monitor the structure of the RPS12 transcript by temperature-dependent UV-spectroscopy and by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE).…”