rna-catalyst-as-reporter-for-screening-drugs-against-rna-editing

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

McGill University

RNA editing regulates mitochondrial gene expression in trypanosomatid pathogens by creating functional mRNAs. It is catalyzed by a multi-protein complex (the editosome), and is found to be essential in both insect stage and mammalian blood stream form of Trypanosoma brucei. This particular form of RNA editing is unique to trypanosomatids, and thus provides a suitable drug target in trypanosomatid pathogens. Here, we demonstrate the feasibility of a rapid and sensitive fluorescence-based reporter assay to monitor RNA editing based on ribozyme activity. We could validate our new assay using previously identified inhibitors against the essential RNA editing ligase. The principle advantages of this assay are: (i) the use of non-radioactively labeled materials, (ii) sensitivity afforded by fluorescence instrumentation applicable to high-throughput screening of chemical inhibitors against the essential editosome and (iii) a rapid and convenient 'mix and measure' type of assay in low volume with a high signal to noise ratio. This assay should enhance rapid identification and characterization of the editosome inhibitors primarily based on the overall composition of the editosomes from T. brucei. These inhibitors could also be tested against the editosomes from the closely related pathogens including T. cruzi and Leishmania species.

A fluorescence-based reporter substrate for monitoring RNA editing in trypanosomatid pathogens.

RNA editing, catalyzed by the multiprotein editosome complex, is an essential step for the expression of most mitochondrial genes in trypanosomatid pathogens. It has been shown previously that Trypanosoma brucei RNA editing ligase 1 (TbREL1), a core catalytic component of the editosome, is essential in the mammalian life stage of these parasitic pathogens. Because of the availability of its crystal structure and absence from human, the adenylylation domain of TbREL1 has recently become the focus of several studies for designing inhibitors that target its adenylylation pocket. Here, we have studied new and existing inhibitors of TbREL1 to better understand their mechanism of action. We found that these compounds are moderate to weak inhibitors of adenylylation of TbREL1 and in fact enhance adenylylation at higher concentrations of protein. Nevertheless, they can efficiently block deadenylylation of TbREL1 in the editosome and, consequently, result in inhibition of the ligation step of RNA editing. Further experiments directly showed that the studied compounds inhibit the interaction of the editosome with substrate RNA. This was supported by the observation that not only the ligation activity of TbREL1 but also the activities of other editosome proteins such as endoribonuclease, terminal RNA uridylyltransferase, and uridylate-specific exoribonuclease, all of which require the interaction of the editosome with the substrate RNA, are efficiently inhibited by these compounds. In addition, we found that these compounds can interfere with the integrity and/or assembly of the editosome complex, opening the exciting possibility of using them to study the mechanism of assembly of the editosome components.

Naphthalene-based RNA editing inhibitor blocks RNA editing activities and editosome assembly in Trypanosoma brucei.

Most mitochondrial mRNAs in trypanosomatid parasites require uridine insertion/deletion RNA editing, a process mediated by guide RNA (gRNA) and catalyzed by multi-protein complexes called editosomes. The six oligonucleotide/oligosaccharide binding (OB)-fold proteins (KREPA1-A6), are a part of the common core of editosomes. They form a network of interactions among themselves as well as with the insertion and deletion sub-complexes and are essential for the stability of the editosomes. KREPA4 and KREPA6 proteins bind gRNA in vitro and are known to interact directly in yeast two-hybrid analysis. In this study, using several approaches we show a minimal interaction surface of the KREPA4 protein that is required for this interaction. By screening a series of N- and C-terminally truncated KREPA4 fragments, we show that a predicted α-helix of KREPA4 OB-fold is required for its interaction with KREPA6. An antibody against the KREPA4 α-helix or mutations of this region can eliminate association with KREPA6; while a peptide fragment corresponding to the α-helix can independently interact with KREPA6, thereby supporting the identification of KREPA4-KREPA6 interface. We also show that the predicted OB-fold of KREPA4; independent of its interaction with gRNA, is responsible for the stable integration of KREPA4 in the editosomes, and editing complexes co-purified with the tagged OB-fold can catalyze RNA editing. Therefore, we conclude that while KREPA4 interacts with KREPA6 through the α-helix region of its OB-fold, the entire OB-fold is required for its integration in the functional editosome, through additional protein-protein interactions.

The oligonucleotide binding (OB)-fold domain of KREPA4 is essential for stable incorporation into editosomes.

The related trypanosomatid pathogens, Trypanosoma brucei spp., Trypanosoma cruzi and Leishmania spp. cause devastating diseases in humans and animals and continue to pose a major challenge in drug development. Mitochondrial RNA editing, catalyzed by multi-protein complexes known as editosomes, has provided an opportunity for development of efficient and specific chemotherapeutic targets against trypanosomatid pathogens. This review will discuss both methods for discovery of RNA editing inhibitors, as well as inhibitors against the T. brucei editosome that were recently discovered through creative virtual and high throughput screening methods. In addition, the use of these inhibitors as agents that can block or perturb one or more steps of the RNA editing process will be discussed. These inhibitors can potentially be used to study the dynamic processing and assembly of the editosome proteins. A thorough understanding of the mechanisms and specificities of these new inhibitors is needed in order to contribute to both the functional studies of an essential gene expression mechanism and to the possibility of future drug development against the trypanosomatid pathogens.

Article	Year
A fluorescence-based reporter substrate for monitoring RNA editing in trypanosomatid pathogens. Nucleic acids research\| PubMed ID: 20444864	2010
Naphthalene-based RNA editing inhibitor blocks RNA editing activities and editosome assembly in Trypanosoma brucei. The Journal of biological chemistry\| PubMed ID: 21378165	2011
The oligonucleotide binding (OB)-fold domain of KREPA4 is essential for stable incorporation into editosomes. PloS one\| PubMed ID: 23056494	2012
Inhibitors of RNA editing as potential chemotherapeutics against trypanosomatid pathogens. International journal for parasitology. Drugs and drug resistance\| PubMed ID: 24533263	2012

Houtan Moshiri

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