Author: Nancy Ritsuko SturmPublisher:
ISBN:
Category :
Languages : en
Pages : 234
Book Description
Mechanistic Studies of RNA Editing in Trypanosomes
Author: Nancy Ritsuko SturmMechanistic Studies of RNA Editing in ̲Ph̲y̲s̲a̲r̲u̲m̲ ̲mitochondria in a Soluble System
Author: Yu-Wei ChengMechanistic Studies of Trypanosoma Brucei Mitochondrial MRNA Editing
Author: Michael E. HarrisDissecting RNA Editing Mechanism "in Vitro" with Genetic and Chemical Genetic Approaches
Author: Vaibhav MehtaPublisher:
ISBN:
Category :
Languages : en
Pages :
Book Description
"Trypanosoma brucei, Trypanosoma cruzi and the Leishmania species are the major pathogenic trypanosomatid organisms that cause devastating diseases in humans and animals, with high incidence and mortality rates. These organisms create functional mitochondrial transcripts by RNA editing, a process catalyzed by multiprotein editosome complexes that entail insertion and/or deletion of uridylates (Us) as dictated by short complementary guide RNAs and associated accessory proteins. RNA editing is essential for the parasite’s viability, making the process an attractive target for drug development. However, how the editosome proteins assemble and function together is not well understood. The focus of this thesis is on identifying small molecule inhibitors of RNA editing as chemical tools to dissect editosome assembly and as lead compounds against the trypanosomatid pathogens. First, using our FRET-based RNA editing assay, we screened the library of 1280 pharmacologically active compounds against RNA editing and identified new inhibitors of this process. Four compounds showed significant potency inhibiting RNA editing, with IC50 values ranging from 1 to 5 [mu]M. We found that to a varying extent, they interfered with RNA-protein interaction. Additionally, we showed for the first time that a compound identified in this screen, NF449, an analog of suramin, could effectively kill T. brucei in vitro. While NF449 does not specifically block a desired catalytic core enzyme and seems to interfere with RNA-protein interaction, these data establish feasibility and proof of concept for finding potent inhibitors of trypanosome RNA editing.In the next part of my thesis, we investigate the naphthalene-based inhibitors that competitively inhibit the auto-adenylylation activity of the recombinant kinetoplastid RNA editing ligase 1 (KREL1). In the context of purified editosomes, the inhibitors block all catalytic editing activities by interfering with RNA-protein interactions, similar to the pilot-scale screen hits. Through mass spectrometry analyses, we identify the accessory mitochondrial RNA binding proteins, MRP1 and MRP2, as potential off-targets for these compounds, and demonstrate their inhibitory activity on recombinant MRP1/2 heterotetramer complex. The compounds affect the association between the core editosomes and MRP1/2 heterotetramer. Functional editosomes purified post-treatment with the compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon addition of recombinant MRP1/2 proteins, directly implicating an essential role for MRP1/2 in the initiation of RNA editing. Next, to evaluate the molecular details of KREL1-inhibitor interactions and binding selectivity, we performed alanine replacement mutagenesis of recombinant KREL1 protein on residues predicted to bind the inhibitors. We identified the residues critical for binding to facilitate the design of more potent inhibitors. Furthermore, as KREL1 activity is enhanced by its interacting protein partner, A2, characterizing the interaction between these two proteins benefits structure-activity exploration of inhibitors. In the last part of my thesis, we examined this interaction using full-length, truncated, and point-mutated KREL1. Our analyses narrow down the point of A2 contact to the terminal 59 residues of KREL1 and identify critical residues not only in this region but also in the ATP binding domain that play a mechanistic role in enhancing ligase activity.The studies described here contribute significantly to functional organization and assembly of the editosome proteins and identify the regulatory mechanisms for the essential mitochondrial gene expression in these organisms. Furthermore, the introduced compounds can be starting points for the development of critically needed anti-trypanosomatid drugs against these disease agents"--
RNA Editing in Trypanosomes
Author: Alfredo J. HernandezPublisher:
ISBN:
Category :
Languages : en
Pages :
Book Description
RNA editing in trypanosomes is the post-transcriptional insertion or deletion of uridylates at specific sites in mitochondrial mRNAs. This process is catalyzed by a multienzyme, multisubunit complex through a series of enzymatic cycles directed by small, trans-acting RNA molecules. Despite impressive progress in our understanding of the mechanism of RNA editing and the composition of the editing complex, fundamental questions regarding RNP assembly and the regulation of catalysis remain. This dissertation presents studies of RNA-protein interactions between RNA editing complexes and substrate RNAs and the determination of substrate secondary structural determinants that govern them. Our results suggest that substrate association, cleavage and full-round editing by RNA editing complexes in vitro obey hierarchical determinants that increase in complexity as editing progresses and we propose a model for substrate recognition by RNA editing complexes. In addition, this dissertation also presents the characterization of a novel mitochondrial RNA helicase, named REH2 and its macromolecular interactions. Our data suggest that REH2 is intimately involved in interactions with macromolecular complexes that integrate diverse processes mediating mitochondrial gene expression. These results have implications for the mechanism of substrate RNA recognition by RNA editing complexes as well as for the integration of RNA editing to other facets of mitochondrial RNA metabolism.
RNA Editing in Trypanosome Mitochondria
Author: Robert S. SabatiniRNA Editing
Author: H. Ulrich GöringerPublisher: Springer Science & Business Media
ISBN: 3540737871
Category : Science
Languages : en
Pages : 239
Book Description
Goringer’s brilliant new work dedicates a chapter to each of the main types of RNA editing – the very first volume to do so. All of the sections here have been written by experts in the various research areas and a specific focus is put on the correlation between RNA structure and function, as well as on the complex cellular machineries that catalyze the different editing reactions. This leads to a "state of the art" compendium of our current knowledge on RNA editing.
RNA Editing
Author: Brenda BassPublisher: Oxford University Press
ISBN: 9780199638147
Category : Medical
Languages : en
Pages : 214
Book Description
The information encoded in DNA is conveyed to the rest of the cell in a molecule called RNA. To diversify this information, as well as repair it when mistakes are made, RNA is modified through a series of reactions known as RNA editing. This book describes the fascinating and unexpectedly diverse ways RNA editing can occur, in organisms ranging from single- celled protozoa to man.
Dissertation Abstracts International
Author: Publisher:
ISBN:
Category : Dissertations, Academic
Languages : en
Pages : 924
Book Description
RNA Editing
Author: Publisher: Elsevier
ISBN: 008055105X
Category : Science
Languages : en
Pages : 591
Book Description
RNA processing plays a critical role in realizing the full potential of a given genome. One means of achieving protein diversity is through RNA editing. A diverse array of editing events has been characterized, affecting gene expression in organisms from viruses and single cell parasites to humans and plants. The variety of editing mechanisms has required the development of many different experimental approaches, many of which are likely to be broadly applicable, particularly given the interplay between editing and other cellular processes, including transcription, splicing, and RNA silencing. RNA Editing not only covers most of the principal methods employed in the field, but also offers innovative solutions to the significant challenges posed by these experimental systems. - Presents newly developed methods - Covers topics ranging from biochemistry to bioinformatics - Includes innovative solutions to potential problems