Trypanosomes are unicellular parasites responsible for major health hazards in developing countries. The causative agent of African sleeping sickness, Trypanosoma brucei, is also an important model organism for several areas of research, including antigenic variation, host-pathogen interaction, developmental reprogramming, and mitochondrial biology. Indeed, some unique gene expression pathways, such as RNA editing, have been discovered in this parasite's giant mitochondrion. Because currently employed treatments against T. brucei are ineffective and unsafe, targeting cellular pathways that are found exclusively in this organism is a promising therapeutic approach. The trypanosome mitochondrion encloses an unusual DNA structure, called the kinetoplast, which is composed of few maxicircles and thousands of minicircles. Mitochondrial genes are encoded in maxicircles, but most are encrypted: an extensive post-transcriptional uridine insertion/deletion RNA editing is required to produce open reading frames. The cascade of editing reactions is catalyzed by enzymes embedded into the ~15-subunit RNA editing core complex, RECC (20S editosome), while each step is directed by minicircle-encoded guide RNAs (gRNAs). Structure-function studies of RECC achieved impressive progress, but little is known about gRNA biogenesis, stabilization, binding to mRNA, mechanism of action, and post-editing metabolic fate. We have discovered that mature gRNAs are stabilized via association with the gRNA binding complex, GRBC, and have identified the two subunits directly responsible for gRNA binding. Preliminary studies indicate that GRBC's complexity likely exceeds that of the RECC and that its functions extend beyond gRNA binding. This proposal focuses on GRBC protein composition and architecture, mechanisms of gRNA-mRNA interaction, and post-editing gRNA displacement. We hypothesize that RNA substrate-dependent RECC-GRBC assembly represents the RNA editing holoenzyme and propose to: 1) delineate protein-protein and RNA-mediated interactions within GRBC; 2) elucidate the functional role of GRBC-RECC interaction; 3) identify GRBC subunits essential for gRNA stability and mRNA binding; and 4) dissect the mechanism of gRNA displacement.

Public Health Relevance

Trypanosomes and closely related organisms are the causative agents of parasitic diseases in developing countries. Available treatments are often toxic and ineffective, and the need for new drugs is pressing. Because mitochondrial gene expression is vital for the parasite, targeting essential parasite-specific ribonucleoprotein complexes, such as the guide RNA binding complex (GRBC), is a potential approach toward developing new trypanocides.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI101057-05
Application #
8827665
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
2012-05-10
Project End
2016-04-30
Budget Start
2015-05-01
Budget End
2016-04-30
Support Year
5
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Dentistry/Oral Hygn
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
Sement, François M; Suematsu, Takuma; Zhang, Liye et al. (2018) Transcription initiation defines kinetoplast RNA boundaries. Proc Natl Acad Sci U S A 115:E10323-E10332
Zhang, Liye; Sement, Francois M; Suematsu, Takuma et al. (2017) PPR polyadenylation factor defines mitochondrial mRNA identity and stability in trypanosomes. EMBO J 36:2435-2454
Aphasizheva, Inna; Aphasizhev, Ruslan (2016) U-Insertion/Deletion mRNA-Editing Holoenzyme: Definition in Sight. Trends Parasitol 32:144-156
Suematsu, Takuma; Zhang, Liye; Aphasizheva, Inna et al. (2016) Antisense Transcripts Delimit Exonucleolytic Activity of the Mitochondrial 3' Processome to Generate Guide RNAs. Mol Cell 61:364-378
Aphasizheva, Inna; Zhang, Liye; Aphasizhev, Ruslan (2016) Investigating RNA editing factors from trypanosome mitochondria. Methods 107:23-33
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Aphasizheva, Inna; Maslov, Dmitri A; Qian, Yu et al. (2016) Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes. Mol Microbiol 99:1043-58
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Aphasizheva, Inna; Zhang, Liye; Wang, Xiaorong et al. (2014) RNA binding and core complexes constitute the U-insertion/deletion editosome. Mol Cell Biol 34:4329-42
Aphasizhev, Ruslan; Aphasizheva, Inna (2014) Mitochondrial RNA editing in trypanosomes: small RNAs in control. Biochimie 100:125-31

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