Diseases caused by Trypanosoma brucei spp. represent health hazards for many disadvantaged countries. Because of failing vector control and the rise of drug-resistance, the re-appearance of African sleeping sickness necessitates a search for new drug targets with emphasis on parasite-specific processes. Trypanosomal mitochondrial RNA processing pathways are extremely divergent from those of humans and represent a potential source of therapeutic intervention points. The mRNA editing has been extensively studied, but the significance of pre- and post-editing processing events emerged only recently. We discovered the mitochondrial poly (A) polymerase (KPAP1) and terminal uridyltransferase (RET1), and showed that intertwined polyadenylation and uridylation processes are critical for mRNA stability, translation and decay. In these studies, I encountered a diverse family of pentatricopeptide (PPR, 35 amino acids) helical repeat- containing RNA binding proteins that populate polyadenylation and translation complexes. By focusing on mRNA 3' end definition, modification and stability, this proposal introduces PPR proteins as key regulators of mitochondrial gene expression. The experimental approaches involve biochemistry, proteomics, crystallography, genetics and deep RNA sequencing. The proposal aims to: 1) Dissect general and transcript-specific mRNA adenylation/uridylation mechanisms. I hypothesize that the two modes of 3' modification, pre-editing addition of a short A-tail and post-editing A/U-tailing, serve discrete purposes of mRNA stabilization and commitment to translation, respectively. 2) Determine principles of uridylation- based mRNA decay. A hypothesis that RET1 TUTase-catalyzed uridylation accelerates mRNA 3'-5' degradation will be tested. I also posit that most transcripts are stabilized by a specific PPR protein that blocks mRNA uridylation. 3) Elucidate functions of a putative mRNA 3' definition and stabilization factor. I suggest that a PPR factor is deposited onto the pre-mRNA prior to polyadenylation to define the 3' end. We focus on select PPR proteins in trypanosomes, but the long term goal is to illuminate the mechanisms of RNA recognition by repeat-containing proteins, which will be of fundamental importance for RNA processing and mitochondrial biology fields.

Public Health Relevance

The causative agent of African sleeping sickness, T. brucei is one of the most ancient mitochondria-containing organisms. Unsurprisingly, fundamental phenomena were discovered in the kinetoplast mitochondrion. Our proposal introduces helical repeat-containing RNA binding proteins as critical regulators of mRNA biogenesis pathways and opens a new area of investigation in organellar gene expression in parasites.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI113157-02
Application #
8991912
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Joy, Deirdre A
Project Start
2015-01-05
Project End
2019-12-31
Budget Start
2016-01-01
Budget End
2016-12-31
Support Year
2
Fiscal Year
2016
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, Fran├žois 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; Zhang, Liye; Aphasizhev, Ruslan (2016) Investigating RNA editing factors from trypanosome mitochondria. Methods 107:23-33
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
Aphasizheva, Inna; Aphasizhev, Ruslan (2016) U-Insertion/Deletion mRNA-Editing Holoenzyme: Definition in Sight. Trends Parasitol 32:144-156