Our long-term goal is to understand the molecular mechanisms that control RNA editing during the life cycle of Trypanosoma brucei. Kinetoplastid protozoa include important pathogenic species of Trypanosoma, Leishmania and Phytomonas. These ancient flagellates undergo a dramatic form of mitochondrial mRNA maturation by insertion and deletion of U residues, which can create over half of the protein-coding sequence. In T. brucei, editing is differentially regulated in the insect vector and the mammalian host (procyclic """"""""Pf"""""""" and bloodstream """"""""Bf"""""""" forms, respectively). Studying unique biological processes, such like this, may suggest ways of halting the development cycle and hence the progress of the disease. While the mechanism of Pf editing is known and the protein composition of Pf editing complexes is starting to emerge, fundamental questions remain unexplored including the function and composition of Bf complexes, and the relevant pre-mRNA/protein interactions during Pf and Bf editing.
Our specific aims are (i) to apply our newly established RNA editing system in bloodstream (Bf) trypanosomes to study regulation in vitro, and (ii) to identify pre-mRNA/ protein interactions during Pf and Bf editing. My lab recently established a long-sought in vitro system to study Bf full editing by U insertion and U deletion. Our preliminary data show significant differences between Pf and Bf complexes and their editing activity. We also began characterizing pre-mRNA/ protein crosslinks that co-purify with editing complexes upon high-stringency affinity chromatography.
In Aim 1 we propose to further compare the protein composition and function of Bf and Pf complexes, search for differential protein components, and identify editing steps and pre-mRNA features involved in regulation.
In Aim 2, we propose to further characterize two current crosslinking proteins, particularly their physical and/or functional relationship with editing complexes. We will similarly characterize additional RNA/protein interactions. Our studies compare stage-specific and constitutive premRNAs (paired or not to gRNA) under catalytic conditions. We expect to characterize RNA/protein contacts relevant for function, which may be stage-specific and/or transcript-specific. Together these studies should provide important insights into the developmental control of RNA editing, and understanding of relevant RNA/protein interactions during this unique maturation process. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067130-04
Application #
7272052
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rhoades, Marcus M
Project Start
2004-09-01
Project End
2009-08-31
Budget Start
2007-09-01
Budget End
2008-08-31
Support Year
4
Fiscal Year
2007
Total Cost
$206,940
Indirect Cost
Name
Texas A&M University
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
078592789
City
College Station
State
TX
Country
United States
Zip Code
77845
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Hernandez, Alfredo; Madina, Bhaskara Reddy; Ro, Kevin et al. (2010) REH2 RNA helicase in kinetoplastid mitochondria: ribonucleoprotein complexes and essential motifs for unwinding and guide RNA (gRNA) binding. J Biol Chem 285:1220-8
Hernandez, Alfredo; Panigrahi, Aswini; Cifuentes-Rojas, Catherine et al. (2008) Determinants for association and guide RNA-directed endonuclease cleavage by purified RNA editing complexes from Trypanosoma brucei. J Mol Biol 381:35-48
Cruz-Reyes, Jorge (2007) RNA-protein interactions in assembled editing complexes in trypanosomes. Methods Enzymol 424:107-25
Cifuentes-Rojas, Catherine; Pavia, Paula; Hernandez, Alfredo et al. (2007) Substrate determinants for RNA editing and editing complex interactions at a site for full-round U insertion. J Biol Chem 282:4265-76
Halbig, Kari; Sacharidou, Anastasia; De Nova-Ocampo, Monica et al. (2006) Preferential interaction of a 25kDa protein with an A6 pre-mRNA substrate for RNA editing in Trypanosoma brucei. Int J Parasitol 36:1295-304
Miller, Melissa M; Halbig, Kari; Cruz-Reyes, Jorge et al. (2006) RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction. RNA 12:1292-303
Sacharidou, Anastasia; Cifuentes-Rojas, Catherine; Halbig, Kari et al. (2006) RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei. RNA 12:1219-28
Cifuentes-Rojas, Catherine; Halbig, Kari; Sacharidou, Anastasia et al. (2005) Minimal pre-mRNA substrates with natural and converted sites for full-round U insertion and U deletion RNA editing in trypanosomes. Nucleic Acids Res 33:6610-20