Trypansome RNA Editing in Two Developmental Forms

Summary

Principal Investigator: Jorge Cruz Reyes
Abstract: [unreadable] DESCRIPTION (provided by applicant): 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. [unreadable] [unreadable]
Funding Period: 2004-09-01 - 2010-08-31
more information: NIH RePORT

Top Publications

  1. pmc RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
    Melissa M Miller
    Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, SUNY Buffalo School of Medicine, Buffalo, NY 14214, USA
    RNA 12:1292-303. 2006
  2. ncbi Preferential interaction of a 25kDa protein with an A6 pre-mRNA substrate for RNA editing in Trypanosoma brucei
    Kari Halbig
    Department of Biochemistry and Biophysics, Texas A and M University, 2128 TAMU, College Station, TX 77843, USA
    Int J Parasitol 36:1295-304. 2006
  3. pmc RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei
    Anastasia Sacharidou
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, TX 77843, USA
    RNA 12:1219-28. 2006
  4. ncbi RNA-protein interactions in assembled editing complexes in trypanosomes
    Jorge Cruz-Reyes
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, Texas, USA
    Methods Enzymol 424:107-25. 2007
  5. ncbi Substrate determinants for RNA editing and editing complex interactions at a site for full-round U insertion
    Catherine Cifuentes-Rojas
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, Texas 77843, USA
    J Biol Chem 282:4265-76. 2007
  6. pmc Determinants for association and guide RNA-directed endonuclease cleavage by purified RNA editing complexes from Trypanosoma brucei
    Alfredo Hernandez
    Department of Biochemistry and Biophysics, Texas A and M University, 2128 TAMU, College Station, TX 77843, USA
    J Mol Biol 381:35-48. 2008
  7. pmc REH2 RNA helicase in kinetoplastid mitochondria: ribonucleoprotein complexes and essential motifs for unwinding and guide RNA (gRNA) binding
    Alfredo Hernandez
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, Texas 77843, USA
    J Biol Chem 285:1220-8. 2010
  8. pmc Guide RNA biogenesis involves a novel RNase III family endoribonuclease in Trypanosoma brucei
    Bhaskara Reddy Madina
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, TX, USA
    RNA 17:1821-30. 2011

Detail Information

Publications8

  1. pmc RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction
    Melissa M Miller
    Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, SUNY Buffalo School of Medicine, Buffalo, NY 14214, USA
    RNA 12:1292-303. 2006
    ..Our results constitute the first report of any putative RNA editing accessory factor eliciting an effect on editing in vitro. Overall, these results support a novel accessory role for RBP16 in U insertion editing...
  2. ncbi Preferential interaction of a 25kDa protein with an A6 pre-mRNA substrate for RNA editing in Trypanosoma brucei
    Kari Halbig
    Department of Biochemistry and Biophysics, Texas A and M University, 2128 TAMU, College Station, TX 77843, USA
    Int J Parasitol 36:1295-304. 2006
    ....
  3. pmc RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei
    Anastasia Sacharidou
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, TX 77843, USA
    RNA 12:1219-28. 2006
    ..This apparent structural selectivity suggests that the RNA-protein interactions we observe may be involved in recognition of editing sites and/or catalysis in assembled complexes...
  4. ncbi RNA-protein interactions in assembled editing complexes in trypanosomes
    Jorge Cruz-Reyes
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, Texas, USA
    Methods Enzymol 424:107-25. 2007
    ..It also illustrates how variations of these assays can be applied to examine the specificity of the editing enzyme/substrate association, and to dissect structural or biochemical requirements of both the substrates and enzyme complex...
  5. ncbi Substrate determinants for RNA editing and editing complex interactions at a site for full-round U insertion
    Catherine Cifuentes-Rojas
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, Texas 77843, USA
    J Biol Chem 282:4265-76. 2007
    ..Importantly, most observed cross-linking interactions are both conserved and relatively stable at ES2 and ES1 in hybrid substrates. However, they were also detected as transient low-stability contacts in a non-edited transcript...
  6. pmc Determinants for association and guide RNA-directed endonuclease cleavage by purified RNA editing complexes from Trypanosoma brucei
    Alfredo Hernandez
    Department of Biochemistry and Biophysics, Texas A and M University, 2128 TAMU, College Station, TX 77843, USA
    J Mol Biol 381:35-48. 2008
    ..A model is proposed whereby one or more RNase III-type endonucleases mediate the initial binding and scrutiny of potential ligands and subsequent catalytic selectivity triggers either insertion or deletion editing enzymes...
  7. pmc REH2 RNA helicase in kinetoplastid mitochondria: ribonucleoprotein complexes and essential motifs for unwinding and guide RNA (gRNA) binding
    Alfredo Hernandez
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, Texas 77843, USA
    J Biol Chem 285:1220-8. 2010
    ..We propose that these putative higher order structures coordinate mitochondrial gene expression...
  8. pmc Guide RNA biogenesis involves a novel RNase III family endoribonuclease in Trypanosoma brucei
    Bhaskara Reddy Madina
    Department of Biochemistry and Biophysics, Texas A and M University, College Station, TX, USA
    RNA 17:1821-30. 2011
    ..The proposed participation of mRPN1 in processing of polycistronic RNA and its specific protein interactions in gRNA expression are discussed...