The trypanosomatid parasites Trypanosoma brucei, T. cruzi and Leishmania spp. (Tritryp) cause the major human diseases African Sleeping Sickness, Chagas'disease, and Leishmaniasis, respectively. Drugs for these diseases are few and toxic and parasite resistance to these drugs is on the rise. Thus, it becomes increasingly important to find new anti-parasitic targets and develop new therapeutic strategies. All trypanosomatids depend on the same unusual mode of gene expression involving polycistronic transcription of protein coding genes and trans splicing of nuclear pre-mRNA. Of central importance to this process is the parasite-specific spliced leader (SL) RNA from which the 5'terminal part is cleaved and fused to the 5'end of each mRNA. Since SL RNA is consumed in this process, the parasites crucially depend on continuously strong SL RNA synthesis throughout their life cycle. We have recently identified and characterized several proteins in T. brucei which are essential for SL RNA gene transcription. These include extremely divergent homologues of the general transcription factors TFIIA, TFIIB, TFIIH, and a candidate TFIIE suggesting that the parasites form a class II transcription pre-initiation complex (PIC) at the SL RNA gene (SLRNA) promoter to recruit RNA polymerase II for accurate transcription initiation. The extraordinary level of sequence divergence between these proteins and their mammalian orthologues, which prevented their identification in the completed Tritryp genome projects, indicates that the trypanosome PIC deviates substantially from its human counterpart. To explore this possibility, we will functionally characterize the PIC which forms at the SLRNA promoter. This will include RNA interference, in vitro transcription experiments and DNA-protein interaction assays to evaluate the identified proteins'importance for parasite growth and role in SLRNA transcription. Finally, we will analyze if the parasites use PIC formation to recruit RNA polymerase II to their long tandem arrays of protein coding genes. Chromatin immunoprecipitation in combination with deep sequencing will identify non-SLRNA PIC formation sites in the T. brucei genome. Together, these experiments may uncover unique and essential structural and/or biochemical features in a fundamentally important process, namely the recruitment of RNA polymerase II to DNA.

Public Health Relevance

The proposed studies of this application will explore the essential process of accurate transcription initiation in trypanosomatid parasites which cause major human diseases. The proteins involved in this process exhibit an unprecedented divergence level to their human counterparts which raises the possibility that we can identify new targets for chemotherapeutic intervention of the parasites. Such targets are urgently needed because drugs for these diseases are few and toxic and parasite resistance to these drugs is on the rise.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI073300-01A2
Application #
7590870
Study Section
Special Emphasis Panel (ZRG1-IDM-R (02))
Program Officer
Mcgugan, Glen C
Project Start
2009-05-15
Project End
2011-04-30
Budget Start
2009-05-15
Budget End
2010-04-30
Support Year
1
Fiscal Year
2009
Total Cost
$363,663
Indirect Cost
Name
University of Connecticut
Department
Genetics
Type
Schools of Medicine
DUNS #
022254226
City
Farmington
State
CT
Country
United States
Zip Code
06030
Srivastava, Ankita; Badjatia, Nitika; Lee, Ju Huck et al. (2018) An RNA polymerase II-associated TFIIF-like complex is indispensable for SL RNA gene transcription in Trypanosoma brucei. Nucleic Acids Res 46:1695-1709
Badjatia, Nitika; Park, Sung Hee; Ambrósio, Daniela L et al. (2016) Cyclin-Dependent Kinase CRK9, Required for Spliced Leader trans Splicing of Pre-mRNA in Trypanosomes, Functions in a Complex with a New L-Type Cyclin and a Kinetoplastid-Specific Protein. PLoS Pathog 12:e1005498
Ambrósio, Daniela L; Badjatia, Nitika; Günzl, Arthur (2015) The spliceosomal PRP19 complex of trypanosomes. Mol Microbiol 95:885-901
Günzl, Arthur; Kirkham, Justin K; Nguyen, Tu N et al. (2015) Mono-allelic VSG expression by RNA polymerase I in Trypanosoma brucei: expression site control from both ends? Gene 556:68-73
Park, Sung Hee; Nguyen, Bao N; Kirkham, Justin K et al. (2014) A new strategy of RNA interference that targets heterologous sequences reveals CITFA1 as an essential component of class I transcription factor A in Trypanosoma brucei. Eukaryot Cell 13:785-95
Badjatia, Nitika; Ambrosio, Daniela L; Lee, Ju Huck et al. (2013) Trypanosome cdc2-related kinase 9 controls spliced leader RNA cap4 methylation and phosphorylation of RNA polymerase II subunit RPB1. Mol Cell Biol 33:1965-75
Badjatia, Nitika; Nguyen, Tu N; Lee, Ju Huck et al. (2013) Trypanosoma brucei harbours a divergent XPB helicase paralogue that is specialized in nucleotide excision repair and conserved among kinetoplastid organisms. Mol Microbiol 90:1293-308
Park, Sung Hee; Nguyen, Tu N; Gunzl, Arthur (2012) Development of an efficient in vitro transcription system for bloodstream form Trypanosoma brucei reveals life cycle-independent functionality of class I transcription factor A. Mol Biochem Parasitol 181:29-36
Gunzl, Arthur (2010) The pre-mRNA splicing machinery of trypanosomes: complex or simplified? Eukaryot Cell 9:1159-70
Lee, Ju Huck; Cai, Gang; Panigrahi, Aswini K et al. (2010) A TFIIH-associated mediator head is a basal factor of small nuclear spliced leader RNA gene transcription in early-diverged trypanosomes. Mol Cell Biol 30:5502-13