The trypanosomatid parasites Trypanosoma brucei, T. cruzi and Leishmania spp. cause the major human diseases African Sleeping Sickness, Chagas'disease, and Leishmaniasis, respectively. Since drugs for these diseases are few, toxic and difficult to administer, and parasite resistance to these drugs is on the rise, it becomes increasingly important to develop new therapeutic strategies. While these parasites have developed unique host-parasite interactions, they all share the same unusual mode of gene expression involving polycistronic transcription of protein coding genes and trans splicing of nuclear pre-mRNA. A key molecule in this process is the 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 trans splicing, parasite viability crucially depends on continuously strong SL RNA synthesis. Therefore, inhibition of SL RNA gene (SLRNA) transcription appears to be a promising broadband strategy against trypanosomatid parasites. So far, we have been able to identify and characterize four transcription factors comprising 25 proteins in T. brucei that are essential for the process: the promoter-binding complex TRF4/SNAPc/TFIIA, TFIIB, a complex of TFIIH and TFIIE, and the trypanosome mediator. Our data show that these proteins form a transcription pre-initiation complex at the SLRNA promoter and recruit RNA polymerase II for accurate transcription initiation. Conversely, we found no evidence that TFIIB binds to divergent strand switch regions (dSSRs) known to initiate RNA polymerase II transcription of the protein coding gene arrays. We therefore propose in Aim 1 to continue our biochemical characterization of RNA polymerase II transcription factors to identify the most promising targets for further analysis;besides the specified factors, these include new proteins that co-purified with RNA pol II as well as a novel TFIIA- associated complex and a CDK-related kinase both of which appear to function specifically in pre-mRNA synthesis. For these factor characterizations we plan to employ a plethora of genetic and biochemical experiments which include tandem affinity purification of protein complexes, conditional gene silencing experiments and in vitro transcription assays.
In Aim 2, we will use a systematic approach to investigate the mechanism of transcription initiation in dSSRs which will be based on a genome-wide analysis of RNA polymerase II occupancy and on a mutational analysis of dSSR-driven reporter gene expression. Overall, these experiments may uncover unique and essential factors or factor domains in a fundamentally important process, namely the recruitment of RNA polymerase II to DNA. Moreover, they will provide a mechanistic understanding of the first step in protein expression which will help to control trypanosomatids in the long term.

Public Health Relevance

The proposed studies of this application will explore mechanism and relevant factors of RNA polymerase II transcription in Trypanosoma brucei, a member of a group of parasites that cause major, devastating diseases in humans. 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 this disease are few and toxic, and parasite resistance to existing drugs is on the rise. Moreover, a thorough understanding of trypanosome gene expression will help to control the parasite in the long term.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI073300-05
Application #
8447031
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
2007-04-01
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2013
Total Cost
$359,603
Indirect Cost
$124,603
Name
University of Connecticut
Department
Genetics
Type
Schools of Medicine
DUNS #
022254226
City
Farmington
State
CT
Country
United States
Zip Code
06030
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
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