Our research is aimed at understanding gene expression in the family Trypanosomatidae, which includes the parasitic protozoa responsible for leishmaniasis, African Sleeping Sickness, and Chagas Disease. The focus is on the genesis and function of the spliced leader (SL) RNA, a small RNA that contributes the 5'-end sequence to every nuclear messenger RNA by a trans-splicing reaction and is conserved extraordinarily at the primary sequence level throughout the Order. Trans-splicing is necessary for the conversion of polycistronic pre-messenger RNA, a likely consequence of the unusual genome organization found in kinetoplastids, into monocistronic mRNA, and is not found in the human host or insect vector, thus representing an ideal therapeutic target. This proposal outlines experiments that detail the maturation pathway of the SL RNA common to Leishmania tarentolae and Trypanosoma brucei at the level of individual proteins and interacting complexes, and probe the role of the SL in translation. 1) We will explore the role of the cap 4 2'-O-ribose methyltransferases in the bloodstream form of T. brucei, and validate their function in procyclic T. brucei using knockout strains. We will investigate the cap-binding properties of TbMTr1, and test models for the role of TbMTAP and TbMTr1 in trafficking and interacting with the pseudouridine synthase complex in the nucleus. 2) To further the characterization of complexes involved in SL RNA biogenesis and function. The trinity of PTP tagging, multidimensional protein identification technology (MudPIT), and the T. brucei genome database enables us to approach complex purification. The kinetoplastid-specific TbMTr1 complex protein TbMTAP is implicated in regulatory and structural interactions. Using the SmD3 protein, the SL RNA RNP complex and associated splicing machinery, both catalytic and core, has been captured. Of the 25 proteins identified, four novel, kinetoplastid-specific proteins will be validated for association and function along with two additional splicing homologs. These goals address discrete steps in the maturation of the SL RNA that determine its subsequent function. Specific inhibition of the trans-splicing pathway is lethal to the parasite and thus a prime target for clinical intervention.

Public Health Relevance

The Order Kinetoplastida contains several pathogens of medical importance including the causative agents of human leishmaniasis, African trypanosomiasis and Chagas disease. These organisms employ unusual mechanisms of gene expression involving polycistronic transcription of protein-coding genes and trans-splicing of a universal 39-nucleotide spliced leader sequence to all nuclear mRNAs. The goal of this study is to continue the identification of spliced leader specific processing components that have the potential to be specific targets for anti-parasite chemotherapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI056034-10
Application #
8507127
Study Section
Special Emphasis Panel (ZRG1-IDM-B (02))
Program Officer
Mcgugan, Glen C
Project Start
2003-07-01
Project End
2014-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
10
Fiscal Year
2013
Total Cost
$350,060
Indirect Cost
$119,736
Name
University of California Los Angeles
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Porcel, Betina M; Denoeud, France; Opperdoes, Fred et al. (2014) The streamlined genome of Phytomonas spp. relative to human pathogenic kinetoplastids reveals a parasite tailored for plants. PLoS Genet 10:e1004007
Freire, Eden R; Malvezzi, Amaranta M; Vashisht, Ajay A et al. (2014) Trypanosoma brucei translation initiation factor homolog EIF4E6 forms a tripartite cytosolic complex with EIF4G5 and a capping enzyme homolog. Eukaryot Cell 13:896-908
Freire, Eden R; Vashisht, Ajay A; Malvezzi, Amaranta M et al. (2014) eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei. RNA 20:1272-86
Zhang, Huan; Campbell, David A; Sturm, Nancy R et al. (2013) Signal recognition particle RNA in dinoflagellates and the Perkinsid Perkinsus marinus. Protist 164:748-61
Zingales, Bianca; Miles, Michael A; Campbell, David A et al. (2012) The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Genet Evol 12:240-53
Tuller, Tamir; Girshovich, Yana; Sella, Yael et al. (2011) Association between translation efficiency and horizontal gene transfer within microbial communities. Nucleic Acids Res 39:4743-55
Ruvalcaba-Trejo, Laura I; Sturm, Nancy R (2011) The Trypanosoma cruzi Sylvio X10 strain maxicircle sequence: the third musketeer. BMC Genomics 12:58
Werner, Maria; Purta, Elzbieta; Kaminska, Katarzyna H et al. (2011) 2'-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family. Nucleic Acids Res 39:4756-68
Zhang, Huan; Campbell, David A; Sturm, Nancy R et al. (2011) Spliced leader RNAs, mitochondrial gene frameshifts and multi-protein phylogeny expand support for the genus Perkinsus as a unique group of alveolates. PLoS One 6:e19933
Sturm, Nancy R; Campbell, David A (2010) Alternative lifestyles: the population structure of Trypanosoma cruzi. Acta Trop 115:35-43

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