African trypanosomes cause disease and suffering in vast regions of sub-Saharan Africa and the drugs available to eliminate the disease are sub-optimal, to say the least. Many laboratories, including our own, have been engaged in studying the biology of Trypanosoma brucei and have contributed to the understanding of several mechanisms that are unique to trypanosomes. The discovery of trypanosome-specific pathways is the first step towards identifying new potential drug targets, which may be exploited against the parasite itself. Since 1998 when we discovered the RNAi pathway in Trypanosoma brucei our research has focused towards understanding the mechanism and components of RNAi. These studies are essential to eventually attempt to use small RNAs as drugs against trypanosomes. This possibility is not without foundation, since there are already several clinical trials in place to use small RNAs to cure certain human diseases. The experiments we propose stem directly from three main findings generated during the last funding period. First, sequencing of small interfering RNAs (siRNAs), from both bloodstream and procyclic trypanosomes has revealed a new target of the RNAi pathway, namely a family of satellite-like repeats (CIR147) located at chromosomal internal sites. These repeats may function in some aspect of chromosome structure and/or regulation of gene expression. Second, we have identified the RNAi genes in Leishmania braziliensis and in collaboration with Steve Beverley have demonstrated that the RNAi pathway is operational. However, L brasiliensis has some limitations for studying pathogenesis because at present there is no suitable small animal model of the disease. In contrast, Diane McMahon-Pratt at Yale has established a mouse model for L. panamensis, a close relative of L. brasiliensis, which also has the RNAi genes. Thus, our findings open the way to establish tools for applying the wonders of RNAi technology to the study of different aspects of the biology and pathogenesis of Leishmania. Third, we have discovered that a second T. brucei Argonaute-like protein (termed PiwiS) is localized in the mitochondrion and forms foci reminiscent of the antipodal sites where some enzymes for kinetoplast DNA (kDNA) replication localize, implicating PiwiS in some aspect of kDNA biogenesis. In the last few years Argonaute proteins have emerged as central regulators of an outstandingly wide range of phenomena, both at the transcriptional and post-transcriptional levels. Thus, we hypothesize that PiwiS functions in some aspect of RNA silencing in the mitochondrion. It should be noted that this protein is the first example of an Argonaute protein that localizes to an organelle.
Our specific aims for the next funding period are: 1. To further mine the biology of trypanosomatids through high-throughput sequencing of small RNAs from T. brucei and from L. braziliensis, and through the functional and structural characterization of the CIR147 satellite-like repeat family. 2. To establish tools for heritable and regulated RNAi in L. panamensis. 3. To establish the function of PiwiS in the T. brucei mitochondrion. Project Narrative: African trypanosomes cause disease and suffering in vast regions of sub-Saharan Africa and the drugs available to eliminate the disease are sub-optimal, to say the least. Our research has focused towards understanding the mechanism and components of RNA interference to eventually attempt to use small RNAs as drugs against trypanosomes. This possibility is not without foundation, since there are already several clinical trials in place to use small RNAs to cure certain human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI056333-10
Application #
8212132
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Joy, Deirdre A
Project Start
2003-07-01
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2014-01-31
Support Year
10
Fiscal Year
2012
Total Cost
$364,966
Indirect Cost
$144,443
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Ericson, Megan; Janes, Michael A; Butter, Falk et al. (2014) On the extent and role of the small proteome in the parasitic eukaryote Trypanosoma brucei. BMC Biol 12:14
Shi, Huafang; Barnes, Rebecca L; Carriero, Nicholas et al. (2014) Role of the Trypanosoma brucei HEN1 family methyltransferase in small interfering RNA modification. Eukaryot Cell 13:77-86
Kolev, Nikolay G; Ullu, Elisabetta; Tschudi, Christian (2014) The emerging role of RNA-binding proteins in the life cycle of Trypanosoma brucei. Cell Microbiol 16:482-9
Atayde, Vanessa D; Shi, Huafang; Franklin, Joseph B et al. (2013) The structure and repertoire of small interfering RNAs in Leishmania (Viannia) braziliensis reveal diversification in the trypanosomatid RNAi pathway. Mol Microbiol 87:580-93
Michaeli, Shulamit; Doniger, Tirza; Gupta, Sachin Kumar et al. (2012) RNA-seq analysis of small RNPs in Trypanosoma brucei reveals a rich repertoire of non-coding RNAs. Nucleic Acids Res 40:1282-98
Atayde, Vanessa D; Ullu, Elisabetta; Kolev, Nikolay G (2012) A single-cloning-step procedure for the generation of RNAi plasmids producing long stem-loop RNA. Mol Biochem Parasitol 184:55-8
Atayde, Vanessa D; Tschudi, Christian; Ullu, Elisabetta (2011) The emerging world of small silencing RNAs in protozoan parasites. Trends Parasitol 27:321-7
Kolev, Nikolay G; Tschudi, Christian; Ullu, Elisabetta (2011) RNA interference in protozoan parasites: achievements and challenges. Eukaryot Cell 10:1156-63
Lye, Lon-Fye; Owens, Katherine; Shi, Huafang et al. (2010) Retention and loss of RNA interference pathways in trypanosomatid protozoans. PLoS Pathog 6:e1001161
Patrick, Kristin L; Shi, Huafang; Kolev, Nikolay G et al. (2009) Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei. Proc Natl Acad Sci U S A 106:17933-8

Showing the most recent 10 out of 15 publications