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Principal Investigator: CHRISTIAN TSCHUDI
Organization: YALE UNIVERSITY
Fiscal Year: 2020
Award: $751,764
Funding agency: National Institute of Allergy and Infectious Diseases
DESCRIPTION (provided by applicant): This application focuses on the protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa. There are no vaccines, and therapeutic drugs have serious side effects and decreasing efficacy. Thus, there is a pressing need for research to better understand the biology of these human pathogens and the mechanisms they use to survive within their hosts. T. brucei undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector, which among others involves changes in cell morphology, surface coat composition, metabolism, signaling pathways and gene expression. Consequently, these parasites have evolved adaptations to allow for their survival in both the gut and salivary glands of the tsetse fly, as well as in the bloodstream of their mammalian host. By overexpressing a single RNA-binding protein (RBP6) in non- infectious trypanosomes, we recapitulated in vitro the events leading to acquisition of infectivity in the insect vector, including the expression of metacyclic variant surface glycoproteins (mVSGs). At present, little is known how mVSG gene expression is activated and how the expression is switched to bloodstream-form VSGs, once the parasite enters a mammalian host. One major goal of this application will be to examine how trypanosomes receive instructions to begin synthesizing the mVSG coat, how each cell expresses a single mVSG, and how mVSG expression is repressed and switched to the expression of bloodstream-form VSGs. We will apply a number of high-throughput approaches to monitor the chromatin structure of mVSG genes during developmental progression and test whether a second RNA-binding protein (RBP10) is a facilitator of [m]VSG expression. Primary transcripts and mature mRNAs will be monitored using RNA-Seq, the position, amount, and orientation of transcriptionally engaged RNA polymerase I will be surveyed by global run-on-sequencing (GRO-Seq) and transcription factor binding will be gauged by chromatin immunoprecipitation (ChIP) coupled with Illumina sequencing (ChIP-Seq). A second emphasis will be on RNA interference (RNAi). Since our discovery of RNAi in T. brucei in 1998, this pathway has been a focus of our investigations, which have led to the finding that RNAi functions both in the nucleus and in the cytoplasm and to the identification of five "core" RNAi genes. We will employ different approaches to address the question what defines the RNA-induced silencing complex (RISC), i.e. what other cellular factors functionally interact with the RNAi machinery, and how the levels of Argonaute are regulated. Finally, we will further address the biological function of RNAi by determining the RNAi targets during the T. brucei developmental cycle, which is now possible with our newly developed differentiation system.
Terms: <5'-Exonuclease><5'-Nucleotidephosphodiesterase><Acceleration><Address><Africa South of the Sahara><African><African Sleeping Sickness><African Trypanosomiasis><Alkaline Phosphodiesterase><Alkaline Phosphodiesterase I><Animals><Arthropoda><Arthropods><Basal Transcription Factor><Basal transcription factor genes><Binding><Biological><Biological Function><Biological Process><Biology><Blood><Blood Circulation><Blood Reticuloendothelial System><Bloodstream><Bovine Species><Cattle><Cell Body><Cell Communication and Signaling><Cell Nucleus><Cell Signaling><Cell Surface Glycoproteins><Cells><Cellular Morphology><ChIP assay><Chromatin Structure><Circulation><Communicable Diseases><Complex><Coupled><Cytoplasm><DNA-Dependent RNA Polymerase I><Development><Disease><Disorder><Domestic Pig><Double-Stranded RNA><Drug Targeting><Drugs><Event><Farm Animal><Future><GRO-seq><GROseq><Gene Down-Regulation><Gene Expression><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Transcription><Genome><Glossina><Goals><Human><Human Biology><In Vitro><Infectious Disease Pathway><Infectious Diseases><Infectious Disorder><Insect Vectors><Instruction><Intermediary Metabolism><Intervention><Intervention Strategies><Intracellular Communication and Signaling><Investigation><Laboratories><Leishmania (Leishmania) major><Leishmania major><Leishmania tropica major><Life Cycle><Life Cycle Stages><Livestock><Medication><Membrane Glycoproteins><Messenger RNA><Metabolic Processes><Metabolism><Modern Man><Molecular><Molecular Interaction><Monitor><Nucleus><Oligonucleate 5'-Nucleotidohydrolase><Orthophosphoric Diester Phosphohydrolase><P falciparum><P. falciparum><P.falciparum><Parasites><Pathogenesis><Pathway interactions><Pharmaceutic Preparations><Pharmaceutical Preparations><Phosphodiesterase I><Plasmodium falciparum><Position><Positioning Attribute><Post-Transcriptional Gene Silencing><Posttranscriptional Gene Silencing><Production><Proteins><Protozoa><Protozoal><Public Health><Quelling><RISC Multicomponent Nuclease><RNA Expression><RNA Interference><RNA Interference Pathway><RNA Polymerase A><RNA Polymerase I><RNA Seq><RNA Silencing><RNA metabolism><RNA sequencing><RNA-Binding Proteins><RNA-Induced Silencing Complex><RNAi><RNAseq><Regulation><Research><Role><Salivary Glands><Salivary Glands Head and Neck><Sequence-Specific Posttranscriptional Gene Silencing><Short interfering RNA><Signal Transduction><Signal Transduction Systems><Signaling><Signaling Factor Proto-Oncogene><Signaling Pathway Gene><Signaling Protein><Site><Small Interfering RNA><Small RNA><Sub-Saharan Africa><Subsaharan Africa><Surface><Surface Glycoproteins><Survey Instrument><Surveys><Sus scrofa domestica><System><T brucei><T. brucei><Testing><Therapeutic><Time><Transcript><Transcription><Transcription Factor Proto-Oncogene><Transcription Repression><Transcription factor genes><Transcriptional Repression><Trypanosoma><Trypanosoma brucei><Trypanosoma brucei brucei><Trypanosome><Tsetse Flies><Vaccines><Validation><Variant><Variation><base><biological signal transduction><bovid><bovine><cell morphology><chromatin immunoprecipitation><cow><developmental><drug/agent><dsRNA><experiment><experimental research><experimental study><feeding><gene repression><global run on sequencing><global run on transcription sequencing><human disease><human pathogen><improved><interventional strategy><life course><mRNA><member><mutant><nagana><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><novel drug treatments><novel drugs><novel therapeutics><novel therapy><overexpress><overexpression><pathogen><pathway><programs><public health relevance><siRNA><side effect><sleeping sickness><social role><tool><transcription factor><transcriptome sequencing><transmission-blocking vaccine><vector><wasting>