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Essential functions of Trypanosoma brucei RAP1

Award Number: R01AI179972
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 12/06/2023
PERIOD OF PERFORMANCE END DATE: 10/31/2028

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Essential functions of Trypanosoma brucei RAP1 - Project summary Trypanosoma brucei causes human sleeping sickness and sequentially expresses immunologically distinct Variant Surface Glycoproteins (VSGs), its major surface antigen, to evade the host’s immune response and establish a long-term infection. VSG is transcribed one at a time (in a monoallelic manner) from regions next to the chromosome end termed telomere. Parasites that express more than one type of VSG are more quickly eliminated by the host. Therefore, monoallelic expression (MAE) of VSG genes is critical for parasite survival. We have shown that T. brucei RAP1, a telomere protein, is essential for telomere integrity, hence parasite proliferation, and VSG MAE. In order for RAP1 to execute these roles, its ability to bind dsDNA is essential but not sufficient. We hypothesize that a high concentration of telomere-localized RAP1 is required. Since RAP1 interacts with TRF that specifically binds the telomeric DNA, we hypothesize that this interaction helps enrich RAP1 at the telomere. We will test this by examining RAP1 chromatin association profile in WT and RAP1 mutants defective of RAP1-TRF interaction or DNA binding. VSG MAE has two key aspects: silencing all but one VSG genes in the T. brucei genome and sustaining high-level expression of the only active VSG, both depending on RAP1. On one hand, RAP1 helps compact the telomeric chromatin, which is critical for VSG silencing, but the underlying mechanisms are unknown. We found that RAP1 is essential for normal level expression of H3v, a histone variant enriched at the telomere and important for complete VSG silencing. RAP1 also interacts with FYRP, a subunit of the chromatin remodeler ISWI that is important for VSG silencing. Therefore, we hypothesize that the RAP1-H3v/FYRP interactions play important roles in RAP1-mediated VSG silencing. We will determine the interfaces of these interactions and examine VSG silencing status in RAP1 mutants defective of these interactions. On the other hand, we found that unlike vertebrate and yeast RAP1 homologs, T. brucei RAP1 has an RRM domain that mediates its binding to the active VSG RNA in vivo. Intriguingly, RAP1’s RNA and DNA binding activities compete with each other in a substrate concentration- dependent manner. At the active VSG locus, RAP1’s binding to the highly concentrated active VSG RNA effectively blocks its binding to local dsDNA and disrupts the RAP1-mediated silencing, enabling high-level expression of the active VSG. However, exactly what RNA sequences can be recognized by RAP1 and how RAP1 specifically regulates VSG MAE is unknown. We will characterize the properties of RAP1’s RNA binding activity using both in vivo iCLIP approach and several in vitro approaches including gel shift, NMR titration, RNA toeprinting and RNase footprinting. Our studies on RAP1 helps build a comprehensive view of RAP1’s pleiotropic functions and will venture into a new paradigm for better understanding mechanisms of antigenic variation in T. brucei. Investigating essential activities uniquely found in kinetoplastid RAP1 homologs will also benefit development of better treatments of infections caused by kinetoplastid parasites including T. brucei.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $437,631 )
2026	2026	CLEVELAND STATE UNIVERSITY	2121 EUCLID AVE	CLEVELAND	OH	44115	CUYAHOGA	USA	Allergy and Infectious Diseases Research	000	3	2/16/2026	NON-COMPETING CONTINUATION	$437,631
														Subtotal = $437,631

Issue Date FY: 2025 ( Subtotal = $431,704 )
2025	2025	CLEVELAND STATE UNIVERSITY	2121 EUCLID AVE	CLEVELAND	OH	44115	CUYAHOGA	USA	Allergy and Infectious Diseases Research	000	2	10/30/2024	NON-COMPETING CONTINUATION	$431,704
														Subtotal = $431,704

Issue Date FY: 2024 ( Subtotal = $455,005 )
2024	2024	CLEVELAND STATE UNIVERSITY	2121 EUCLID AVE	CLEVELAND	OH	44115	CUYAHOGA	USA	Allergy and Infectious Diseases Research	000	1	12/6/2023	NEW	$409,505
2024	2024	CLEVELAND STATE UNIVERSITY	2121 EUCLID AVE	CLEVELAND	OH	44115	CUYAHOGA	USA	Allergy and Infectious Diseases Research	001	1	9/4/2024	NEW	$45,500
														Subtotal = $455,005

Grand Total All Awards = $1,324,340

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Essential functions of Trypanosoma brucei RAP1

Award Number: R01AI179972

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 12/06/2023

PERIOD OF PERFORMANCE END DATE: 10/31/2028

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