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Base J and transcription termination in Leishmania

Award Number: R01AI103858
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: 06/11/2013
PERIOD OF PERFORMANCE END DATE: 05/31/2019

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Base J and transcription termination in Leishmania - DESCRIPTION (provided by applicant): Leishmania parasites infect humans worldwide, leading to substantial morbidity and mortality. Transcription of protein-coding genes in Leishmania and other trypanosomatids initiates at the 5' end of a small number of large polycistronic transcription units (PTUs), and mature mRNAs are generated by trans-splicing of the primary transcript. However, little is known about how transcription is terminated in Leishmania. Base J is a hyper-modified DNA base that replaces ~1% of T in the nuclear DNA in Leishmania and related organisms. When one of the genes (JBP2) involved in J biosynthesis is disrupted in L. tarentolae promastigotes, they lose ~70% of their J, leading to massive transcriptional read-through at the convergent strand-switch regions between PTUs (and an increase in false-starts where transcription initiates). Furthermore, JPB2 dKO parasites become hypersensitive to growth in bromodeoxyuridine (BrdU), leading to a further reduction in J, even more read-through, and cell death. We postulate that iJ serves as a critical signal for transcription termination, and that loss of proper termination in J-deficient cells kills the paraste. In this project, we will critically assess this hypothesis by using a combination of state-of-the-at approaches to further elucidate J biosynthesis and function in Leishmania. In Aim 1, we will use RNA-seq to analyze samples isolated at various times after disruption of the JBP2 and JBP1 genes, as well as BrdU and DMOG treatment, to identify mRNAs whose levels increase or decrease after iJ loss (with potentially lethal consequences), as well compensatory changes in gene expression that enable L. tarentolae to cope with this insult. Aim 2 will utilize single-molecule real-time (SMRT) sequencing to identify the exact T residues that are converted to J, and will use systematic modification of these sequences to identify the minimal information required for de novo J synthesis, as well as for J "spreading". In Aim 3, we will use in vivo and i vitro transcription assays, genome-wide chromatin mapping, as well as fluorescence in situ hybridization and chromosome conformation capture experiments to determine whether J terminates transcription either by directly blocking the RNA polymerase, by inducing chromatin changes, or by recruitment of chromatin to specific sub-nuclear "silencing domains". Aim 4 will use DNA affinity and PTP-tagged protein affinity approaches, coupled with quantitative mass spectrometry-based proteomics, to identify proteins associated with iJ insertion and those recruited by iJ at transcription termination sites. The results from these experiments will be combined to build a more complete model of the molecular processes associated with J-mediated transcription termination. The proposed studies will increase our understanding of the role of J in controlling transcription in these organisms, and may provide insight into a possible role for similar base modifications in other eukaryotes. Furthermore, since base J is not found in the mammalian hosts of Leishmania (including humans), this information will provide a new opportunity for the development of novel chemotherapeutic agents against these important parasites.


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: 2022 ( Subtotal = $144,285 )
2022	2017	SEATTLE CHILDREN'S HOSPITAL	4800 SAND POINT WAY NE	SEATTLE	WA	98105	KING	USA	Allergy and Infectious Diseases Research	001	6	9/26/2022	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	-$144,285
2022	2017	SEATTLE CHILDREN'S HOSPITAL	4800 SAND POINT WAY NE	SEATTLE	WA	98105	KING	USA	Allergy and Infectious Diseases Research	002	6	9/26/2022	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$144,285
2022	2017	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	000	5	9/26/2022	NON-COMPETING CONTINUATION	$144,285
														Subtotal = $144,285

Issue Date FY: 2020 ( Subtotal = -$144,285 )
2020	2017	SEATTLE CHILDREN'S HOSPITAL	4800 SAND PT WAY NE	SEATTLE	WA	98105	KING	USA	Allergy and Infectious Diseases Research	000	6	11/4/2019	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	-$144,285
														Subtotal = -$144,285

Issue Date FY: 2019 ( Subtotal = $0 )
2019	2017	SEATTLE CHILDREN'S HOSPITAL	4800 SAND POINT WAY NE	SEATTLE	WA	98105	KING	USA	Allergy and Infectious Diseases Research	002	6	2/28/2019	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$144,285
2019	2017	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	003	5	7/23/2019	NON-COMPETING CONTINUATION	-$144,285
2019	2017	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	001	5	2/28/2019	NON-COMPETING CONTINUATION	$0
2019	2017	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	000	5	12/17/2018	NON-COMPETING CONTINUATION	-$144,285
2019	2017	SEATTLE CHILDREN'S HOSPITAL	4800 SAND POINT WAY NE	SEATTLE	WA	98105	KING	USA	Allergy and Infectious Diseases Research	004	6	7/23/2019	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$144,285
														Subtotal = $0

Issue Date FY: 2017 ( Subtotal = $565,909 )
2017	2017	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	002	5	5/13/2017	NON-COMPETING CONTINUATION	$565,909
2017	2016	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	001	4	1/23/2017	EXTENSION WITH OR WITHOUT FUNDS	$598
2017	2015	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	000	3	1/23/2017	NON-COMPETING CONTINUATION	-$598
														Subtotal = $565,909

Issue Date FY: 2016 ( Subtotal = $565,909 )
2016	2016	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N, STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	000	4	5/13/2016	EXTENSION WITH OR WITHOUT FUNDS	$565,909
														Subtotal = $565,909

Issue Date FY: 2015 ( Subtotal = $565,909 )
2015	2015	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N, STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	000	3	4/29/2015	NON-COMPETING CONTINUATION	$565,909
														Subtotal = $565,909

Issue Date FY: 2014 ( Subtotal = $565,909 )
2014	2014	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N, STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	000	2	5/27/2014	NON-COMPETING CONTINUATION	$565,909
														Subtotal = $565,909

Issue Date FY: 2013 ( Subtotal = $557,335 )
2013	2013	SEATTLE BIOMEDICAL RESEARCH INSTITUTE	307 WESTLAKE AVE N, STE 500	SEATTLE	WA	98109	KING	USA	Allergy and Infectious Diseases Research	000	1	6/11/2013	NEW	$557,335
														Subtotal = $557,335

Grand Total All Awards = $2,820,971

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Base J and transcription termination in Leishmania

Award Number: R01AI103858

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: 06/11/2013

PERIOD OF PERFORMANCE END DATE: 05/31/2019

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