# Mitochondrial DNA of Normal and Mutant Trypanosomes

> **NIH NIH R01** · SEATTLE CHILDREN'S HOSPITAL · 2024 · $1,131,890

## Abstract

Abstract
We will elucidate how three RNA editing Catalytic Complexes (CCs) interact with Substrate Complexes (SCs),
Accessory Factors (AFs), mRNAs and gRNAs to generate translatable mitochondrial mRNAs via precise U
insertion and deletion and do so differentially between bloodstream (BF) and procyclic (PF) forms in
Trypanosoma brucei. We hypothesize that this occurs via specific molecular interactions between domains of
CC, SC and AF proteins and the mRNA/gRNA substrate. We will in: Aim 1. Identify these interactions in PFs
and determine which are RNA dependent by a combination of in vivo proximity labelling, protein-protein cross-
linking, and affinity purification methods. We will build structural models to identify similarities and differences
between the three CCs. Aim 2. Determine the effects of loss of function (LOF) mutations of editing steps on
these interactions, interactions with RNA by cross-linking immunoprecipitation and on RNA editing by RT-PCR
sequencing. We will prioritize mutations that affect initiation of editing, subsequent catalytic steps and the
functions of other essential proteins that are in all three CCs. This will validate and extend results from Aim 1,
link the interactions to functional steps of editing and advance the structural models. Aim 3. Identify physical and
functional interactions in BF cells by approaches used in Aims 1 and 2 and extended using a MGA knock-in
approach which eliminates dependence on RNA editing in BFs and enables additional analyses of LOF
mutations and interactions with RNA. This aim will identify similar and different molecular interactions between
BFs and PFs. Aim 4. Determine the detailed characteristics of protein domains that function differently between
BFs and PFs. We will determine the detailed chemical, structural, and functional characteristics of CC protein
domains that affect editing differently in BFs vs. PFs using a focused saturation mutagenesis approach.
Overall, this project will advance the understanding of how multiprotein complexes generate translatable mRNAs
for essential components of the mt energy generating system in protozoan pathogens and do so differently
between their developmental stages. The results may aid identification of targets for novel drug targets that are
needed and provide insights into mechanisms of protein/nucleic acid interactions that are widespread and
important in eukaryotes and which may lead to practical applications.

## Key facts

- **NIH application ID:** 10800497
- **Project number:** 2R01AI014102-47A1
- **Recipient organization:** SEATTLE CHILDREN'S HOSPITAL
- **Principal Investigator:** KENNETH D STUART
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $1,131,890
- **Award type:** 2
- **Project period:** 1978-09-01 → 2029-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10800497

## Citation

> US National Institutes of Health, RePORTER application 10800497, Mitochondrial DNA of Normal and Mutant Trypanosomes (2R01AI014102-47A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10800497. Licensed CC0.

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