mitochondria; Trypanosoma brucei; developmental regulation; RNA editing; mitochondrial biogenesis; systems biology; parallel sequencing
Siegel T Nicolai, Gunasekera Kapila, Cross George A M, Ochsenreiter Torsten (2011), Gene expression in Trypanosoma brucei: lessons from high-throughput RNA sequencing., in Trends in parasitology
, 27(10), 434-41.
Ekanayake Dilrukshi K, Minning Todd, Weatherly Brent, Gunasekera Kapila, Nilsson Daniel, Tarleton Rick, Ochsenreiter Torsten, Sabatini Robert (2011), Epigenetic regulation of transcription and virulence in Trypanosoma cruzi by O-linked thymine glucosylation of DNA., in Molecular and cellular biology
, 31(8), 1690-700.
Nilsson Daniel, Gunasekera Kapila, Mani Jan, Osteras Magne, Farinelli Laurent, Baerlocher Loic, Roditi Isabel, Ochsenreiter Torsten (2010), Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei., in PLoS pathogens
, 6(8), 1001037-1001037.
Zhang Xiaobai, Cui Juan, Nilsson Daniel, Gunasekera Kapila, Chanfon Astrid, Song Xiaofeng, Wang Huinan, Xu Ying, Ochsenreiter Torsten (2010), The Trypanosoma brucei MitoCarta and its regulation and splicing pattern during development., in Nucleic acids research
, 38(21), 7378-87.
Rettig Jochen, Wang Yimu, Schneider André, Ochsenreiter Torsten, Dual targeting of isoleucyl-tRNA synthetase in Trypanosoma brucei is mediated through alternative trans-splicing., in Nucleic acids research
RNA editing in trypanosomes is a remarkable post-transcriptional process that results in the formation of mitochondrial
mRNAs differing from their genes by the insertion or deletion of uridylyl (U) nucleosides. The information for RNA
editing is provided by small guide RNAs (gRNAs) that basepair with their cognate pre-mRNAs to direct the precise sites
for U-insertion or U-deletion. While considerable progress has been made in the elucidation of the components of the
editing machinery and the general mechanism of RNA editing little is known about the regulation and function of this
process. Simplistically, it has been thought that the sole function for RNA editing was to correct mistakes in
mitochondrial protein coding genes thus allowing the translation of edited mRNAs to produce components of the
mitochondrial respiratory system. While this is certainly an important function for RNA editing we recently discovered
that primary mRNAs are differentially edited and translated to produce novel mitochondrial proteins. Our initial studies
focused on the mRNA for cytochrome c oxidase III (COIII), which we discovered was alternatively edited in
Trypanosoma brucei. A gRNA for the alternatively edited COIII transcript was identified and antibodies against the
predicted novel coding sequence of the alternatively edited protein (AEP-1) reacted with a mitochondrial membrane
protein. We have recently expanded the analysis of alternative mRNA editing to four additional genes, NADH
dehydrogenase subunits 7, 8, 9 (ND7, 8, 9) and ATP synthase subunit 6 (A6). Alternatively edited mRNAs, creating
novel open reading frames, were found for each of these genes.
The overall goals of this proposal are to determine the role of alternative mRNA editing in the developmental
regulation of mitochondrial biogenesis and how the translation products interact with the nuclear encoded mitochondrial
proteins. To accomplish these goals the following specific aims are proposed. In Specific Aim 1, we predict the nuclear
encoded mitochondrial proteome in T. brucei. In order to do so we will identify a list of features such as transmembrane
domains, signal peptides, disordered regions, secondary structural content, hydrophobicity and polarity measures that
show relevance to protein localization to the mitochondrion. Using these features, we will train a recently developed
Support Vector Machine (SVM) based classifier to predict mitochondrial protein localization. In specific aim 2 we will
test our bioinformatics model. For this we will select 20 proteins from our prediction and the localization of these
proteins will be verified in vivo. In specific aim 3 we will evaluate the mitochondrial encoded proteome and with it the
extent of alternative mRNA editing using cDNA sequencing of mitochondrial transcripts. In specific aim 4 we will then
verify the translation products of the alternatively edited transcripts directly testing our hypothesis that alternative
editing leads to a diversification of the mitochondrial proteome. In order to establish co-regulation patterns among
nuclear encoded mitochondrial genes and mitochondrial transcripts during development of the parasite we will employ
an expression profiling approach using 454 and Solexa sequencing technology. Analyzing the expression patterns will
then allow us integrate this data in specific aim 5 in order to build a model of mitochondrial nuclear protein interaction
which will then be tested using RNAi and dominant negative genetic strategies.
Together these studies will provide the first analysis of the role of alternative RNA editing in mitochondrial
biogenesis in trypanosomes and are likely to lead to the discovery of novel mitochondrial proteins and novel
protein-protein interaction data.