Project

mitochondria; Trypanosoma brucei; developmental regulation; RNA editing; mitochondrial biogenesis; systems biology; parallel sequencing

Lead
Lay summary
The overall goal of this proposal is to understand how the mitochondrion, an organelle inside almost every cell is formed during the live cycle of Trypanosoma brucei. Why is this important? The mitochondrion is known as the powerhouse of the cell. In recent years however, we have learnt it is much more. Integration of signals is the key phrase here. One could compare the mitochondrion to an information hub where the cell combines many different signals in order to respond to the environment. Furthermore, many human diseases like neurological disorders or cancer involve the mitochondrion. Why in Trypanosoma brucei? This organism offers several advantages. It only contains one single mitochondrion and this mitochondrion is tightly regulated during the different lifestages. This allows us to basically follow the making of a mitochondrion from scratch. There are two major areas that need attention. Since the majority of the proteins required for the mitochondrion are encoded in the nuclear genome the first aim is to establish a mitochondrial parts list that contains the proteins directly involved in mitochondrial formation as well as their interacting partners. This parts list will be predicted based on biological data using informatics techniques. The prediction will then be tested and verified through the use of cell biological methods that allow visualizing where a particular protein localizes in the cell. Only a small number of the proteins required for a functional mitochondrion are encoded in the mitochondrial genome. However, in trypanosomes mitochondrial transcripts are modified prior to translation by a process called RNA editing. This process changes the information content of the RNA through the addition or deletion of a large number of individual bases. We have recently shown that this process creates diversity within the transcript pool such that from one initial gene you can generate a large number of alternatively edited messages. We could also show that these messages are then translated into proteins. We are now interested how these proteins contribute in order to form a proper mitochondrion. To address this question we will establish what sequences are formed and if they are translated into proteins. In our last aim we will build a model of how the proteins involved in the making of a mitochondrion interact using bioinformatics. This will be done for example using information on how the proteins are regulated during the different stages of the parasite.

Direct link to Lay Summary

Last update: 21.02.2013

Self-organised

Number	Title	Start	Funding scheme

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.