The Trypanosoma brucei MitoCarta and its regulation and splicing pattern during development

Abstract

It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational method for genome-wide prediction of mitochondrial proteins using a support vector machine-based classifier with ∼90% prediction accuracy. Using this method, we predicted the mitochondrial localization of 468 proteins with high confidence and have experimentally verified the localization of a subset of these proteins. We then applied a recently developed parallel sequencing technology to determine the expression profiles and the splicing patterns of a total of 1065 predicted MitoCarta transcripts during the development of the parasite, and showed that 435 of the transcripts significantly changed their expressions while 630 remain unchanged in any of the three life stages analyzed. Furthermore, we identified 298 alternatively splicing events, a small subset of which could lead to dual localization of the corresponding proteins.

Figure 1.

Statistical relationship between the prediction accuracy and the SVM score. The relationship is based on the analysis of 597 (positive set) and 1290 (negative set). The x-axis is the absolute SVM score and the y-axis represents prediction precision.

Figure 2.

Localization of predicted MitoCarta proteins. The fluorescence microscopy images from nine GFP tagged proteins that were randomly picked from our prediction are shown. First column GFP, second column Mitotracker, third column, overlay including the DAPI and differential interference contrast image, fourth column western blotts of total cell protein fractionated in a mitochondrial fraction (M) and a cytosolic fraction (C) and probed with antibodies against the cytosolic HSP70, the mitochondrial iron sulfur protein and GFP, fifth column the SDS–PAGE from the fractionated protein extract.

Figure. 3.

Two examples of alternative splice variants potentially leading to changes in localization. Screenshot of a region from chromosome of 2.5 kbp of two genes with SLT data in TPM from procyclic (green), bloodstream long slender (red), short stumpy forms (orange) and potential mitochondrial targeting signals (MTS, white box) and signal peptide sequence (SP, grey box) on the corresponding protein sequences (black line). (A) Tb927.4.4910, the upstream splice site potentially lead to a N-terminal extension of the open reading frame now containing a MTS of 58 amino acids as predicted by MITOPROT (confidence 0.75). The downstream splice site excludes the MTS, instead leading to a SP of 34 amino acids (confidence 0.98, SingnalP). (B) Tb11.01.1040, the uptstream splice site would allow translation from the currently annotated AUG leading to a protein containing a 30 amino acid MTS (confidence 0.99, MITOPROT), while the downstream, internal splice site would exclude the MTS leading to a 345 amino acid protein.