Arguably, the most bizarre mitochondrial DNA (mtDNA) is that of the euglenozoan eukaryote Diplonema papillatum. The genome consists of numerous small circular chromosomes none of which appears to encode a complete gene. For instance, the cox1 coding sequence is spread out over nine different chromosomes in non-overlapping pieces (modules), which are transcribed separately and joined to a contiguous mRNA by trans-splicing. Here, we examine how many genes are encoded by Diplonema mtDNA and whether all are fragmented and their transcripts trans-spliced. Module identification is challenging due to the sequence divergence of Diplonema mitochondrial genes. By employing most sensitive protein profile search algorithms and comparing genomic with cDNA sequence, we recognize a total of 11 typical mitochondrial genes. The 10 protein-coding genes are systematically chopped up into three to 12 modules of 60-350 bp length. The corresponding mRNAs are all trans-spliced. Identification of ribosomal RNAs is most difficult. So far, we only detect the 3'-module of the large subunit ribosomal RNA (rRNA); it does not trans-splice with other pieces. The small subunit rRNA gene remains elusive. Our results open new intriguing questions about the biochemistry and evolution of mitochondrial trans-splicing in Diplonema.

Biology Centre Institute of Parasitology Czech Academy of Science and Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Department of Biochemistry Université de Montréal 2900 Edouard Montpetit Montreal Quebec H3T 1J4 Canada

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Department of Genomics and Bioinformatics 142 20 Prague Czech Republic

Robert Cedergren Centre for Bioinformatics and Genomics Université de Montréal Montreal Quebec Canada

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