Dual use of Toxoplasma gondii mitochondrial DNA sequence block boundaries gives rise to short non-coding RNAs

Abstract text

1. Introduction
Mitochondrial genomes of apicomplexan parasites have only three open reading frames (ORFs) encoding subunits of the mitochondrial electron transport chain and in addition several genes for highly fragmented ribosomal RNAs. How Apicomplexan mitochondrial RNAs are expressed and processed, in particular how rRNA fragments are produced from precursor RNAs, is largely unknown. Toxoplasma gondii displays one of the most striking and poorly understood Apicomplexan mitogenome organization, built of 21 sequence blocks arranged in different combinations. How these blocks are combined has not been quantitatively analyzed so far.
2. Objectives
Generate a full-genome map of the mitochondrial genome of T. gondii.
Use the map to allow a comprehensive analysis of short RNAs in T. gondii – the first Apicomplexan transcriptome-wide sRNA analysis outside of Plasmodium.
3. Materials & methods
We established a protocol based on differential membrane permeabilization that allows purification of organellar DNA of T. gondii. Using single molecule Nanopore sequencing of mitochondrial DNA, we generated a comprehensive genome map of the variety of existing mitochondrial DNA molecules. Illumina-based short RNA (sRNA) sequencing was used to determine the first mitochondrial transcriptome of T. gondii.
4. Results
Our Nanopore sequencing efforts lead to a quantitative understanding of sequence block combinations in the T. gondii mitochondrial genome. We show that there are striking differences in the frequencies of block combinations and that 5"-ends of coding regions are linked to each other within the genome. sRNA sequencing data confirmed the transcription of previously predicted rRNAs and in addition identified a large number of novel noncoding short RNAs. Several of the newly discovered transcripts are spanning the borders of sequence blocks. This leads to the intriguing situation that - depending on the different DNA block combinations - chimeric sRNAs are generated at block borders. sRNAs with fusions of coding sequences with rRNA sequences are observed.
5. Conclusion
T. gondii is using block borders to produce short RNAs with overlapping but not identical sequences. This may lead to dual use of sequence elements as coding region and at the same time also for ribosome production.