Long-read direct RNA sequencing of the mitochondrial transcriptome of Saccharomyces cerevisiae

Mitochondria fulfil many essential roles in eukaryotic cells, yet some of their molecular mechanisms are still unexplored. Although 99% of the mitochondrial proteins are imported from the cytosol, mitochondria have their own DNA, transcription and translation machinery. The Saccharomyces cerevisiae mitochondrial DNA contains 11 polycistronic transcripts that encode 2 ribosomal subunits, 24 tRNAs and 9 genes, which can be spliced in alternative ways to yield different proteins.There are still many unresolved questions about mitochondrial genes and their splicing, including how gene expression and splicing is affected by different growth conditions and what role introns play in mitochondrial physiology. In the present study, we aimed to elucidate this by developing an RNA-sequencing method for mitochondrial RNA using Nanopore technology.A robust method was developed based on isolation of S. cerevisiae mitochondria and direct long-read sequencing of the mitochondrial RNA. Furthermore, a bioinformatics pipeline was developed to tackle challenges presented by mitochondrial RNA-seq, such as detection of polycistronic genes or alternative splicing events and faithful quantification of spliced sequences. The developed method was used to successfully capture the mitochondrial transcriptome and splicing variants of cultures grown on glucose or ethanol as sole carbon source and revealed remarkable differences in splicing pattern of the mitochondrial transcriptome. This phenotype was characterized in an intronless strain background.This study demonstrates that long-read RNA-seq combined with a customized bioinformatics pipeline is a robust and powerful method to investigate transcriptomic responses and splicing of polycistronic (mitochondrial) genomes.