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Abstract
Mitochondria are essential organelles that harbor a reduced genome, and expression of that genome requires regulated metabolism of its transcriptome by nuclear-encoded proteins. Despite extensive investigation, a comprehensive map of the yeast mitochondrial transcriptome has not been developed and all of the RNA-metabolizing proteins have not been identified, both of which are prerequisites to elucidating the basic RNA biology of mitochondria. Here, we present a mitochondrial transcriptome map of the yeast S288C reference strain. Using RNAseq and bioinformatics, we show the expression level of all transcripts, revise all promoter, origin of replication, and tRNA annotations, and demonstrate for the first time the existence of alternative splicing, mirror RNAs, and a novel RNA processing site in yeast mitochondria. The transcriptome map has revealed new aspects of mitochondrial RNA biology and we expect it will serve as a valuable resource. As a complement to the map, we present our compilation of all known yeast nuclear-encoded ribonucleases (RNases), and a screen of this dataset for those that are imported into mitochondria. We sought to identify RNases that are refractory to recovery in traditional mitochondrial screens due to an essential function or eclipsed accumulation in another cellular compartment. Using this in silico approach, the essential RNase of the nuclear and cytoplasmic exosome, Dis3p, emerges as a strong candidate. Bioinformatics and in vivo analyses show that Dis3p has a conserved and functional mitochondrial-targeting signal (MTS). A clean and marker-less chromosomal deletion of the Dis3p MTS results in a defect in the decay of intron and mirror RNAs, thus revealing a role for Dis3p in mitochondrial RNA decay.
Highlights
Mitochondrial inheritance was first discovered in 1949 in the yeast, Saccharomyces cerevisiae, by the group of Ephrussi and Slonimski[1]
Purified mitochondrial RNA from each culture was subjected one time to Illumina Hiseq2000 deep sequencing, which resulted in approximately 9 million mapped reads for each RNA sample (~18 million total reads)
To create a reference for mapping RNAseq data, we built a model of the S288C mitochondrial genome using DNA sequence information from the Saccharomyces Genome Database (SGD; www.yeastgenome.org), the public repository for this organism [35]
Summary
Mitochondrial inheritance was first discovered in 1949 in the yeast, Saccharomyces cerevisiae, by the group of Ephrussi and Slonimski[1] (see 2 for a review). The availability of a sequenced genome, and the subsequent creation of largescale isogenic collections, such as the deletion of every open reading frame (ORF)[6], has made S288C the cornerstone of contemporary yeast genetics. The mitochondrial genome of S288C contains 35 genes required for oxidative phosphorylation. There are 11 ORFs that show sequence similarity to homing endonuclease genes (HEG)[10]. These ORFs have been shown in some cases to encode active homing endonuclease enzymes, and in other cases the protein appears to have lost DNase activity, or has gained maturase (intron splicing), or reverse transcriptase activity. The exception is Q0255, which is a HEG-related ORF that overlaps the 3’ end of the COX2 ORF, and is not associated with an intron
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