Pervasive transcription of the mitochondrial genome in Candida albicans is revealed in mutants lacking the mtEXO RNase complex

Abstract

The mitochondrial genome of the pathogenic yeast Candida albicans displays a typical organization of several (eight) primary transcription units separated by noncoding regions. Presence of genes encoding Complex I subunits and the stability of its mtDNA sequence make it an attractive model to study organellar genome expression using transcriptomic approaches. The main activity responsible for RNA degradation in mitochondria is a two-component complex (mtEXO) consisting of a 3ʹ-5ʹ exoribonuclease, in yeasts encoded by the DSS1 gene, and a conserved Suv3p helicase. In C. albicans, deletion of either DSS1 or SUV3 gene results in multiple defects in mitochondrial genome expression leading to the loss of respiratory competence. Transcriptomic analysis reveals pervasive transcription in mutants lacking the mtEXO activity, with evidence of the entire genome being transcribed, whereas in wildtype strains no RNAs corresponding to a significant fraction of the noncoding genome can be detected. Antisense ('mirror') transcripts, absent from normal mitochondria are also prominent in the mutants. The expression of multiple mature transcripts, particularly those translated from bicistronic mRNAs, as well as those that contain introns is affected in the mutants, resulting in a decreased level of proteins and reduced respiratory complex activity. The phenotype is most severe in the case of Complex IV, where a decrease of mature COX1 mRNA level to ~5% results in a complete loss of activity. These results show that RNA degradation by mtEXO is essential for shaping the mitochondrial transcriptome and is required to maintain the functional demarcation between transcription units and non-coding genome segments.

The Pet127 protein is a mitochondrial 5′-to-3′ exoribonuclease from the PD-(D/E)XK superfamily involved in RNA maturation and intron degradation in yeasts

“…Pathways that shape the transcriptome show great evolutionary diversity, yet they share the common theme of interplay between transcription and RNA degradation. In general, transcriptional activity in the known genomes extends beyond the annotated gene sequences, often covering the majority of the genome ( Kapranov et al 2007a , b ; Wade and Grainger 2014 ; Tudek et al 2015 ; Łabędzka-Dmoch et al 2021 ). Intronic RNAs, as well as transcripts originating from noncoding intergenic sequences are quickly degraded by diverse ribonucleases.…”

“…Mitochondrial genes are transcribed as polycistronic primary transcripts (from two to dozens, depending on the species) that undergo extensive processing ( Ojala et al 1981 ; Lipinski et al 2010 ; Turk et al 2013 ; Kolondra et al 2015 ; Łabędzka-Dmoch et al 2021 ) by simple single-subunit polymerases of bacteriophage origin ( Masters et al 1987 ). In wild-type Candida albicans, there are eight primary transcripts transcribed from separate (but similar) promoters ( Kolondra et al 2015 ).…”

“…In wild-type Candida albicans, there are eight primary transcripts transcribed from separate (but similar) promoters ( Kolondra et al 2015 ). Transcripts corresponding to the entire mtDNA sequence, including intergenic regions, can however be observed in RNA degradation impaired strains ( Łabędzka-Dmoch et al 2021 ), suggesting pervasive transcription encompassing intergenic regions. RNA degradation is also responsible for the varying final abundance of mitochondrial RNAs originating from a single primary transcript ( Mercer et al 2011 ; Turk et al 2013 ; Kolondra et al 2015 ; Shang et al 2018 ; Łabędzka-Dmoch et al 2021 ).…”

“…The main ribonuclease of mitochondria is the mtEXO (mitochondrial degradosome) complex, exhibiting a 3′-to-5′ exoribonucleolytic activity that is composed of the conserved Suv3 helicase, and either the hydrolytic Dss1 RNase in fungi ( Dziembowski et al 2003 ; Hoffmann et al 2008 ) and in trypanosomes ( Mattiacio and Read 2008 ), or the phosphorolytic polynucleotide phosphorylase (PNPase) in animals ( Borowski et al 2013 ) and plants ( Holec et al 2006 ). This activity is essential for the functioning of the organellar gene expression system, and its dysfunction results in the loss of respiratory capacity in S. cerevisiae ( Dmochowska et al 1995 ; Golik et al 1995 ; Rogowska et al 2006 ), S. pombe ( Hoffmann et al 2008 ), and C. albicans ( Łabędzka-Dmoch et al 2021 ). The yeast mtEXO complex shapes the 3′ ends of protein coding transcripts that are protected from further degradation by the Rmd9 protein ( Hillen et al 2021 ), and degrades a variety of junk RNAs ( Dziembowski et al 2003 ; Rogowska et al 2006 ; Łabędzka-Dmoch et al 2021 ).…”

“…This activity is essential for the functioning of the organellar gene expression system, and its dysfunction results in the loss of respiratory capacity in S. cerevisiae ( Dmochowska et al 1995 ; Golik et al 1995 ; Rogowska et al 2006 ), S. pombe ( Hoffmann et al 2008 ), and C. albicans ( Łabędzka-Dmoch et al 2021 ). The yeast mtEXO complex shapes the 3′ ends of protein coding transcripts that are protected from further degradation by the Rmd9 protein ( Hillen et al 2021 ), and degrades a variety of junk RNAs ( Dziembowski et al 2003 ; Rogowska et al 2006 ; Łabędzka-Dmoch et al 2021 ). In mammalian mitochondria it is also responsible for RNA degradation and surveillance ( Szczesny et al 2010 ; Dhir et al 2018 ; Pietras et al 2018a , b ).…”