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

Abstract

Pet127 is a mitochondrial protein found in multiple eukaryotic lineages, but absent from several taxa, including plants and animals. Distant homology suggests that it belongs to the divergent PD-(D/E)XK superfamily which includes various nucleases and related proteins. Earlier yeast genetics experiments suggest that it plays a nonessential role in RNA degradation and 5’ end processing. Our phylogenetic analysis suggests that it is a primordial eukaryotic invention that was retained in diverse groups, and independently lost several times in the evolution of other organisms. We demonstrate for the first time that the fungal Pet127 protein in vitro is a processive 5’-to-3’ exoribonuclease capable of digesting various substrates in a sequence nonspecific manner. Mutations in conserved residues essential in the PD-(D/E)XK superfamily active site abolish the activity of Pet127. Deletion of the PET127 gene in the pathogenic yeast Candida albicans results in a moderate increase in the steady state levels of several transcripts and in accumulation of unspliced precursors and intronic sequences of three introns. Mutations in the active site residues result in a phenotype identical to that of the deletant, confirming that the exoribonuclease activity is related to the physiological role of the Pet127 protein. Pet127 activity is, however, not essential for maintaining the mitochondrial respiratory activity in C. albicans.

RNA processing and degradation mechanisms shaping the mitochondrial transcriptome of budding yeasts

“…84 Impaired RNA degradation leads to the accumulation of unprocessed precursors, introns and splicing intermediates, antisense (mirror) RNAs, and transcripts from intergenic regions of the genome. 11,12,[84][85][86] The mitochondrial genome of C. albicans, which is more compact and has shorter intergenic regions compared with S. cerevisiae, was shown to be transcribed in its entirety (pervasive transcription) in mutants deficient in the function of the main RNA degradation complex (mtEXO). 11 A range of ribonucleases, in mitochondria mostly those exhibiting exoribonuclease activities, act as general degradation factors involved in RNA turnover and quality control.…”

“…The role played by the second mitochondrial exoribonuclease, 12 belonging to the PD-(D/E)XK superfamily and encoded by the PET127 gene, is far less clear. In S. cerevisiae, it trims the 5 0 end of the COB mRNA precursor (Figure 3), 78 as well as several other mRNAs and the 15S rRNA, 92 whereas in C. albicans it is also involved in degradation of introns.…”

“…Advances in economical high-throughput sequencing next generation sequencing (NGS), as well as in genome editing techniques, provide the opportunity to move the study of biological mechanisms, including mitochondrial gene expression, beyond the few model species studied in the past decades. In the field of yeast mitochondrial genome research, Candida albicans emerged as an attractive alternative model organism for the study of mitochondrial replication, 9 RNA metabolism, [10][11][12] and protein import, 13 among others. In addition to the well-known significance of this yeast as an opportunistic human pathogen, 14 its potential to complement the knowledge of mitochondrial genome function obtained in S. cerevisiae is related to the presence of a typical Complex I including mitochondrially encoded subunits, 15 active respiration on glucose as the carbon source (Crabtree-negativity), 16 and a stable mitochondrial genome that cannot be lost (petite-negativity).…”

How RNases Shape Mitochondrial Transcriptomes

“…It remained to be formally proved that it has a catalytic function [ 177 , 178 , 179 ], but a recent article showed that the pet127 PD-(D/E)XK nuclease domain is responsible for its activity in vitro. In addition, pet127 deletion and catalytic site mutants harbor the same molecular phenotype, showing that the function is linked to the catalysis of RNAs [ 180 ]. The RNA-seq study showed some intron accumulation, as well as changes in the steady-state transcriptome.…”

“…The RNA-seq study showed some intron accumulation, as well as changes in the steady-state transcriptome. In vitro results suggest that the enzyme is processive and is non-selective toward ss- or dsRNAs [ 180 ]. Lastly, the authors showed that pet127 was present in LECA and is present in different phylogenetic branches (as stated in Table 1 ), but lost in animals and plants.…”