Mitochondria of the Yeasts Saccharomyces cerevisiae and Kluyveromyces lactis Contain Nuclear rDNA-Encoded Proteins

Abstract

In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a Nin-Cout topology. Plasmid-versions of 5′ end or 3′ end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5′ flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5′ and 3′ ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats.

A positive role for yeast extrachromosomal rDNA circles?

“…Our proposal derives from findings that TAR1 is under the control of the retrograde response and that Tar1p is targeted to mitochondria 1, 4, 5. Like other rDNA pol-II transcripts 6, 7, TAR1 expression is pol-II-dependent and normally silenced via Sir2p 1.…”

“…Selection would then have favoured retrograde response-dependent control of ERC production/rDNA pol-II transcription. Interestingly, TAR1 is present in Kluyveromyces lactis 4, which cannot form petites. However, ability to form petites is highly labile across hemiascomycetous yeasts 32, and we therefore suggest TAR1 evolved in an ancestral petite-forming lineage.…”

“…Oddly, TAR1 is normally silenced by Sir2p, a repressor of RNA polymerase II (pol-II)-transcribed genes 3. Recent reports suggest Tar1p protein is localised to the inner mitochondrial membrane 4, interacts with Coq5p (a protein involved in coenzyme Q synthesis [5]), and can maintain oxidative phosphorylation capacity 5. Direct elucidation of TAR1 function is lacking however, as available observations derive from monitoring a single, modified TAR1 copy 5.…”

Condensin and Hmo1 Mediate a Starvation-Induced Transcriptional Position Effect within the Ribosomal DNA Array

“…The rDNA of many organisms, including yeast and humans, encodes RNAPII-expressed transcripts that must be expressed at certain stages without causing general instability (Coelho et al, 2002; Kobayashi and Ganley, 2005; McStay and Grummt, 2008). In yeast, the TAR1 gene, encoded in the anti-sense direction of the 35S gene, is of particular interest because it is specifically expressed upon nutrient depletion (Bonawitz et al, 2008; Galopier and Hermann-Le Denmat, 2011), coincident with positional desilencing. Strikingly, nutrient-dependent regulation of TAR1 is lost when the gene is analyzed on a plasmid (Galopier and Hermann-Le Denmat, 2011), suggesting that rDNA structure contributes regulatory control.…”

“…In yeast, the TAR1 gene, encoded in the anti-sense direction of the 35S gene, is of particular interest because it is specifically expressed upon nutrient depletion (Bonawitz et al, 2008; Galopier and Hermann-Le Denmat, 2011), coincident with positional desilencing. Strikingly, nutrient-dependent regulation of TAR1 is lost when the gene is analyzed on a plasmid (Galopier and Hermann-Le Denmat, 2011), suggesting that rDNA structure contributes regulatory control.…”