Mito-nDNA interactions require active mitochondrial reverse transcriptase machinery.

<p>A) Illustration of <i>COX1</i> gene arrangement in the WT (161-U7), intron a15γ (161-U7 GII-0 a15γ), and no mitochondrial group II introns (161-U7 GII-0) strains. Group II introns within the <i>COX1</i> gene encode functional reverse transcriptase. The region of <i>COX1</i> that participates in the <i>COX1</i>-<i>MSY1</i> interaction is indicated (qPCR probe). Strain 161-U7 GII-0 was included as a control to rule out a nuclear sequence, originating from a mitochondrial integration within the nuclear genome (NUMT), being responsible for the observed interaction. B) <i>COX1-MSY1</i> interaction frequencies for wt and intron mutants, illustrated in A), grown in glucose or galactose. C) <i>Q0182</i>-<i>RSM7</i> interaction frequencies for mitochondrial reverse transcriptase mutant 161-U7 GII-0 a15γ, illustrated in A), grown in glucose or galactose. Interaction frequencies are expressed as percentages of the wild-type <i>S. cerevisiae</i> strain 161-U7 for each carbon source (set at 100%) +/− standard error of the mean (n = 3). Interaction values in B) and C) were corrected for nuclear genome copy number to facilitate direct comparison.</p

A functional electron transport chain is required to maintain the interaction between the mitochondrial <i>COX1</i> and nuclear <i>MSY1</i> loci.

<p>Uncoupling of the electron transport chain was achieved by 2,4-dinitrophenol (5 mM) treatment of exponentially growing <i>S. cerevisiae</i> in synthetic complete media, containing glucose or galactose, for the indicated time (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030943#pone.0030943.s004" target="_blank">Figure S4</a>). <i>COX1-MSY1</i> A) and nDNA-nDNA B) interaction frequencies were determined by quantitative 3 C analyses using fluorescent probes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030943#pone.0030943.s014" target="_blank">Methods S1</a>). Interaction values in A) were corrected for mitochondrial genome copy number while those in B) were corrected for nuclear genome copy number (see Methods). Interaction values were expressed as percentages of the untreated sample (set at 100%) +/− standard error of the mean (n = 3).</p

The number of Mito-nDNA interactions correlates with chromosome length, except chromosome X.

<p>Statistically significant Mito-nDNA interactions, occurring above the expected noise level (selected to have a false positive rate of between 1 and 3%), have been summed for each nuclear chromosome and expressed as a percentage of the total number of interactions for the particular sample before being plotted according to chromosome length in base pairs. Interactions included in this analysis are between the mitochondrial genome and nuclear chromosomes, with the 2-micron plasmid and rDNA interactions removed. The length of chromosome XII has been reduced to account for the rDNA interactions being removed.</p

Inter-organelle interactions are carbon source dependent.

<p>Inter-organelle interactions are carbon source dependent.</p

Knocking out mitochondrial encoded reverse transcriptase activity results in increased transcript levels of nuclear genes that are involved in Mito-nDNA interactions.

<p>A) Nuclear encoded <i>MSY1</i> transcript levels were determined by qRT-PCR in WT (strain 161-U7), 161-U7 GII-0 (lacks both the mitochondrial group II introns and the <i>COX1</i> interacting region; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030943#pone-0030943-g004" target="_blank">Figure 4A</a>), and 161-U7 GII-0 a15γ (contains the interacting region and lacks the group II introns; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030943#pone-0030943-g004" target="_blank">Figure 4A</a>) cells. B) Nuclear encoded <i>RSM7</i> transcript levels were determined by qRT-PCR in: WT (strain 161-U7); 161-U7 GII-0; and 161-U7 GII-0 a15γ cells. Neither 161-U7 GII-0 nor 161-U7 GII-0 a15γ has any alteration within the <i>Q0182</i> open reading frame. C) Deletion of <i>MRS1</i> (BY4741 Δ<i>mrs1</i>), a nuclear gene involved in splicing mitochondrial type-I introns, has no effect on i) <i>MSY1</i> or ii) <i>RSM7</i> transcript levels. All transcript levels were standardized to nuclear <i>ACT1</i> and expressed as percentage of wild-type (set at 100%) +/− standard error of the mean (n = 2).</p

Deletion of <i>yme1</i> causes a significant reduction in the frequency of the mitochondrial-nuclear <i>COX1</i>-<i>MSY1</i> interaction.

<p>Interaction frequency between the mitochondrial <i>COX1</i> and nuclear <i>Msp</i>I fragments was assayed by quantitative 3 C (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030943#pone.0030943.s014" target="_blank">Methods S1</a>) in wild-type (<i>S. cerevisiae</i> BY4741), Δ<i>yme1</i> (BY4741 Δ<i>yme1</i>), Δ<i>yme2</i> (BY4741 Δ<i>yme2</i>), Δ<i>ade2</i> (BY4741 Δ<i>ade2</i>) and Δ<i>mdv1</i> (BY4741, Δ<i>mdv1</i>) strains. Interaction values were corrected for mitochondrial genome copy number (see Methods) and are expressed as percentages of wild-type (set at 100%) +/− standard error of the mean (n = 4). Deletion of an unconnected gene (<i>ade2</i>) did not significantly affect interaction frequency. T-tests (paired P(T< = t) one-tail, n = 4) were performed to determine the significance of observed variations: wild-type: Δ<i>yme1</i> p = 0.01; wild-type: Δ<i>yme2</i> p = 0.377; wild-type: Δ<i>ade2</i> p = 0.103; wild-type: Δ<i>mdv1</i> p = 0.143; Δ<i>yme1</i>: Δ<i>mdv1</i> p = 0.210. Only Δ<i>yme1</i> demonstrated a significant difference.</p