Decreasing mitochondrial RNA polymerase activity reverses biased inheritance of hypersuppressive mtDNA

Abstract

Faithful inheritance of mitochondrial DNA (mtDNA) is crucial for cellular respiration/oxidative phosphorylation and mitochondrial membrane potential. However, how mtDNA is transmitted to progeny is not fully understood. We utilized hypersuppressive mtDNA, a class of respiratory deficient Saccharomyces cerevisiae mtDNA that is preferentially inherited over wild-type mtDNA (rho+), to uncover the factors governing mtDNA inheritance. We found that some regions of rho+ mtDNA persisted while others were lost after a specific hypersuppressive takeover indicating that hypersuppressive preferential inheritance may partially be due to active destruction of rho+ mtDNA. From a multicopy suppression screen, we found that overexpression of putative mitochondrial RNA exonuclease PET127 reduced biased inheritance of a subset of hypersuppressive genomes. This suppression required PET127 binding to the mitochondrial RNA polymerase RPO41 but not PET127 exonuclease activity. A temperature-sensitive allele of RPO41 improved rho+ mtDNA inheritance over a specific hypersuppressive mtDNA at semi-permissive temperatures revealing a previously unknown role for rho+ transcription in promoting hypersuppressive mtDNA inheritance.

Fig 1. Generation and Characterization of HS Alleles.

Mitochondria DNA from HS1b (ORI5-1), HS3b (ORI5-2), HS6a (ORI3-1), HS11b (ORI2-1), and rho+ control cells was sequenced using pacific biosciences sequencing technology and mapped back to reference genome. Reads from HS ORI5-1 mapped to base pairs 82,101 to 82,615 of the wild type reference genome. HS ORI3-1 mapped to 53,495 to 55,836. HS ORI5-2 mapped to 82,095 to 82,817. HS ORI2-1 mapped to 30,305 to 32,497. Scale bar ranges are 0 to 1619 for HS ORI5-1, 0 to 1474 for HS ORI5-1, 0 to 1111 for HS ORI3-1, 0 to 660 for HS ORI2-1 and 0 to 305 for rho+.

Fig 2. Colony PCR after HS x rho+ Reveals Remnants of rho+ mtDNA.

The indicated strains were mated and plated on diploid selection medium. The parent strains were plated to single colonies. After two days, individual colonies were scraped off the plates, and DNA was isolated. Genomic DNA was normalized, and PCRs were performed with primer sets amplifying the indicated loci. Reactions were run on agarose gels and stained with ethidium bromide. Arrows indicate the size of the intended product.

Fig 3. High-copy PET127 is a Suppressor of HS rho- Preferential Inheritance.

A. Rho+ cells were transformed with Yep13 high-copy library [45] and 10,967 colonies were plated. Plasmid containing colonies were mated with lawns of HS Ori5-1, replica plated to diploid selection medium and replica plated again to YEPD with no additional adenine. We identified 154 red colonies initially, however only 5 colonies were red after repetition of the assay. Four of the five colonies showed suppression and were sanger sequenced using Yep13 sequencing primers. B. Quantitative mtDNA inheritance assay validating plasmid 20.4, identified by the screen, and the two candidate genes carried by 20.4. Significance was determined using one-way ANOVA separately on the HS (N = 4) and rho0 (N = 3) crosses with means compared to the vector control and using Dunnett’s multiple comparison’s test. **** indicates adjusted P value less than 0.0001. All other comparisons were not significantly different. C. Quantitative mtDNA inheritance assay as in b testing high-copy PET127 on the other isolated HS alleles. Significance was determined by student’s unpaired T test on the vector and high-copy PET127 pair for each HS or rho0 set (N = 3). * indicates a P value between less than 0.05. ** indicates a P value less than 0.01. *** indicates a P value between 0.001. All other comparisons were not significantly different.

Fig 4. PET127 Binds to RPO41 and the Binding Region is Responsible for Suppression of HS Biased Inheritance.

A. Coimmunoprecipitation of Rpo41 and Pet127. Cells expressing either PET127-HA, V5-RPO41, or PET127-HA and V5-RPO41 were collected, lysed, and V5-Rpo41 was immunoprecipitated using anti-V5 conjugated beads. Samples were immunoblotted as indicated. B. Bacterial expression and lysate mixing of recombinant RPO41 and PET127 alleles. The indicated alleles were expressed in BL21 by the addition of IPTG. Cells were collected and lysed in a French press. For IPs, equal amounts of lysate from Pet127 construct expressing cells was mixed with lysate from either GST-Rpo41 or GST only expressing cells and precipitated with glutathione agarose beads. Samples were immunoblotted as indicated. C. Diagram of PET127 alleles. The predicted mitochondrial targeting sequence is amino acids 1–47 in gray [54]. Amino acids 48–215 contain the Rpo41 binding region in yellow, and amino acids 216–800 contain the region lacking Rpo41 binding activity in blue. pet127-nd is a nuclease dead allele of PET127 with amino acids predicted to be conserved active site residues changed to alanines: E346, D378, D391, and K393 [50]. D. Co-immunoprecipitation of PET127-HA alleles and V5-RPO41 as in a. Samples were immunoblotted as indicated. Anti-porin used as a negative pulldown control. E. Quantitation of IP enrichment in d. F. Quantitative mtDNA inheritance assay with rho+ high copy PET127 alleles x HS ORI5-1, HS ORI5-2, or rho0. Significance was determined using one-way ANOVA separately on each HS and rho0 cross with means compared to the vector control using Dunnett’s multiple comparison’s test (N = 3). **** indicates adjusted P-value less than 0.0001 and ** indicates an adjusted P-value of 0.0025. All other comparisons were not significantly different.

Fig 5. Reduction of Transcription from the Mitochondrial RNA pol, RPO41, Suppresses HS Biased Inheritance.

A. Quantitative mtDNA inheritance assay on strains carrying all combinations of high-copy PET127, high-copy V5-RPO41, and their respective empty vector controls. Significance was determined using one-way ANOVA separately on each HS and rho0 cross with means compared to all other samples and using Tukey’s multiple comparison’s test (N = 3). **** indicates adjusted P value less than 0.0001. * indicates adjusted P-value less than 0.05. All other comparisons were not significantly different. B. RT-qPCR of COX3/ACT1 RNA divided by qPCR of COX3/ACT1 DNA on the high-copy PET127 high-copy V5-RPO41 combination strains collected at high cell density to increase respiration. Significance was determined using one-way ANOVA comparing all values with each other using Tukey’s multiple comparison’s test (N = 2). **** indicates adjusted P-value less than 0.0001, *** indicates an adjusted P-value of 0.0008 to 0.0001, ** indicates an adjusted P-value of 0.0057, * indicates an adjusted P-value of 0.0275. C. Five-fold serial dilution of strains containing RPO41 alleles beginning at 1.1 x 10⁷ cells/ml plated on glycolysis (YEPD) or respiration (YEPG) utilizing media. Plates were incubated at either 30°C or 34°C for 2 days or 37°C for 3 days. D. Quantitative mtDNA inheritance assay of rho+ V5-RPO41 x either HS ORI5-1 or rho0 V5-RPO41 and rho+ v5-rpo41-EI x either HS ORI5-1 or rho0 v5-rpo41-EI at 30°C, 34°C, and 37°C. v5-rpo41-EI cross at 37°C was not determined as there was synthetic lethality on the diploid selection plates (see S11 Fig). Significance was determined using student’s T-test on each temperature between V5-RPO41 and v5-rpo41-EI in HS (N = 4) and rho0 (N = 3) cross. ** indicates a P-value of 0.0015. All other comparisons were not significantly different.