Contingency and selection in mitochondrial genome dynamics

  1. Christopher J Nunn
  2. Sidhartha Goyal

  • Curated by eLife

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    Evaluation Summary:

    This study uses long-read sequencing to investigate the origin of spontaneous petite mutants in S. cerevisiae. The results illustrate how the S. cerevisiae mitochondrial genome is prone to recombination events that lead to the formation of complex concatemers of fragments of the mitochondrial DNA that contain a high density of replication origins and, as a result, may outcompete the full mitochondrial genome. Apart from confirming existing hypotheses about the nature of petite mutants and revealing the structural diversity of rho-mitochondrial DNA, the results also allow drawing parallels to the origins of mitochondrial mutations in other organisms.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

High frequencies of mutant mitochondrial DNA (mtDNA) in human cells lead to cellular defects that are associated with aging and disease. Yet much remains to be understood about the dynamics of the generation of mutant mtDNAs and their relative replicative fitness that informs their fate within cells and tissues. To address this, we utilize long-read single-molecule sequencing to track mutational trajectories of mtDNA in the model organism Saccharomyces cerevisiae . This model has numerous advantages over mammalian systems due to its much larger mtDNA and ease of artificially competing mutant and wild-type mtDNA copies in cells. We show a previously unseen pattern that constrains subsequent excision events in mtDNA fragmentation in yeast. We also provide evidence for the generation of rare and contentious non-periodic mtDNA structures that lead to persistent diversity within individual cells. Finally, we show that measurements of relative fitness of mtDNA fit a phenomenological model that highlights important biophysical parameters governing mtDNA fitness. Altogether, our study provides techniques and insights into the dynamics of large structural changes in genomes that we show are applicable to more complex organisms like humans.

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  1. Version published to 10.7554/elife.76557 on eLife
  2. eLife

    Evaluation Summary:

    This study uses long-read sequencing to investigate the origin of spontaneous petite mutants in S. cerevisiae. The results illustrate how the S. cerevisiae mitochondrial genome is prone to recombination events that lead to the formation of complex concatemers of fragments of the mitochondrial DNA that contain a high density of replication origins and, as a result, may outcompete the full mitochondrial genome. Apart from confirming existing hypotheses about the nature of petite mutants and revealing the structural diversity of rho-mitochondrial DNA, the results also allow drawing parallels to the origins of mitochondrial mutations in other organisms.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    In this paper, Nunn and Goyal use long-read sequencing technology to investigate the origins and evolution of petite mutations in the S. cerevisiae mitochondrial genome. In brief, their results confirm much of what had been shown or hypothesized in a large number of older studies, many dating back to the 19070s and 1980s. Specifically, the results show that petite cells result from recombination events within the mtDNA that result in the development of small fragments, often complex concatemers, that have a higher density of replication origins compared to the WT mitochondrial DNA. The recombination events often involve short repeated sequences within the mtDNA and seem randomly distributed within the mt genome (as opposed to being enriched near replication origins). In a second part of the study, the authors find indirect evidence for the presence of both homoplasmic and heteroplasmic cells (i.e. cells that contain different species of mtDNA).

    Overall, the major strength of this paper is that it uses modern technology to confirm many of the conclusions and hypotheses of previous studies.

    The main problem with the text in its current format is that the general relevance of the findings is not explained well. In many organisms, mtDNA is indispensable or at least important for fitness, and mutants that lose the mitochondrial function are rapidly selected against. Moreover, the authors focus on the laboratory strain W303, which, like its more commonly used sibling S288C/BY4741, may have a higher petite frequency than feral yeasts because of mutations in genes like SAL1, MIP1, CAT5 (see https://doi.org/10.1016/S0076-6879(02)50954-X and 10.1534/genetics.109.104497). This makes one wonder whether the findings have a more broad relevance apart from laboratory S. cerevisiae strains. I believe there are possible links with mitochondrial diseases, but the authors do not explore these. The broader relevance and novelty are not always clear, but if the authors can draw very strong parallels with other systems, showing how their results help understand more general phenomena, it would increase the impact of the work. Interesting questions to discuss include: Do we observe similar phenomena in other species (e.g., mitochondrial human diseases?) Which general conclusions can be drawn?

  4. eLife

    Reviewer #2 (Public Review):

    This work seeks to answer long-time outstanding questions in the field of yeast mitochondrial genetics using sequence analysis and biophysical modeling. The article uses new sequencing techniques to answer questions about the Petite genomes and by highlighting the significance of these findings outside the specific question of petite genome dynamics, the work could also be made more appealing to a wider audience of evolutionary geneticists.

    The authors investigated the structural changes in yeast mitochondrial genomes by twice passaging spontaneous Petite phenotype colonies and comparing the long-read sequencing results of these cultured families to Grande (wild-type) colonies. Petite genomes were found to have a higher percentage of breakpoints than Grande genomes and these breakpoints are often clustered around replication origins. The authors also found that alternate structures, those that occur in lower frequencies than the primary mtDNA sequence, can give rise to "excision cascades" that in turn can result in new evolutionary pathways by bringing into close proximity areas of homology. Furthermore, using the percentage of alternate structure breakpoints within a colony, the contributions of heteroplasmic vs homoplasmic cells to the frequency of the alternate structure is able to be somewhat elucidated. Lastly, using crosses of Petite colonies with Grande colonies, the authors developed a model of suppressivity based on the biophysical parameters of mtDNA fitness.

    Some questions and areas of clarification still remain within the paper to be addressed:

    1. The heterogenous nature of mtDNA content within family 1 is not addressed. Is there some reason why these colonies remain heterogenous even after being twice passaged?
    2. How does the mtDNA coverage of the sequenced Grande genomes compare to the coverage of Petite genomes shown in figure 1b? Adding similar sequencing coverage of the Grande genomes in this figure would be helpful for a wild-type comparison.
    3. In figure 3a, please clarify if there is a difference between the black and grey circles on this plot. If these are just more densely occupied points, please make this clear in your plot.
    4. In figure 3c, please clarify the objective of the circled diagrams in the green and orange alignments. How do these reflect what is going on in the larger model?
    5. Figure 4e seems to directly reflect the criteria applied for the different types of alternate structures. For example, since the criteria for type III alternate repeats is that they share an existing primary alignment edge, they will have a shorter distance from the closest primary/alternate alignment edge as diagramed in figure 4e than the type I alternate repeats that occur within primary alignments. This plot seems unnecessary due to this circular logic. If the purpose of this figure is just to show that overall, the alternate repeats tend to occur close to alignment edges, this could be diagramed as a plot of all the breakpoints rather than broken down by categorical types.
    6. On page 12, you use the term "non-periodic" primary structures. Please clarify if this is the same as "non-tandemly duplicated".
    7. Please define the terms "heteroplasmic limit" and "homoplasmic limit". Are these the same as intracellular heteroplasmy and intercellular heteroplasmy.
    8. Please clarify how it is known that the fraction of alternate structures found in cells grown on non-fermentable YPG media directly correlates with the heteroplasmic contribution and the fermentable YPD media correlates with the additive effect of the homoplasmic contribution.
    9. How was mtDNA replication speed of the Petite families in figure 6b measured for use in the suppressivity rate calculation? Please add this to the accompanying methods section.

  5. eLife

    Reviewer #3 (Public Review):

    This work reports the innovative sequencing of individual molecules to access the diversity of mitochondrial genomes in yeast cells and mechanisms driving the emergence and evolution of such diversity. The huge amount of data generated (needs to be made fully accessible to the reader with sufficient annotation) will certainly benefit the field and conceptual analyses performed already are elegant and convincing. The interest of this study to the general reader is in potential analogies between yeast and mammalian mtDNA rearrangements, recombination, and their relation to the aging process, but this issue is barely discussed in the manuscript.

    I fully support and praise the idea of using nanopore sequencing in single-molecule analysis of mtDNA. The data are great and the analysis looks sound and convincing. Making analogies with animals (essentially human mtDNA, because this is where most of the research has been conducted) would benefit the manuscript.

    There are similar phenomena in humans, and I can recommend a few publications that are closely related to what is reported in the current manuscript:

    - Mapping of recombinants in human mtDNA and evidence for mtDNA recombination as an abundant ongoing intracellular process in humans (DOI: 10.1126/science.1096342),
    - Distribution and nature of mtDNA rearrangement breakpoints (in a very similar format to that of Fig 3 of the manuscript) - https://doi.org/10.1016/j.tig.2010.05.006);
    - Perhaps the most convincing ever study on the involvement of clonal expansions of mtDNA rearrangements in human aging pertaining to substantia nigra neurons (https://doi.org/10.1038/ng1778).

  6. Version published to 10.1101/2021.11.15.468706v2 on bioRxiv
  7. Version published to 10.1101/2021.11.15.468706v1 on bioRxiv