The multi-tissue landscape of somatic mtdna mutations indicates tissue specific accumulation and removal in aging
Abstract
Accumulation of somatic mutations in the mitochondrial genome (mtDNA) has long been proposed as a possible mechanism of mitochondrial and tissue dysfunction that occurs during aging. A thorough characterization of age-associated mtDNA somatic mutations has been hampered by the limited ability to detect low frequency mutations. Here, we used Duplex Sequencing on eight tissues of an aged mouse cohort to detect >89,000 independent somatic mtDNA mutations and show significant tissue-specific increases during aging across all tissues examined which did not correlate with mitochondrial content and tissue function. G→A/C→T substitutions, indicative of replication errors and/or cytidine deamination, were the predominant mutation type across all tissues and increased with age, whereas G→T/C→A substitutions, indicative of oxidative damage, were the second most common mutation type, but did not increase with age regardless of tissue. We also show that clonal expansions of mtDNA mutations with age is tissue and mutation type dependent. Unexpectedly, mutations associated with oxidative damage rarely formed clones in any tissue and were significantly reduced in the hearts and kidneys of aged mice treated at late age with Elamipretide or nicotinamide mononucleotide. Thus, the lack of accumulation of oxidative damage-linked mutations with age suggests a life-long dynamic clearance of either the oxidative lesions or mtDNA genomes harboring oxidative damage.
Data availability
The Duplex-Seq-Pipeline is written in Python and R, but has dependencies written in other languages. The DuplexSeq-Pipeline software has been tested to run on Linux, Windows WSL1, Windows WSL2 and Apple OSX. The software can be obtained at https://github.com/KennedyLab-UW/Duplex-Seq-Pipeline. Raw mouse sequencing data used in this study are available at SRA accension PRJNA727407. The data from Arbeithuber et al. are available at SRA accension PRJNA563921. The final post-processed data, including variant call files, depth information, data summaries, and mutation frequencies, as well as the scripts to perform reproducible production of statistics and figure generation (with the exception of Figure 5C-E) are available at https://github.com/Kennedy-Lab-UW/Sanchez_Contreras_etal_2022.
Article and author information
Author details
Funding
National Institute on Aging (P01AG001751)
- David J Marcinek
- Peter S Rabinovitch
National Institute on Aging (K01AG062757)
- Mariya T Sweetwyne
National Institute of Diabetes and Digestive and Kidney Diseases (R21DK128540)
- Monica Sanchez-Contreras
- Mariya T Sweetwyne
Congressionally Directed Medical Research Programs (W81XWH-16-1-0579)
- Scott R Kennedy
National Human Genome Research Institute (R21HG011229)
- Scott R Kennedy
National Cancer Institute (R21CA259780)
- Scott R Kennedy
National Institute on Aging (T32AG000057)
- Kristine A Tsantilas
- Jeremy A Whitson
National Institute on Aging (T32AG066574)
- Kristine A Tsantilas
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: This study was performed in accordance to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to an approved institutional animal care and use committee (IACUC) protocol (2174-23) at the University of Washington.
Copyright
© 2023, Sanchez-Contreras et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,629
- views
-
- 405
- downloads
-
- 46
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Chromosomes and Gene Expression
- Genetics and Genomics
Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here, we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Although gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.
-
- Chromosomes and Gene Expression
- Genetics and Genomics
Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA structure and nucleotide modifications can each interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We show that OTTR successfully captures both full-length tRNAs and tRNA fragments in budding yeast and in mouse reproductive tissues without any prior enzymatic treatment, and that tRNA cloning efficiency can be further enhanced via AlkB-mediated demethylation of modified nucleotides. As with other recent tRNA cloning protocols, we find that a subset of nucleotide modifications leave misincorporation signatures in OTTR datasets, enabling their detection without any additional protocol steps. Focusing on tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRNA fragments - including both 5′ and 3′ tRNA halves derived from the majority of tRNAs – than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.