bims-midmar 2022-03-20 papers

J Biol Chem. 2022 Mar 09. pii: S0021-9258(22)00255-1. [Epub ahead of print] 101815

Phosphorylation and acetylation of mitochondrial transcription factor A promote transcription processivity without compromising initiation or DNA compaction.

Sean D Reardon, Tatiana V Mishanina.

Mitochondrial transcription factor A (TFAM) plays important roles in mitochondrial DNA (mtDNA) compaction, transcription initiation, and in the regulation of processes like transcription and replication processivity. It is possible that TFAM is locally regulated within the mitochondrial matrix via such mechanisms like phosphorylation by protein kinase A (PKA) and non-enzymatic acetylation by acetyl-CoA. Here we demonstrate that DNA-bound TFAM is less susceptible to these modifications. We confirmed using electrophoretic mobility shift assays that phosphorylated or acetylated TFAM compacted circular double-stranded DNA just as well as unmodified TFAM and provide an in-depth analysis of acetylated sites on TFAM. We show that both modifications of TFAM increase the processivity of mitochondrial RNA polymerase during transcription through TFAM-imposed barriers on DNA, but that TFAM bearing either modification retains its full activity in transcription initiation. We conclude that TFAM phosphorylation by PKA and non-enzymatic acetylation by acetyl-CoA are unlikely to occur at the mtDNA and that modified free TFAM retains its vital functionalities like compaction and transcription initiation while enhancing transcription processivity.

Keywords: Acetylation; DNA compaction; Mitochondrial transcription; Phosphorylation

DOI: https://doi.org/10.1016/j.jbc.2022.101815

J Cell Sci. 2022 Mar 17. pii: jcs.258937. [Epub ahead of print]

Expression analysis and function of mitochondrial genome-encoded microRNAs.

Raviprasad Kuthethur, Vaibhav Shukla, Sandeep Mallya, Divya Adiga, Shama Prasada Kabekkodu, Lingadakai Ramachandra, Prakash Saxena Pu, Kapaettu Satyamoorthy, Sanjiban Chakrabarty.

MicroRNAs play a significant role in nuclear and mitochondrial anterograde and retrograde signaling. Most of the miRNAs found inside mitochondria are nuclear genome encoded, with few mitochondrial genome encoded non-coding RNAs have been reported. In this study, we have identified 13 mitochondrial genome-encoded microRNAs (mitomiRs), which were differentially expressed in breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231), non-malignant breast epithelial cell line (MCF-10A), and normal and breast cancer tissue specimens. We found that mitochondrial DNA depletion and inhibition of mitochondrial transcription leads to reduced expression of mitomiRs in breast cancer cells. MitomiRs physically interact with Ago2, an RNA-induced silencing complex (RISC) protein, in the cytoplasm and inside mitochondria. MitomiRs regulate the expression of both nuclear and mitochondrial transcripts in breast cancer cells. We showed that mitomiR-5 targets PPARGC1A and regulates mtDNA copy number in breast cancer cells. MitomiRs identified in the present study may be a promising tool for expression and functional analysis in patients with a defective mitochondrial phenotype, including cancer and metabolic syndromes.

Keywords: PPARGC1A ; MicroRNAs; Mitochondria; mitomiRs; mtDNA copy number

DOI: https://doi.org/10.1242/jcs.258937

Nat Plants. 2022 Mar 17.

Targeted introduction of heritable point mutations into the plant mitochondrial genome.

Joachim Forner, Dennis Kleinschmidt, Etienne H Meyer, Axel Fischer, Robert Morbitzer, Thomas Lahaye, Mark A Schöttler, Ralph Bock.

The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.

DOI: https://doi.org/10.1038/s41477-022-01108-y

Cell Cycle. 2022 Mar 17. 1-16

POLG mutations lead to abnormal mitochondrial remodeling during neural differentiation of human pluripotent stem cells via SIRT3/AMPK pathway inhibition.

Anbin Chen, Cecilie Katrin Kristiansen, Lena Elise Høyland, Mathias Ziegler, Jian Wang, Gareth John Sullivan, Xingang Li, Laurence A Bindoff, Kristina Xiao Liang.

We showed previously that POLG mutations cause major changes in mitochondrial function, including loss of mitochondrial respiratory chain (MRC) complex I, mitochondrial DNA (mtDNA) depletion and an abnormal NAD+/NADH ratio in both neural stem cells (NSCs) and astrocytes differentiated from induced pluripotent stem cells (iPSCs). In the current study, we looked at mitochondrial remodeling as stem cells transit pluripotency and during differentiation from NSCs to both dopaminergic (DA) neurons and astrocytes comparing the process in POLG-mutated and control stem cells. We saw that mitochondrial membrane potential (MMP), mitochondrial volume, ATP production and reactive oxygen species (ROS) changed in similar ways in POLG and control NSCs, but mtDNA replication, MRC complex I and NAD+ metabolism failed to remodel normally. In DA neurons differentiated from NSCs, we saw that POLG mutations caused failure to increase MMP and ATP production and blunted the increase in mtDNA and complex I. Interestingly, mitochondrial remodeling during astrocyte differentiation from NSCs was similar in both POLG-mutated and control NSCs. Further, we showed downregulation of the SIRT3/AMPK pathways in POLG-mutated cells, suggesting that POLG mutations lead to abnormal mitochondrial remodeling in early neural development due to the downregulation of these pathways. [Figure: see text].

Keywords: DA neurons; Mitochondrial remodeling; NSCs; POLG; astrocytes; iPSCs

DOI: https://doi.org/10.1080/15384101.2022.2044136

Hum Mol Genet. 2022 Mar 14. pii: ddac059. [Epub ahead of print]

Mitochondrial DNA variants segregate during human preimplantation development into genetically different cell lineages that are maintained postnatally.

Joke Mertens, Marius Regin, Neelke De Munck, Edouard Couvreu Deckersberg, Florence Belva, Karen Sermon, Herman Tournaye, Christophe Blockeel, Hilde Van de Velde, Claudia Spits.

Humans present remarkable diversity in their mitochondrial DNA (mtDNA) in terms of variants across individuals as well as across tissues and even cells within one person. We have investigated the timing of the first appearance of this variant-driven mosaicism. For this, we deep-sequenced the mtDNA of 254 oocytes from 85 donors, 158 single blastomeres of 25 day-3 embryos, 17 inner cell mass and trophectoderm samples of 7 day-5 blastocysts, 142 bulk DNA and 68 single cells of different adult tissues. We found that day-3 embryos present blastomeres that carry variants only detected in that cell, showing that mtDNA mosaicism arises very early in human development. We classified the mtDNA variants based on their recurrence or uniqueness across different samples. Recurring variants had higher heteroplasmic loads and more frequently resulted in synonymous changes or were located in non-coding regions than variants unique to one oocyte or single embryonic cell. These differences were maintained through development, suggesting that the mtDNA mosaicism arising in the embryo is maintained into adulthood. We observed a decline in potentially pathogenic variants between day-3 and day-5 of development, suggesting early selection. We propose a model in which closely clustered mitochondria carrying specific mtDNA variants in the ooplasm are asymmetrically distributed throughout the cell divisions of the preimplantation embryo, resulting in the earliest form of mtDNA mosaicism in human development.

DOI: https://doi.org/10.1093/hmg/ddac059