bims-mitran 2025-07-13 papers

Issue of 2025–07–13
five papers selected by
Andreas Kohler, Umeå University

Nucleic Acids Res. 2025 Jul 08. pii: gkaf634. [Epub ahead of print]53(13):

A molecular switch at the yeast mitoribosomal tunnel exit controls cytochrome b synthesis.

Andreas Carlström, Joseph B Bridgers, Mary Couvillion, Abeer Prakash Singh, Ignasi Forné, Axel Imhof, L Stirling Churchman, Martin Ott.

Mitochondrial gene expression needs to be balanced with cytosolic translation to produce oxidative phosphorylation complexes. In yeast, translational feedback loops involving lowly expressed proteins called translational activators help to achieve this balance. Synthesis of cytochrome b (Cytb or COB), a core subunit of complex III in the respiratory chain, is controlled by three translational activators and the assembly factor Cbp3-Cbp6. However, the molecular interface between the COB translational feedback loop and complex III assembly is yet unknown. Here, using protein-proximity mapping combined with selective mitoribosome profiling, we reveal the components and dynamics of the molecular switch controlling COB translation. Specifically, we demonstrate that Mrx4, a previously uncharacterized ligand of the mitoribosomal polypeptide tunnel exit, interacts with either the assembly factor Cbp3-Cbp6 or with the translational activator Cbs2. These reciprocal interactions determine whether the translational activator complex with bound COB messenger RNA (mRNA) can interact with the mRNA channel exit on the small ribosomal subunit for translation initiation. Organization of the feedback loop at the tunnel exit therefore orchestrates mitochondrial translation with respiratory chain biogenesis.

DOI: https://doi.org/10.1093/nar/gkaf634

Nucleic Acids Res. 2025 Jul 08. pii: gkaf665. [Epub ahead of print]53(13):

FASTKD5 processes mitochondrial pre-mRNAs at noncanonical cleavage sites.

Hana Antonicka, Ana Vučković, Woranontee Weraarpachai, Seungwoo Hong, Michele Brischigliaro, Ahram Ahn, Antoni Barrientos, Hauke S Hillen, Eric A Shoubridge.

The first post-transcriptional step in mammalian mitochondrial gene expression, required for the synthesis of the 13 polypeptides encoded in mitochondrial DNA (mtDNA), is endonucleolytic cleavage of the primary polycistronic transcripts. Excision of the mtDNA-encoded transfer RNAs (tRNAs) releases most mature RNAs; however, processing of three noncanonical messenger RNAs (mRNAs) not flanked by tRNAs (CO1, CO3, and CYB) requires FASTKD5. To investigate the molecular mechanism involved, we created knockout human cell lines to use as assay systems. The absence of FASTKD5 produced a severe OXPHOS assembly defect due to the inability to translate two unprocessed noncanonical mRNAs and predicted altered folding patterns specifically at the 5'-end of the CO1 coding sequence. Structural features 13-15 nt upstream of the CO1 and CYB cleavage sites suggest FASTKD5 recognition mechanisms. Remarkably, a map of essential FASTKD5 amino acid residues revealed RNA substrate specificity; however, a key, putative active site residue was required for processing all three noncanonical pre-RNAs. Mutating this site did not significantly alter the binding of any client RNA substrate. A reconstituted in vitro system showed that wild-type, but not mutant, FASTKD5, was able to cleave client substrates correctly. These results establish FASTKD5 as the missing piece of biochemical machinery required to completely process the primary mitochondrial transcript.

DOI: https://doi.org/10.1093/nar/gkaf665

PLoS Genet. 2025 Jul 07. 21(7): e1011773

Clu1/Clu form mitochondria-associated granules upon metabolic transitions and regulate mitochondrial protein translation via ribosome interactions.

Leonor Miller-Fleming, Wing Hei Au, Laura Raik, Pedro Rebelo-Guiomar, Jasper Schmitz, Ha Yoon Cho, Aron Czako, Alexander J Whitworth.

Mitochondria perform essential metabolic functions and respond rapidly to changes in metabolic and stress conditions. As the majority of mitochondrial proteins are nuclear-encoded, intricate post-transcriptional regulation is crucial to enable mitochondria to adapt to changing cellular demands. The eukaryotic Clustered mitochondria protein family has emerged as an important regulator of mitochondrial function during metabolic shifts. Here, we show that the Drosophila melanogaster and Saccharomyces cerevisiae Clu/Clu1 proteins form dynamic, membraneless, mRNA-containing granules adjacent to mitochondria in response to metabolic changes. Yeast Clu1 regulates the translation of a subset of nuclear-encoded mitochondrial proteins by interacting with their mRNAs while these are engaged in translation. We further show that Clu1 regulates translation by interacting with polysomes, independently of whether it is in a diffuse or granular state. Our results demonstrate remarkable functional conservation with other members of the Clustered mitochondria protein family and suggest that Clu/Clu1 granules isolate and concentrate ribosomes engaged in translating their mRNA targets, thus, integrating metabolic signals with the regulation of mitochondrial protein synthesis.

DOI: https://doi.org/10.1371/journal.pgen.1011773

Nat Commun. 2025 Jul 10. 16(1): 6391

Super-resolution microscopy of mitochondrial mRNAs.

Stefan Stoldt, Frederike Maass, Michael Weber, Sven Dennerlein, Peter Ilgen, Jutta Gärtner, Aysenur Canfes, Sarah V Schweighofer, Daniel C Jans, Peter Rehling, Stefan Jakobs.

Mitochondria contain their own DNA (mtDNA) and a dedicated gene expression machinery. As the mitochondrial dimensions are close to the diffraction limit of classical light microscopy, the spatial distribution of mitochondrial proteins and in particular of mitochondrial mRNAs remains underexplored. Here, we establish single-molecule fluorescence in situ hybridization (smFISH) combined with STED and MINFLUX super-resolution microscopy (nanoscopy) to visualize individual mitochondrial mRNA molecules and associated proteins. STED nanoscopy reveals the spatial relationships between distinct mRNA species and proteins such as the RNA granule marker GRSF1, demonstrating adaptive changes in mRNA distribution and quantity in challenged mammalian cells and patient-derived cell lines. Notably, STED-smFISH shows the release of mRNAs during apoptosis, while MINFLUX reveals the folding of the mRNAs into variable shapes, as well as their spatial proximity to mitochondrial ribosomes. These protocols are transferable to various cell types and open new avenues for understanding mitochondrial gene regulation in health and disease.

DOI: https://doi.org/10.1038/s41467-025-61577-5

J Cell Sci. 2025 Jul 01. pii: jcs263638. [Epub ahead of print]138(13):

Mitochondrial topoisomerases, nucleoid architecture and mtDNA repair in human disease.

Sangheeta Bhattacharjee, Benu Brata Das.

DNA topoisomerases are essential for maintaining DNA topology, gene expression and the accurate transmission of genetic information. Mitochondria possess circular DNA (mtDNA), which, unlike nuclear chromosomes, lacks protective histones and exists in nucleoprotein complexes called nucleoids, which are vital for mtDNA stability. Although the mitochondrial genome encodes essential genes involved in ATP production via oxidative phosphorylation, it does not encode crucial mtDNA maintenance genes and depends entirely on nuclear-encoded proteins for mtDNA maintenance. These include nuclear-encoded topoisomerases (i.e. Top1mt, Top2α, Top2β and Top3α), which alleviate topological stress during mtDNA transcription and replication, and mitochondrial transcription factor A (TFAM), are crucial for ensuring proper nucleoid structure and mtDNA packaging. Furthermore, tyrosyl-DNA phosphodiesterase 1 and 2 (TDP1 and TDP2) participate in the repair of mtDNA damage associated with trapped topoisomerase-mtDNA complexes, which can compromise mtDNA integrity and contribute to neurodegeneration, cancer and premature aging. Drugs that stabilize these protein-DNA adducts (PDAs) to induce mtDNA damage and mitochondrial dysfunction are promising new strategies for cancer therapy. This Review explores the essential roles of mitochondrial topoisomerases, overviews mechanisms involved in mtDNA repair and discusses how mitochondrial fission and mitophagy are employed as a survival strategy for clearing damaged mtDNA.

Keywords: DNA repair; Mitochondria; Mitochondrial DNA; Neurological diseases; TDP1; TFAM; Topoisomerase 1

DOI: https://doi.org/10.1242/jcs.263638