bims-mirnam Biomed News
on Mitochondrial RNA metabolism
Issue of 2025–12–28
twenty-one papers selected by
Hana Antonicka, McGill University
  1. Turnover and Quality Control of Mitochondrial DNA.
  2. Mechanisms of Intracellular Selection of Mitochondrial DNA.
  3. Structural Basis of TACO1-Mediated Efficient Mitochondrial Translation.
  4. Proximity Labeling Reveals RNA-Binding Proteins Associating with the Human Mitochondrial Import Receptor TOMM20.
  5. A systematic analysis of mitochondrial aminoacyl tRNA synthetase variants in a rare disease cohort.
  6. Advances in gene therapy for mitochondrial genetic disorders: current status and clinical implementation challenges.
  7. Improved long-transcript representation in Oxford Nanopore direct RNA sequencing with UltraMarathonRT.
  8. Suboptimal codon pairs trigger ribosome collisions and cellular quality control responses in tRNA modification mutants.
  9. Pseudouridine increases ribosome stability in a thermophilic eukaryote.
  10. Investigation of TRMT61B methyltransferase activity on mRNA and its effects on translation.
  11. RNA Modifications in T cell Immunity: Mechanisms, Disease Relevance, and Therapeutic Potential.
  12. A Small Cationic Probe for Accurate, Punctate Discovery of RNA Tertiary Structure.
  13. StructRMDB: A database of RNA modification sites that affect RNA secondary structure.
  14. TAG-PL: A Universal Proximity Labeling Platform for Mapping Mitochondrial Proteomes and Organelle Interactions under Stress.
  15. Deciphering RNA modification and post-transcriptional regulation with NetRNApan.
  16. RNA modifications in health and disease: from mechanistic insights to therapeutic applications.
  17. Designer RNA nanostructures co-transcribed and self-assembled inside human cell nuclei.
  18. Transient muscle expression of mitoARCUS in mice leads to a sustained reduction in pathogenic mtDNA content and improves muscle function.
  19. MsipNet: a multi-scale representation learning framework for predicting protein-RNA interaction.
  20. Stroke-like lesion and status epilepticus in a child with NARS2-related combined oxidative phosphorylation deficiency 24.
  21. Molecular basis of tRNA substrate recognition and modification by the atypical SPOUT methyltransferase Trm10.
  1. Biochemistry (Mosc). 2025 Dec;90(12): 1849-1861
    Turnover and Quality Control of Mitochondrial DNA.
    Wolfram S Kunz.
      The quantitative content of mitochondrial DNA (mtDNA) - a multicopy circular genome - is an important parameter relevant for function of mitochondrial oxidative phosphorylation (OxPhos) in cells, since mtDNA encodes 13 essential OxPhos proteins, 22 tRNAs, and 2 rRNAs. In contrast to the nuclear genome, where almost all lesions have to be repaired, the multicopy nature of mtDNA allows the degradation of severely damaged genomes. Therefore, cellular mtDNA maintenance and its copy number not only depend on replication speed and repair reactions. The speed of intramitochondrial mtDNA degradation performed by a POLGexo/MGME1/TWNK degradation complex and the breakdown rate of entire mitochondria (mitophagy) are also relevant for maintaining the required steady state levels of mtDNA. The present review discusses available information about the processes relevant for turnover of mitochondrial DNA, which dysbalance leads to mtDNA maintenance disorders. This group of mitochondrial diseases is defined by pathological decrease of cellular mtDNA copy number and can be separated in diseases related to decreased mtDNA synthesis rates (due to direct replication defects or mitochondrial nucleotide pool dysbalance) or diseases related to increased breakdown of entire mitochondria (due to elevated mitophagy rates).
    Keywords:  determinants of cellular mtDNA content; mtDNA degradation; mtDNA maintenance; mtDNA maintenance disorders; mtDNA replication
    DOI:  https://doi.org/10.1134/S0006297925602485
  2. Biochemistry (Mosc). 2025 Dec;90(12): 1919-1928
    Mechanisms of Intracellular Selection of Mitochondrial DNA.
    Georgii Muravyov, Dmitry A Knorre.
      Eukaryotic cells contain multiple mitochondrial DNA (mtDNA) molecules. Heteroplasmy is coexistence in the same cell of different mtDNA variants competing for cellular resources required for their replication. Here, we review documented cases of emergence and spread of selfish mtDNA (i.e., mtDNA that has a selective advantage in a cell but decreases cell fitness) in eukaryotic species, from humans to baker's yeast. The review discusses hypothetical mechanisms enabling preferential proliferation of certain mtDNA variants in heteroplasmy. We propose that selfish mtDNAs have significantly influenced the evolution of eukaryotes and may be responsible for the emergence of uniparental inheritance and constraints on the mtDNA copy number in germline cells.
    Keywords:  heteroplasmy; intracellular selection; mitochondrial DNA; mitophagy; mtDNA quality control; selfish gene
    DOI:  https://doi.org/10.1134/S0006297925603296
  3. bioRxiv. 2025 Dec 19. pii: 2025.12.18.695269. [Epub ahead of print]
    Structural Basis of TACO1-Mediated Efficient Mitochondrial Translation.
    Shuhui Wang, Michele Brischigliaro, Yuekang Zhang, Chunxiang Wu, Wei Zheng, Antoni Barrientos, Yong Xiong.
      Translation elongation is a universally conserved step in protein synthesis, relying on elongation factors that engage the ribosomal L7/L12 stalk to mediate aminoacyl-tRNA delivery, accommodation, and ribosomal translocation. Using in organello cryo-electron microscopy, we reveal how the mitochondrial translation accelerator TACO1 promotes efficient elongation on human mitoribosomes. TACO1 binds the mitoribosomal region typically bound by elongation factor Tu (mtEF-Tu), bridging the large and small subunits via contacts with 16S rRNA, bL12m, A-site tRNA, and uS12m. While active throughout elongation, TACO1 is especially critical when translating polyproline motifs. Its absence prolongs mtEF-Tu residence in A/T states, causes persistent mitoribosomal stalling and premature subunit dissociation. Structural analyses indicate that TACO1 competes with mtEF-Tu for mitoribosome binding, stabilizes A-site tRNA, and enhances peptidyl transfer through a mechanism distinct from EF-P and eIF5A. These findings suggest that bacterial TACO1 orthologs may serve analogous roles, highlighting an evolutionarily conserved strategy for maintaining elongation efficiency during challenging translation events.
    DOI:  https://doi.org/10.64898/2025.12.18.695269
  4. J Proteome Res. 2025 Dec 22.
    Proximity Labeling Reveals RNA-Binding Proteins Associating with the Human Mitochondrial Import Receptor TOMM20.
    Saira Akram, Katharina I Zittlau, Karan Sharma, Julia C Fitzgerald, Nisha Rafiq, Boris Maček, Ralf-Peter Jansen.
      The import of most mitochondrial proteins requires that their precursor proteins be bound by the peripheral receptor proteins TOM20, TOM22, and TOM70. Budding yeast TOM20 and TOM70 have been extensively studied regarding their interaction partners and recognized substrates; however, little data is available for metazoan cells. Using APEX2-based proximity labeling, we created association profiles for human TOMM20 and TOMM70 in HeLa cells. We focused particularly on their interactions with RNA-binding proteins (RBPs) because there is evidence of RNA association with the mitochondrial outer membrane (MOM) and of local translation at the mitochondrial surface, however, these processes are poorly understood. Our results demonstrate that several RBPs and translation factors preferentially associate with TOMM20 rather than TOMM70. These include SYNJ2BP, a previously identified membrane-bound RBP that binds and protects mRNA encoding mitochondrial proteins. Inhibiting translation with puromycin increased the association of these RBPs with TOMM20 compared to TOMM70. This suggests that TOMM20, but not TOMM70, may play a role in maintaining cellular homeostasis during translation stress by retaining protective RBPs and translation-related proteins at the MOM.
    Keywords:  APEX2; SYNJ2BP; TOMM20; TOMM70; mitochondrial import; proteomics; proximity labeling
    DOI:  https://doi.org/10.1021/acs.jproteome.5c00905
  5. Eur J Hum Genet. 2025 Dec 27.
    A systematic analysis of mitochondrial aminoacyl tRNA synthetase variants in a rare disease cohort.
    Thiloka E Ratnaike, M Eren Kule, Ida Paramonov, Leslie Matalonga, Kiran Polavarapu, Catarina Olimpio, Rita Horváth.
      Mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are a group of proteins encoded by nuclear DNA that play a crucial role in mitochondrial protein synthesis. Mitochondrial diseases caused by mt-aaRS variants are phenotypically heterogenous but often present with significant neurological features such as childhood-onset encephalopathy and seizures. As such, these conditions are a diagnostic challenge. We present an approach that systematically quantifies phenotypic similarity of individuals with an mt-aaRS variant to published cases, to aid variant interpretation, in RD-Connect-a large Europe-wide rare disease cohort. Across 98 individuals with a mt-aaRS gene of interest, we prioritised 38 individuals with 63 variants following bioinformatic and manual analyses. We additionally reviewed Exomiser prioritisation using a pre-defined gene list for neurological disorders within the RD-Connect Genome-Phenome Analysis Platform (GPAP). We were able to generate likely diagnoses in 11 individuals and VUS findings in 13 individuals, following careful phenotype similarity analysis using a phenotype-genotype dataset generated from 234 published individuals. Four of these 24 individuals did not have an Exomiser-ranked gene variant in the GPAP. Therefore, this approach, using individual-level curated phenotype-genotype data to support variant interpretation, can highlight potentially significant variants that may not be captured by current pipelines. This workflow can be replicated in other heterogeneous rare diseases to support clinical practice.
    DOI:  https://doi.org/10.1038/s41431-025-01990-y
  6. J Transl Med. 2025 Dec 23. 23(1): 1415
    Advances in gene therapy for mitochondrial genetic disorders: current status and clinical implementation challenges.
    Lei Lyu, Beibei Qie, Yanjie He, Feilong Chen, Bao Liu.
      Mitochondria function as the primary energy hubs of cells and possess semi-autonomous genetic characteristic. Mutations in mitochondrial DNA (mtDNA) frequently lead to severe illness and even premature death. The rapid advancement of gene therapy offers promising potential for correcting such disorders. This review first aims to delineate the mechanisms of gene therapy strategies applicable to mitochondrial diseases, including the allotopic expression of mtDNA in the nucleus, mitochondrial-targeted nuclease cleavage, and mtDNA-targeted base editing. It also discusses in detail the clinical efficacy of mtDNA allotopic expression and the preclinical progress of other strategies. Furthermore, the unique physiological features of mitochondria, such as heteroplasmy and independent molecular transport mechanisms, pose distinct challenges for the clinical implementation of mitochondrial gene therapy strategies. Accordingly, this review elaborates on the current limitations of each approach. Finally, it highlights potential optimization directions to address these challenges, emphasizing that understanding heteroplasmy dynamics and their corresponding phenotypes, ensuring the safe delivery and tissue-specific expression of therapeutic elements, and maintaining long-term therapeutic specificity and efficiency are essential for the clinical translation of mitochondrial gene therapy.
    Keywords:  Allotopic expression; Base editing; Mitochondrial DNA; Mitochondrial disorders; Nuclease
    DOI:  https://doi.org/10.1186/s12967-025-07420-3
  7. bioRxiv. 2025 Dec 12. pii: 2025.12.10.693495. [Epub ahead of print]
    Improved long-transcript representation in Oxford Nanopore direct RNA sequencing with UltraMarathonRT.
    George Maio, Li-Tao Guo, Sara Olson, Brenton R Graveley, Jason G Underwood.
      While most RNA-seq methods sequence amplified cDNA molecules, the advent of direct RNA sequencing (DRS) empowered the scientific community to read native RNA. This technology unlocked characterization of natural RNA modifications and long RNA isoforms without the inherent biases of PCR amplification. In the library preparation prior to Oxford Nanopore (ONT) sequencing, polyadenylated RNAs are copied by a reverse transcriptase (RT) to generate an RNA-cDNA hybrid. The step aims to eliminate the secondary and tertiary structure inherent to most RNA sequences prior to presentation of the RNA strand to the pore for sequencing. The current recommended protocol for DRS utilizes Induro ® RT and requires reverse transcription at 60°C. We demonstrate that these RT conditions promote hydrolysis of the RNA strand. We further show that UltraMarathonRT ® (uMRT), an ultraprocessive reverse transcriptase with intrinsic helicase activity that works optimally at 30°C, can be incorporated into a new uMRT-based DRS method that results in longer RNA reads in ONT DRS and longer final isoform predictions. We optimize this reaction along with other molecular biology steps and demonstrate the performance improvements of this new workflow on the benchmark sample, Universal Human Reference RNA, along with human brain RNA. This improved DRS protocol should empower new discoveries by the scientific community.
    DOI:  https://doi.org/10.64898/2025.12.10.693495
  8. Nucleic Acids Res. 2025 Nov 26. pii: gkaf1311. [Epub ahead of print]53(22):
    Suboptimal codon pairs trigger ribosome collisions and cellular quality control responses in tRNA modification mutants.
    Jie Wu, Cristian Eggers, Olga Sin, Łukasz Koziej, Hector Mancilla, Fabienne Mollet, Hans R Schöler, Hannes C A Drexler, Tristan Ranff, Christian Fufezan, Claudine Kraft, Sebastian Glatt, Jan M Bruder, Sebastian A Leidel.
      Transfer RNA (tRNA) modifications tune translation rates and codon optimality, thereby optimizing co-translational protein folding. However, the mechanisms by which tRNA modifications modulate codon optimality and trigger phenotypes remain unclear. Here, we show that ribosomes stall at specific modification-dependent codon pairs in wobble uridine modification (U34) mutants. This triggers ribosome collisions and a coordinated hierarchical response of cellular quality control pathways. High-resolution ribosome profiling reveals an unexpected functional diversity of U34 modifications during decoding. For instance, 5-carbamoylmethyluridine (ncm5U) exhibits distinct effects at the A and P sites. Importantly, ribosomes only slow down at a fraction of codons decoded by hypomodified tRNA, and the decoding speed of most codons remains unaffected. However, the translation speed of a codon largely depends on the identity of A- and P-site codons. Stalling at modification-dependent codon pairs induces ribosome collisions, triggering ribosome-associated quality control (RQC) and preventing protein aggregation by degrading aberrant nascent peptides and messenger RNAs. Inactivation of RQC stimulates the expression of molecular chaperones that remove protein aggregates. Our results demonstrate that loss of tRNA modifications primarily disrupts translation rates of suboptimal codon pairs, showing the coordinated regulation and adaptability of cellular surveillance systems. These systems ensure efficient and accurate protein synthesis and maintain protein homeostasis.
    DOI:  https://doi.org/10.1093/nar/gkaf1311
  9. Nucleic Acids Res. 2025 Nov 26. pii: gkaf1368. [Epub ahead of print]53(22):
    Pseudouridine increases ribosome stability in a thermophilic eukaryote.
    Klemens Wild, Marius Klein, Alicia Burkard, Virginie Marchand, Nikola Kellner, Antonio Paez, Stefan Pastore, Tamer Butto, Yuri Motorin, Mark Helm, Irmgard Sinning.
      RNA modifications alter stability, folding space, and interaction network of RNA molecules. Ribosomal RNA (rRNA) modifications stabilize the structure of ribosomes and cluster around functionally important sites such as the peptidyl transferase center, ribosomal subunit bridges, and the polypeptide tunnel. Here, we investigate the rRNA modifications of the thermophilic fungus Chaetomium thermophilum (ct), a model organism for eukaryotic thermophily and structural stability. Using LC-MS/MS, orthogonal second and third generation RNA-sequencing and high-resolution cryo-electron microscopy, we describe a cross-correlating method to assign and quantify all ct rRNA modifications. Overall, a doubling of rRNA modifications to 4% explains ribosomal thermostability with an extended distribution towards peripheral functional sites. The 2.4 Å structure of the idle ct60S ribosome, retaining nascent chains and including metal ions, polyamines, and water molecules, allows for a comprehensive structure-function analysis. Comparison with mesophilic ribosomes from Chaetomium globosum, yeast, and human highlights the significant increase of pseudouridines (Ψs). The number of Ψs linearly correlates with growth temperature, suggesting statistical modification. A ct-specific Ψ substitution forming a 'Ψ-turn' at the polypeptide tunnel exit close to the third constriction exemplifies mechanistic adaptations of the ribosome at elevated temperatures.
    DOI:  https://doi.org/10.1093/nar/gkaf1368
  10. bioRxiv. 2025 Dec 08. pii: 2025.12.08.692954. [Epub ahead of print]
    Investigation of TRMT61B methyltransferase activity on mRNA and its effects on translation.
    Dorthy Fang, John M Babich, Ryan Stanton, Isaac W Vock, Kyrillos Abdallah, Mingyi Zhu, Richard Li, Matthew D Simon, Wendy V Gilbert, Sigrid Nachtergaele.
      Despite recent advances in technology to map RNA chemical modifications transcriptome-wide, the distribution of N¹-methyladenosine (m¹A) in mRNA remains contested, hindering a clear understanding of its function. Additionally, the enzyme(s) that installs the majority of reported mRNA m 1 A sites has yet to be identified. In this study, we characterized TRMT61B, an m 1 A methyltransferase known to methylate mitochondrial RNAs, but whose sequence preferences have been underexplored. By integrating cellular overexpression of TRMT61B and in vitro methylation of a synthetic pool of diverse human RNA sequences, we identified a preference for a YMR A consensus motif in single stranded RNA regions. In these experiments, TRMT61B methylated thousands of novel human mRNA sites, revealing activity on cytosolic mRNAs. We used these novel m 1 A-modifiable sequences to test the effects of m 1 A on translation of luciferase reporters and on ribosome recruitment to modified transcripts in the pool. We found that m 1 A addition can significantly affect translation and ribosome recruitment, but that these effects are vary by transcript. Taken together, our results can inform future studies of TRMT61B and mRNA, and emphasize that studies of m 1 A regulation of mRNA must be carried out and interpreted in a highly context-aware manner.
    DOI:  https://doi.org/10.64898/2025.12.08.692954
  11. Theranostics. 2026 ;16(5): 2576-2597
    RNA Modifications in T cell Immunity: Mechanisms, Disease Relevance, and Therapeutic Potential.
    Xinyuan Zhao, Meiyan Zou, Weiyao Feng, Nina Li, Xu Chen, Pei Lin, Zihao Zhou, Yunfan Lin, Li Cui.
      RNA modifications constitute a versatile and dynamic layer of post-transcriptional regulation that enables T lymphocytes to fine-tune gene expression programs in response to developmental, environmental, and pathogenic cues. Chemical marks such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ) shape transcript stability, splicing, localization, and translation through coordinated actions of writer, reader, and eraser proteins. Emerging evidence reveals that these pathways orchestrate T cell lineage specification, activation thresholds, effector-memory balance, and immune tolerance, while their dysregulation contributes to infection, autoimmunity, malignancy, and graft rejection. Integrating findings across m6A and other epitranscriptomic marks-including m5C, Ψ, N7-methylguanosine (m7G), N1-methyladenosine (m1A), N4-acetylcytidine (ac4C), and N6-2'-O-methyladenosine (m6Am) -this review delineates how distinct RNA modifications converge on shared molecular circuits controlling transcriptional, metabolic, and signaling networks in T cell immunity. Aberrant modification patterns reshape cytokine profiles, mitochondrial metabolism, and antigen-driven responses, thereby influencing disease trajectories across diverse pathological contexts. Collectively, these insights establish RNA modification as a central regulatory axis linking transcriptomic plasticity to immune function and therapeutic responsiveness. We further highlight unresolved challenges-such as defining spatiotemporal modification landscapes and achieving selective pharmacological modulation-and propose integrative multi-omics and in vivo perturbation approaches to translate epitranscriptomic mechanisms into targeted immunotherapies.
    Keywords:  RNA modifications; T cell immunity; epitranscriptomics; immune regulation; therapeutic potential
    DOI:  https://doi.org/10.7150/thno.124482
  12. JACS Au. 2025 Dec 22. 5(12): 6287-6297
    A Small Cationic Probe for Accurate, Punctate Discovery of RNA Tertiary Structure.
    Jeffrey E Ehrhardt, David Y Qiu, Shouhong Jin, Mark A Boerneke, Caroline J Aufgebauer, Stacy M Horner, Kevin M Weeks.
      RNA molecules fold into intricate three-dimensional tertiary structures that are central to their biological functions. Yet reliably discovering new motifs that form true tertiary interactions remains a major challenge. Here we show that RNA tertiary folding occasionally generates electronegative motifs that react selectively with the small, positively charged probe trimethyloxonium (TMO). Sites with enhanced reactivity to TMO, compared with the neutral reagent dimethyl sulfate (DMS), are indicative of tertiary structure and define T-sites. These positions share a structural signature in which a reactive nucleobase is adjacent to nonbridging phosphate oxygens, creating a localized region of negative charge. T-sites consistently map to the cores of higher-order structural interactions and functional centers across diverse RNAs, including distinct states in conformational ensembles. In the 10,723-nt dengue virus genome, three strong T-sites were detected, each within a complex structure required for viral replication. Cation-based covalent chemistry enables high-confidence discovery and analysis of functional RNA tertiary motifs across long and complex RNAs, opening new opportunities for transcriptome-wide structural analysis.
    Keywords:  RNA electrostatics; RNA tertiary structure; RNA virus; mutational profiling; transcriptome-wide probing; trimethyloxonium cation
    DOI:  https://doi.org/10.1021/jacsau.5c01280
  13. Comput Struct Biotechnol J. 2025 ;27 5493-5502
    StructRMDB: A database of RNA modification sites that affect RNA secondary structure.
    Ziyan Zhang, Xuan Wang, Jingxian Zhou, Jia Meng, Bowen Song, Yuxin Liang, Yuheng Cai, Jiongming Ma.
      Post-transcriptional RNA modifications, prevalent in multiple RNA species such as mRNA, rRNA, and tRNA, play a significant role in biological processes by altering RNA structures. With recent advancements in prediction algorithms, it is possible to predict RNA secondary structure for sequences containing modified bases. In this study, we introduce StructRMDB, the first database designed to characterize the impact of chemical modifications on RNA secondary structure. StructRMDB comprises more than 880,000 RNA modification sites and their structural impacts, including N 6 -Methyladenosine (m6A), pseudouridine (Ψ), and adenosine-to-inosine editing (A-to-I) from nine species in both pre-RNA and mature RNA. Two RNA secondary structure prediction tools (RNAstructure and ViennaRNA), along with four scoring methods (Similarity Score, Relative Score, Distance, and SMC Score), were adopted to assess structural changes induced by these modifications. Additionally, we visualized RNA secondary structures with and without modifications to highlight structural alterations. A user-friendly graphical interface is provided to facilitate the querying, downloading, and sharing of modified site evaluation and annotation data, offering novel insights into the effects of RNA modifications on secondary structure. StructRMDB serves as a valuable resource for studying the structural impact of RNA modifications and is available at: http://www.rnamd.org/StructRMDB/index.html.
    Keywords:  Adenosine-to-inosine editing; N6-methyladenosine; Pseudouridine; RNA modification; RNA secondary structure
    DOI:  https://doi.org/10.1016/j.csbj.2025.11.058
  14. J Proteome Res. 2025 Dec 23.
    TAG-PL: A Universal Proximity Labeling Platform for Mapping Mitochondrial Proteomes and Organelle Interactions under Stress.
    Enming Miao, Xuyang Yue, Zhixiong Tao, Hongqiang Qin, Mingliang Ye.
      Mitochondrial dysfunction induces numerous diseases, yet current proximity labeling methods require gene transfection and membrane potential-sensitive probes, limiting their use in hard-to-transfect cells and disease models. We developed TAG-PL (Tailored Antibody-Guided Proximity Labeling), a transfection-free approach for in-depth mapping of the mitochondrial proteome, achieving >90% specificity and identifying >450 mitochondrial proteins─more than the coverage of existing nontransfection methods. Applied to heat-stressed macrophages, TAG-PL revealed dynamic mitochondrial proteome remodeling, including antioxidant responses and metabolic shifts during heat stress. Notably, we discovered physical interactions between stress granules and mitochondria, identifying 10 interaction mediators (including MSRA and UBA1). These findings establish stress granules as regulatory hubs for organelle dynamics and immune responses. TAG-PL's high performance and broad applicability across diverse sample types, particularly immune cells and tissues, make it a powerful tool for dissecting mitochondrial function in disease models without genetic manipulation.
    Keywords:  SG−mitochondria interaction; antibody-guided proximity labeling; mitochondrial proteome
    DOI:  https://doi.org/10.1021/acs.jproteome.5c00855
  15. Brief Bioinform. 2025 Nov 01. pii: bbaf690. [Epub ahead of print]26(6):
    Deciphering RNA modification and post-transcriptional regulation with NetRNApan.
    Haodong Xu, Wankun Deng, Ruifeng Hu, Binfeng Liu, Wenchao Zhang, Lujuan Wang, Lin Qi, Xiaolei Ren, Chao Tu, Zhihong Li, Zhongming Zhao.
      RNA modification, which is evolutionarily conserved, is crucial for modulating various biological functions and disease pathogenesis. High resolution transcriptome-wide mapping of RNA modifications has facilitated both data resources and computational prediction of RNA modification. While these prediction algorithms are promising, they are limited in interpretability or generalizability, or the capacity for discovering novel post-transcriptional regulations. Here, we present NetRNApan, a deep learning framework for RNA modification site prediction, motif discovery and trans-regulatory factor identification. Using m5U profiles generated by FICC-seq and miCLIP-seq technologies and single-base resolution m6A sites from multiple experiments as cases, we demonstrated the accuracy of NetRNApan with more efficient and interpretive feature representations. For m5U modification, we uncovered five representative clusters with consensus motifs that may be essential by decoding the informative characteristics detected by NetRNApan. Furthermore, NetRNApan revealed interesting trans-regulatory factors and provided a protein-binding perspective for investigating the function of RNA modifications. Specifically, we discovered 21 potential functional RNA-binding proteins (RBPs) whose binding sites were significantly linked to the extracted top-scoring motifs for m5U modification. Two examples are ANKHD1 and RBM4 with potential regulatory function of m5U modifications. Meanwhile, the analysis of convolution layer parameters within the model offers valuable insights into the regulation of m6A in humans. Collectively, NetRNApan demonstrated high accuracy, interpretability and generalizability for study of RNA modification and mRNA regulation. NetRNApan is freely available at https://github.com/bsml320/NetRNApan.
    Keywords:  RNA binding protein; RNA modification; deep learning; epitranscriptomics; m5U; m6A; motif discovery
    DOI:  https://doi.org/10.1093/bib/bbaf690
  16. Precis Clin Med. 2025 Dec;8(4): pbaf035
    RNA modifications in health and disease: from mechanistic insights to therapeutic applications.
    Yiting Chen, Dulin Ding, Xing Tang, Rui Ma, Jian-Kang Zhou.
      RNA modifications encompass a series of dynamic chemical changes and editing events on RNA molecules, playing a pivotal role in essential physiological processes such as embryonic development, immune response, and the maintenance of cell homeostasis. By influencing RNA stability, splicing, translation, and intermolecular interactions, RNA modifications serve as crucial mechanisms regulating gene expression at the post-transcriptional level. Dysregulation of the modification machineries or aberrant modification patterns is closely associated with the onset and progression of various diseases, including tumors, metabolic disorders, cardiovascular diseases, and neurological and immune conditions, making them potential biomarkers for disease diagnosis, prognosis, and treatment. In this review, we summarize the molecular mechanisms of major RNA modifications, emphasize their functions in health and disease, and discuss their diagnostic and therapeutic value in pathological contexts.
    Keywords:  RNA editing; RNA modification; ac4C; m6A; methylation; pseudouridine
    DOI:  https://doi.org/10.1093/pcmedi/pbaf035
  17. Nat Commun. 2025 Dec 26.
    Designer RNA nanostructures co-transcribed and self-assembled inside human cell nuclei.
    Xu Chang, Maciej Jeziorek, Qi Yang, Edward M Bonder, Hao Yan, Jean-Pierre Etchegaray, Fei Zhang.
      The use of nucleic acid-based nanostructures as synthetic biological tools to interface with and regulate cell processes remains challenging. A major obstacle lies in nuclear delivery and retention within live eukaryotic cells. Here, we present a platform of single-stranded RNAs that can co-transcriptionally fold into defined nanostructures and assemble into rings, ribbons, and nanonet-like architectures. We validate the formation of these structures in vitro using atomic force microscopy. Then, we demonstrate the functional integration of fluorescent aptamers and RNA sensing capability within the single chain by co-folding with these structures. Notably, we show that the RNA nanonets can be co-transcriptionally produced and assembled directly inside the nucleus of live human cells. We use confocal live-cell imaging and transmission electron microscopy to reveal well-defined nanostructure patterns retained in the nucleus. Together, these results establish a genetically encoded, self-assembling RNA nanostructure system with programmable geometry and localization, providing a foundation for the development of RNA-based nanodevices to examine biological properties in live cells and tissues.
    DOI:  https://doi.org/10.1038/s41467-025-67817-y
  18. Mol Ther. 2025 Dec 24. pii: S1525-0016(25)01064-0. [Epub ahead of print]
    Transient muscle expression of mitoARCUS in mice leads to a sustained reduction in pathogenic mtDNA content and improves muscle function.
    Sandra R Bacman, Wendy Shoop, C Spencer Henley-Beasley, Rhese Thompson, Jose Domingo Barrera-Paez, Lise-Michelle Theard, Monica Rodriguez-Silva, Cassandra Gorsuch, Elisabeth R Barton, Carlos T Moraes.
      Mitochondrial myopathies are often caused by heteroplasmic mutations in the mitochondrial DNA (mtDNA). In muscle, biochemical, pathological, and clinical impairments are observed only when the ratios of mutant/wild-type mtDNA are high. Because reductions in mutant mtDNA loads are essentially permanent, we reasoned that transient expression of a therapeutic mitochondrial nuclease could be sufficient to permanently alter heteroplasmy. We expressed a mitochondrial targeted gene editing nuclease (mitoARCUS) via intramuscular injection of lipid nanoparticle (LNP)/mRNA complexes in a mouse model of mtDNA disease (m.5024C>T in the mt-tRNAAla gene). Transient expression of mitoARCUS in the tibialis anterior (TA) led to a robust decrease in mtDNA mutation load which was maintained up to forty-two weeks after injection. A molecular marker of the mitochondrial defect in this model, namely low levels of mt-tRNAAla, were markedly improved in treated muscles. Muscle force assessment in situ after repeated stimulation showed that fatigability was improved in the treated TA. Finally, we showed that multi-muscle injections can alter mtDNA heteroplasmy essentially in whole limbs. These results demonstrate that transient expression of mitoARCUS via LNP/mRNA intramuscular injections have long-lasting positive effects in muscles afflicted with mitochondrial myopathy.
    DOI:  https://doi.org/10.1016/j.ymthe.2025.12.041
  19. Int J Biol Macromol. 2025 Dec 23. pii: S0141-8130(25)10440-6. [Epub ahead of print] 149883
    MsipNet: a multi-scale representation learning framework for predicting protein-RNA interaction.
    Nan Song, Zhijin Li, Yang Deng, Jinhu Huang, Zhiwei Ji.
      Protein-RNA interactions (PRIs) are fundamental to post-transcriptional regulation, influencing key processes such as RNA splicing, stability, translation, and broader cellular functions. Understanding these interactions is essential for uncovering gene regulatory mechanisms and discerning how disease-associated mutations disrupt cellular homeostasis, thereby providing insights that can inform the development of novel therapeutic strategies. Consequently, accurate identification of PRIs represents a critical link between basic biological research and biomedical applications. To advance PRI prediction, we introduce MsipNet, a multi-scale representation learning framework that integrates global and local RNA sequence features with structural information via a multimodal learning strategy. MsipNet employs a hybrid architecture combining Long Short-Term Memory (LSTM) networks with U-shaped convolution-dilated convolution (UCDC) modules, enabling fine-grained feature refinement and improving prediction accuracy. This design facilitates the capture of intricate binding patterns while maintaining high computational efficiency. Experimental evaluations show that MsipNet consistently outperforms eight state-of-the-art (SOTA) methods across 42 RNA-binding proteins (RBPs) from six cell lines, demonstrating superior performance in predicting binding preferences. Furthermore, MsipNet reliably identifies biologically validated binding motifs and exhibits strong generalizability when applied to unseen data. Collectively, these findings position MsipNet as a robust and interpretable tool for PRI prediction and functional mutation prioritization, with broad potential for mechanistic studies and biomedical applications.
    Keywords:  Convolution; Motif; Protein-RNA interaction; RNA structure
    DOI:  https://doi.org/10.1016/j.ijbiomac.2025.149883
  20. Front Neurol. 2025 ;16 1731858
    Stroke-like lesion and status epilepticus in a child with NARS2-related combined oxidative phosphorylation deficiency 24.
    Song Su, Wandong Hu, Yong Liu, Qiong Lang, Hongwei Zhang.
       Introduction: The NARS2 gene encodes mitochondrial asparaginyl-tRNA synthetase, and biallelic pathogenic variants have been associated with combined oxidative phosphorylation deficiency 24 (COXPD24), an autosomal recessive mitochondrial disorder characterized by highly heterogeneous clinical manifestations. This study retrospectively analyzed the clinical and genetic findings of a Chinese infant presenting with status epilepticus and explored potential genotype-phenotype correlations.
    Methods: Clinical data, laboratory tests, neuroimaging, and disease course of the proband were reviewed. Whole-exome sequencing (WES) and copy-number variation (CNV) analysis were performed to identify causative variants in NARS2. Candidate variants were assessed by population database screening and literature review.
    Results: The proband, a 9-month-old girl, presented with status epilepticus, global developmental delay, increased muscle tone, elevated serum lactate and myocardial enzyme levels. Brain magnetic resonance imaging (MRI) revealed a focal cerebral lesion consistent with a metabolic or stroke-like infarction, as well as delayed myelination. WES identified compound heterozygous NARS2 variants: a large exon 6-11 deletion and a novel missense variant c.467T>C (p.Leu156Ser), inherited in an autosomal recessive manner. Both variants were absent from public population databases and published literature. Notably, cerebral infarction has not been previously reported in NARS2-related disorders, suggesting a potential expansion of the clinical spectrum.
    Discussion: Review of previously reported NARS2 variants indicates that both missense and loss-of-function mutations can lead to variable disease severity depending on residual enzyme activity. This case broadens the phenotypic and mutational spectrum of NARS2-associated COXPD24 and highlights the importance of evaluating large exon deletions and novel variants in infants with early-onset mitochondrial encephalopathy and epileptic manifestations.
    Keywords:  COXPD24; NARS2; infarction-like lesion; mitochondrial disease; status epilepticus
    DOI:  https://doi.org/10.3389/fneur.2025.1731858
  21. bioRxiv. 2025 Dec 14. pii: 2025.12.12.694034. [Epub ahead of print]
    Molecular basis of tRNA substrate recognition and modification by the atypical SPOUT methyltransferase Trm10.
    Suparno Nandi, Sarah E Strassler, Debayan Dey, Aiswarya Krishnamohan, George M Harris, Lindsay R Comstock, Jane E Jackman, Graeme L Conn.
      The evolutionarily conserved methyltransferase Trm10 modifies the N1 position of guanosine 9 (G9) in some tRNAs, but how the enzyme recognizes and modifies its substrate tRNAs remains unclear. Here, we used an S-adenosyl-L-methionine (SAM) analog to trap the Trm10-tRNA Gly complex and enable determination of its structure in a post-catalytic state by cryogenic electron microscopy (cryo-EM). We observed three distinct complexes: two with a single Trm10 bound to tRNA that differ in their tRNA acceptor stem orientation ("closed" and "open") and a minor population with two Trm10s engaging the same tRNA. The monomeric complexes reveal a positively charged surface that guides the G9 into the catalytic site with key conserved residues forming "pincer"-like interactions that stabilize the flipped methylated nucleotide. In the open tRNA conformation, the acceptor stem is rotated away from the enzyme, weakening the tRNA-protein contacts, consistent with a product-release conformation. The dimeric complex, which is supported by tRNA-dependent protein crosslinking, reveals one Trm10 positioned similarly to the monomeric complexes and engaged with G9, while the other Trm10 contacts distal tRNA regions, suggesting a potential role in facilitating a key conformational transition during the process of catalysis or modified tRNA release. Finally, molecular dynamics simulations comparing G9- and A9-containing complexes reveal that G9 is efficiently stabilized in the binding pocket unlike A9, identifying the structural basis for guanosine selectivity. Overall, these findings reveal the structural determinants of G9-specific tRNA methylation by Trm10 and suggest a unique mechanism of action among RNA-modifying SPOUT methyltransferases.
    DOI:  https://doi.org/10.64898/2025.12.12.694034
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