bims-mirnam Biomed News
on Mitochondrial RNA metabolism
Issue of 2025–12–07
twenty-two papers selected by
Hana Antonicka, McGill University
  1. Structural insights into cauliflower mitoribosome in translation state and in association with a late assembly factor.
  2. Trans-generational maintenance of mitochondrial DNA integrity in oocytes during early folliculogenesis.
  3. Mitochondrial Genetics in Cardiovascular Health and Disease: A Scientific Statement From the American Heart Association.
  4. Roles and related underlying mechanisms of mitochondrial RNA modification in cancer progression.
  5. Mitochondrial DNA A3243G variant: Current perspectives and clinical implications.
  6. Slirp2 modulates oogenesis via regulating mitochondrial protein translation.
  7. Mitochondria-targeted gene delivery using fluorinated lipid nanoparticles to alleviate Leber's hereditary optic neuropathy.
  8. Translational activators align mRNAs at the small mitoribosomal subunit for translation initiation.
  9. Enzymes in high-throughput RNA sequencing: Applications and challenges.
  10. Systematic analysis of noncanonical ribosomal protein paralogs does not provide evidence for specialized functions in Drosophila.
  11. m6A Immunoprecipitation from Ribosome-Bound mRNA.
  12. Induro-seq to analyze subcellular enrichment of small RNAs.
  13. Molecular mechanisms of mitochondrial function in neurodegenerative diseases.
  14. RNA structure analyzed by chemical probing and mutational profiling with sequencing (MaPseq).
  15. High-throughput mapping of modular regulatory domains in human RNA-binding proteins.
  16. Detecting tRNA modifications with fluorescent-labeled DNA probes.
  17. Preparation of enzymes and libraries for MapID-tRNA-seq to identify chemical modifications in human tRNAs.
  18. Age-associated G-quadruplex accumulation and DDX5 loss shape chromatin landscapes in human astrocytes.
  19. m6AConquer: a consistently quantified and orthogonally validated database for the N6-methyladenosine (m6A) epitranscriptome.
  20. RNAsolo 2.0: multimodal database to study RNAs, their structural families and intermolecular interfaces.
  21. Implications of Codon Usage, tRNA Gene Redundancy and tRNA Gene Clustering in Experimental Models of Mistranslation.
  22. Capturing Translation in Action with Protein Synthesis Profiling.
  1. Nat Commun. 2025 Dec 02. 16(1): 10839
    Structural insights into cauliflower mitoribosome in translation state and in association with a late assembly factor.
    Vasileios Skaltsogiannis, Philippe Wolff, Tan-Trung Nguyen, Nicolas Corre, David Pflieger, Todd Blevins, Yaser Hashem, Philippe Giegé, Florent Waltz.
      Ribosomes are key molecular machines that translate mRNA into proteins. Mitoribosomes are specific ribosomes found in mitochondria, which have been shown to be remarkably diverse across eukaryotic lineages. In plants, they possess unique features, including additional rRNA domains stabilized by plant-specific proteins. However, the structural specificities of plant mitoribosomes in translation state remained unknown. We used cryo-electron microscopy to provide a high-resolution structural characterization of the cauliflower mitoribosome, in translating and maturation states. The structure reveals the mitoribosome bound with a tRNA in the peptidyl site, along with a segment of mRNA and a nascent polypeptide. Moreover, using structural data, nanopore sequencing and mass spectrometry, we identify a set of 19 ribosomal RNA modifications. Additionally, we observe a late assembly intermediate of the small ribosomal subunit, in complex with the RsgA assembly factor. This reveals how a plant-specific extension of RsgA blocks the mRNA channel to prevent premature mRNA association before complete small subunit maturation. Our findings elucidate key aspects of translation in angiosperm plant mitochondria, revealing its distinct features compared to other eukaryotic lineages.
    DOI:  https://doi.org/10.1038/s41467-025-65864-z
  2. PLoS Genet. 2025 Dec 03. 21(12): e1011562
    Trans-generational maintenance of mitochondrial DNA integrity in oocytes during early folliculogenesis.
    Qin Xie, Haibo Wu, Jiaxin Qiu, Junbo Liu, Xueyi Jiang, Huihui Wu, Qifeng Lyu, Hui Long, Wenzhi Li, Shuo Zhang, Yuxiao Zhou, Yining Gao, Aaron J W Hsueh, Yanping Kuang, Lun Suo.
      Mutations in mitochondrial DNA (mtDNA) can lead to mitochondrial and cellular dysfunction. However, recent studies suggest that purifying selection acts against mutant mtDNAs during transgenerational transmission. We investigated the mtDNA dynamics during ovarian follicle development. Using base-editing, we generated mice harboring a 3177 G > A mutation corresponding to the human Leber hereditary optic neuropathy (LHON)-related mtDNA mutation and confirmed a transgenerational reduction of the mutant mtDNA. Utilizing a mouse follicle culture system in which pathogenic mtDNA mutations were introduced in vitro, followed by mtDNA sequencing and digital PCR, we found that the germline heteroplasmy shift during early folliculogenesis was driven by a decrease in mutant mtDNA along with compensatory replication of wild-type mtDNA. In contrast, synonymous mtDNA mutations did not affect mtDNA dynamics. These findings demonstrate that mice can eliminate certain pathogenic mtDNA mutations in the germline during early folliculogenesis, thus advancing our understanding of mtDNA purifying selection during oogenesis. Furthermore, our use of mtDNA editing in in vitro-cultured follicles provides a novel approach to create and monitor mitochondrial DNA mutations.
    DOI:  https://doi.org/10.1371/journal.pgen.1011562
  3. Circulation. 2025 Dec 02.
    Mitochondrial Genetics in Cardiovascular Health and Disease: A Scientific Statement From the American Heart Association.
    American Heart Association Council on Genomic and Precision Medicine Council on Cardiopulmonary, Critical Care, Perioperative and Resuscitation Council on Cardiovascular and Stroke Nursing Council on Peripheral Vascular Disease
      Metabolic and genetic abnormalities have long been noted in cardiovascular diseases, but the contribution of mitochondrial genetic (mitochondrial DNA [mtDNA]) variation is understudied. Mitochondrial genetics is complex in that each mitochondrion contains multiple mtDNA copies that may carry different variants, which is called heteroplasmy. Heteroplasmic variation is dynamic, increases with advancing age, and may contribute to aging-related cardiovascular diseases. Pathogenic variants in mitochondrial genes of the mtDNA or nuclear genome cause mitochondrial diseases, often with cardiac involvement, particularly in patients with adult-onset disease. Population-level studies have identified mtDNA variants associated with cardiovascular risk factors and disease, but evaluation of mtDNA genetic variation is often limited to only a handful of variants and small sample sizes. Studies in animal models have linked several mtDNA variants to cardiac remodeling and dysfunction and suggest a role for mitochondrial-nuclear genetic interactions in disease penetrance. The objective of this scientific statement is to outline the current state of understanding of the role of mitochondrial genetics in cardiovascular pathobiology and highlight important gaps in knowledge. The intended audience of this scientific statement is meant to be broad, spanning clinical, translational, and basic researchers and health care professionals. Despite remaining limitations and barriers, recent advances in genomic sequencing, mtDNA gene editing modalities, and the directed differentiation of stem cells to cardiovascular cell types are creating new opportunities to advance understanding of mitochondrial genetics in cardiovascular pathophysiology.
    Keywords:  AHA Scientific Statements; DNA, mitochondrial; cardiovascular diseases; genes, mitochondrial; mitochondria
    DOI:  https://doi.org/10.1161/CIR.0000000000001393
  4. Acta Pharmacol Sin. 2025 Dec 01.
    Roles and related underlying mechanisms of mitochondrial RNA modification in cancer progression.
    Ling-Zhi Wang, Tao-Yu Yang, Zi-Hui Xiong, Jian-Xin Peng, Jia-Wang Zhou, Hong-Sheng Wang, Jun-Ming He.
      Mitochondrial RNA modifications refer to modifications that occur across various RNA species within the mitochondria. While the incidence of such modifications is lower than that in the cytoplasm, substantial evidence highlights their pivotal role in facilitating mitochondrial gene expression and precise protein synthesis. Emerging research suggests that these mitochondrial RNA modifications are also significantly involved in the progression of cancer. In this review, we first delve into the diversity of mitochondrial RNA modifications, their potential sites of occurrence, and the enzymes involved in these processes. We evaluate the existing evidence regarding mitochondrial RNA modifications and their significance in cancer progression and investigate the potential of these modifications as promising targets for both preventative strategies and early diagnostic screening. Finally, we deliberate on the trajectory of mitochondrial RNA modifications in the context of cancer progression and how these RNA modifications might pave the way for novel diagnostic and therapeutic approaches for preventive medicine.
    Keywords:  RNA modifications; cancer; methylation; mitochondria
    DOI:  https://doi.org/10.1038/s41401-025-01682-9
  5. Intractable Rare Dis Res. 2025 Nov 30. 14(4): 249-257
    Mitochondrial DNA A3243G variant: Current perspectives and clinical implications.
    Kuan-Yu Chu.
      The mitochondrial DNA A3243G variant, located in the MT-TL1 gene encoding tRNALeu(UUR), represents one of the most clinically significant pathogenic mitochondrial mutations. This point mutation accounts for approximately 80% of Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS) syndrome cases and is the primary cause of Maternally Inherited Diabetes and Deafness (MIDD) syndrome. The clinical spectrum associated with this mutation ranges from asymptomatic carriers to severe multisystem disease with early mortality. The pathophysiology involves impaired mitochondrial protein synthesis leading to respiratory chain dysfunction, with phenotypic expression determined by heteroplasmy levels and tissue-specific energy demands. Understanding the complex inheritance patterns, genetic bottleneck effects during oogenesis, and heteroplasmy variations is crucial for comprehending the variable clinical presentations observed in affected families. Histological examination reveals characteristic features including ragged-red fibers, cytochrome c oxidase-deficient fibers, and abnormal mitochondrial proliferation. Current therapeutic approaches focus on metabolic support, antioxidant therapy, and management of specific complications, with L-arginine showing promise for stroke-like episodes. However, careful attention to drug safety profiles and potential mitochondrial toxicity is essential in treatment planning. Understanding the diverse clinical manifestations and implementing appropriate screening strategies are crucial for early diagnosis and optimal patient management. This review synthesizes current knowledge regarding the A3243G variant's pathophysiology, clinical features, diagnostic approaches, and therapeutic interventions.
    Keywords:  MELAS syndrome; heteroplasmy; lactic acidosis; mitochondrial DNA (mtDNA); point mutation; transfer RNA (tRNALeu(UUR))
    DOI:  https://doi.org/10.5582/irdr.2025.01051
  6. J Mol Cell Biol. 2025 Dec 02. pii: mjaf047. [Epub ahead of print]
    Slirp2 modulates oogenesis via regulating mitochondrial protein translation.
    Jinguo Cao, Jiting Zhang, Zhaoqi Wu, Wei Luan, Yue Zhou, Huihui Huang, Lingling Li, Wen Hu.
      Mitochondria are essential organelles responsible for generating ATP through oxidative phosphorylation (OXPHOS). Despite having their own genome, mitochondria rely on a complex interplay with nuclear-encoded proteins to maintain their function, as mutations in these proteins can lead to mitochondrial dysfunction and associated diseases. Mutations in the SLIRP (stem-loop interacting RNA-binding protein) gene are known to cause severe human mitochondrial diseases, and loss of SLIRP function can impair mitochondrial mRNA stability and translation. However, in vivo roles of the SLIRP protein remain inadequately understood. Drosophila melanogaster serves as a powerful model for studying mitochondrial function, particularly in the context of reproductive system development and gametogenesis. In this study, we focus on the role of the fly Slirp2 in oogenesis. Loss of Slirp2 impairs mitochondrial protein synthesis, leading to reduced OXPHOS efficiency, diminished ATP production, and disrupted insulin/mTOR signaling. These defects ultimately promote reactive oxygen species-induced programmed cell death, resulting in infertility. Our findings provide novel insights into the mechanistic role of Slirp2 in mitochondrial function and reproductive biology in vivo. We demonstrate that Slirp2 exhibits species-specific regulation of mitochondrial translation, revealing its complex, context-dependent function. These results have broader implications for understanding mitochondrial diseases, suggesting that the effects of Slirp2 mutations may vary across different organisms and tissue types.
    Keywords:  SLIRP; Slirp2; mitochondrial diseases; oogenesis
    DOI:  https://doi.org/10.1093/jmcb/mjaf047
  7. Nat Commun. 2025 Dec 04. 16(1): 10891
    Mitochondria-targeted gene delivery using fluorinated lipid nanoparticles to alleviate Leber's hereditary optic neuropathy.
    Yi Wang, Min Zhao, Hai-Xin Xie, Hao-Yuan Yu, Jing-Song Yang, Lu-Xin Qie, Na-Hui Liu, Jia-Qi Chen, Zi-Juan Yi, Tian-Jiao Zhou, Lei Xing, Xian Wu Cheng, Hu-Lin Jiang.
      Mutations in mitochondrial DNA (mtDNA) lead to various mitochondrial diseases for which no cure is currently available. Despite the promising potential of mtDNA correction to treat these disorders, the double mitochondrial membranes have proven to be a tough barrier to overcome. Here, we develop fluorinated lipid nanoparticles with a mitochondrial targeting sequence (F-M-LNP) to overcome the mitochondrial barrier by virtue of their high affinity for mitochondrial membranes, thereby effectively introducing gene into mitochondria. Through the rational design of ionizable lipid structures, we synthesize 16 lipid nanoparticles (LNPs) with varying degrees of fluorination and investigate the key structural features required for efficient mitochondria-targeted gene delivery. As fluorinated ionizable lipid-mediated mitochondrial transport is independent of mitochondrial membrane potential (MMP), F-M-LNPs deliver gene to mitochondria under pathological conditions where MMP is impaired, resulting in a 3.8-fold increase in functional protein expression compared to non-fluorinated LNPs. In a male mouse model of genetically induced mitochondrial disease, F-M-LNP demonstrate functional complementation of mutant mtDNA, alleviating disease symptoms. Together, our results show that modifying vectors with fluorinated groups offers valuable tools for correcting mitochondrial genome defects.
    DOI:  https://doi.org/10.1038/s41467-025-65874-x
  8. Nat Struct Mol Biol. 2025 Dec 03.
    Translational activators align mRNAs at the small mitoribosomal subunit for translation initiation.
    Joseph B Bridgers, Andreas Carlström, Dawafuti Sherpa, Mary T Couvillion, Urška Rovšnik, Jingjing Gao, Bowen Wan, Sichen Shao, Martin Ott, L Stirling Churchman.
      Mitochondrial gene expression is essential for oxidative phosphorylation. Mitochondrial-encoded mRNAs are translated by dedicated mitochondrial ribosomes (mitoribosomes), whose regulation remains elusive. In Saccharomyces cerevisiae, nuclear-encoded mitochondrial translational activators (TAs) facilitate transcript-specific translation by a yet unknown mechanism. Here, we investigated the function of TAs containing RNA-binding pentatricopeptide repeats using selective mitoribosome profiling and cryo-electron microscopy (cryo-EM) structural analysis. These analyses show that TAs exhibit strong selectivity for mitoribosomes initiating on their target transcripts. Moreover, TA-mitoribosome footprints indicate that TAs recruit mitoribosomes proximal to the start codon. Two cryo-EM structures of mRNA-TA complexes bound to mitoribosomes stalled in the post-initiation, pre-elongation state revealed the general mechanism of TA action. Specifically, the TAs bind to structural elements in the 5' untranslated region of the client mRNA and the mRNA channel exit to align the mRNA in the small subunit during initiation. Our findings provide a mechanistic basis for understanding how mitochondria achieve transcript-specific translation initiation without relying on general sequence elements to position mitoribosomes at start codons.
    DOI:  https://doi.org/10.1038/s41594-025-01726-y
  9. Methods Enzymol. 2025 ;pii: S0076-6879(25)00399-4. [Epub ahead of print]725 51-75
    Enzymes in high-throughput RNA sequencing: Applications and challenges.
    Yuko Nakano, Howard Gamper, Ya-Ming Hou.
      High-throughput RNA sequencing provides genome-wide information on the dynamics of RNA in each cell and how the dynamics responds to environmental changes. Next-generation sequencing by the Illumina platform currently provides the highest information output as compared to other platforms. A key component of next generation sequencing of each RNA is the successful end-to-end reverse-transcription into a cDNA strand. This can be highly challenging given the propensity of each RNA to adopt ordered structures and to contain post-transcriptional modifications. While many reverse transcriptase (RT) enzymes have been developed over the years to maximize read-through of an RNA, their processivity and efficiency varies, raising the question of how to select the RT for the experiment at hand. Here, we use tRNA as a model for genome-wide sequencing, as tRNA has a stable secondary and tertiary structure and has a high density and wide variety of post-transcriptional modifications, presenting one of the most challenging problems of sequencing RNA. We compare the efficiency of end-to-end cDNA synthesis of tRNA among several recent RT enzymes and provide a general sequencing workflow that is applicable to most of these enzymes.
    Keywords:  BoMoC; Induro; Maxima H minus; Superscript IV; TGIRT; UMarathon
    DOI:  https://doi.org/10.1016/bs.mie.2025.10.007
  10. Proc Natl Acad Sci U S A. 2025 Dec 09. 122(49): e2506642122
    Systematic analysis of noncanonical ribosomal protein paralogs does not provide evidence for specialized functions in Drosophila.
    Katarina Z A Grobicki, Daniel Gebert, Carol Sun, Felipe Karam Teixeira.
      Ribosomes catalyze all protein synthesis, and mutations altering their levels and function underlie many developmental diseases and cancer. Historically considered to be invariant machines, ribosomes differ in composition between tissues and developmental stages, incorporating a diversity of ribosomal proteins (RPs) encoded by duplicated paralogous genes. Here, we use Drosophila to systematically investigate the origins and functions of noncanonical RP paralogs. We show that new paralogs mainly originated through retroposition and that only a few new copies retain coding capacity over time. Although transcriptionally active noncanonical RP paralogs often present tissue-specific expression, we show that the majority of those are not required for either viability or fertility in Drosophila melanogaster. The only exception, RpS5b, which is required for oogenesis, is functionally interchangeable with its canonical paralog, indicating that the RpS5b-/- phenotype results from insufficient ribosomes rather than the absence of an RpS5b-specific, functionally specialized ribosome. Altogether, our results provide evidence that instead of new functions, RP gene duplications provide a means to regulate ribosome levels during development.
    Keywords:  germline; ribosome heterogeneity; translational control
    DOI:  https://doi.org/10.1073/pnas.2506642122
  11. Methods Enzymol. 2025 ;pii: S0076-6879(25)00401-X. [Epub ahead of print]725 225-253
    m6A Immunoprecipitation from Ribosome-Bound mRNA.
    Nora T Kiledjian, Morgan J McGrath, Crystal S Conn.
      Here we describe a protocol for correlating a transcript's translational state to its N6-methyladenosine (m6A) status using polysome profiling and m6A immunoprecipitation (IP). Polysome profiling is a technique used to separate out cellular components by density, allowing the visualization of transcripts based on the number of ribosomes bound. The technique uses high ribosome occupancy as a proxy for high translational activity. Transcripts bound by many ribosomes can be isolated from lowly translated transcripts and from those unbound by translational machinery. It has been demonstrated that the RNA modification m6A can alter the stability, localization, and splicing of a transcript. The role that m6A plays in translational selectivity is an established, but still highly debated area in the field. m6A IP is a technique developed to isolate m6A-containing RNA by using an antibody against the m6A modification itself. We have developed this protocol, which pairs polysome profiling with m6A IP in order to broadly characterize the relationship between the presence of m6A modifications and the extent of a transcript's translation.
    Keywords:  Epitranscriptomics; N6-Methyladenosine; Polysome profiling; Protein synthesis; RNA modification; m(6)A immunoprecipitation; mRNA translation
    DOI:  https://doi.org/10.1016/bs.mie.2025.10.009
  12. Methods Enzymol. 2025 ;pii: S0076-6879(25)00406-9. [Epub ahead of print]725 155-173
    Induro-seq to analyze subcellular enrichment of small RNAs.
    Monima Anam, Xisheng Liu, Taylor L Schanel, Christopher D Willey, Zhangli Su.
      Small non-coding RNAs, including microRNAs (miRNAs) and tRNA fragments (tRFs), play critical roles in gene regulation across diverse biological contexts. Although miRNAs and tRFs are traditionally viewed as cytoplasmic effectors, recent studies suggest they may also adopt functions within the nucleus. However, accurately mapping the subcellular localization of small RNAs remains technically challenging due to inherent biases in RNA yield between the nuclear and cytoplasmic compartments, as well as the presence of base-pair-disrupting RNA modifications. Here, we present a method for small RNA sequencing in example glioblastoma (GBM) cell lines that enables accurate subcellular localization by integrating defined synthetic spike-in controls and Induro-RT, a highly processive reverse transcriptase capable of reading through modified nucleotides. Spike-in controls correct for input disparities across compartments, while Induro-RT allows for the transcription of modified small RNAs, which are often overlooked by conventional reverse transcriptases. This approach enables unbiased detection of both canonical and modified small RNAs, providing a more accurate and comprehensive view of miRNA and tRF distribution between the nucleus and cytoplasm.
    Keywords:  Fractionation; Genomics; MicroRNA; Next-generational sequencing; Small RNA; TRNA-derived RNA
    DOI:  https://doi.org/10.1016/bs.mie.2025.10.014
  13. Mol Biol Rep. 2025 Dec 01. 53(1): 143
    Molecular mechanisms of mitochondrial function in neurodegenerative diseases.
    Heather M Wilkins, Simone Patergnani.
      Mitochondria regulate cellular homeostasis and function in both neurons and glial cells, but molecular mechanisms are not fully understood. Recent advances have expanded our understanding of how mitochondrial dynamics, quality control, bioenergetics, redox regulation, and proteostasis contribute to neurodegenerative processes. The collection "Neuroscience: Mitochondrial Function in Neurons and Glia" highlights the pivotal role of mitochondria in energy production, redox signaling, calcium buffering, and apoptosis. Articles within this collection discuss the effects of mitochondria in neurodegeneration. Together, these studies emphasize ongoing challenges in defining cell type specific mitochondrial responses and point to the need for improved strategies to target mitochondrial dysfunction in neurological disease.
    DOI:  https://doi.org/10.1007/s11033-025-11303-7
  14. Methods Enzymol. 2025 ;pii: S0076-6879(25)00400-8. [Epub ahead of print]725 301-337
    RNA structure analyzed by chemical probing and mutational profiling with sequencing (MaPseq).
    Eva Edelson, Nicholas M Forino, Michael D Stone.
      RNA chemical probing, coupled with mutational profiling read out by sequencing (MaPseq), is a powerful approach for investigating the folding properties of complex RNA molecules. For over half a century, structure probing methods have advanced our understanding of the diverse functions of cellular RNAs. Depending on the analytical goal, these methods can generate models that reflect either the ensemble average or, more recently, deconvoluted structures within heterogeneous folding populations. The integration of chemical probing with next-generation sequencing has transformed the field by greatly accelerating experimental workflows and enabling analysis of complex mixtures of RNA conformations. The latter capability is particularly important, as RNA structural heterogeneity is central to biological function. Here, we present detailed protocols for RNA chemical probing, library preparation, sequencing, and bioinformatic analysis. Using factor VIII pre-mRNA and human telomerase RNA as case studies, we illustrate and compare different experimental strategies. We hope this protocol will provide a flexible framework for researchers seeking to investigate the structural properties of any RNA.
    Keywords:  Deconvolution; Mutational profiling; Next-generation sequencing; RNA probing; RNA structure
    DOI:  https://doi.org/10.1016/bs.mie.2025.10.008
  15. Cell Syst. 2025 Dec 01. pii: S2405-4712(25)00283-2. [Epub ahead of print] 101450
    High-throughput mapping of modular regulatory domains in human RNA-binding proteins.
    Abby R Thurm, Yaara Finkel, Cecelia Andrews, Xiangmeng S Cai, Colette Benko, Lacramioara Bintu.
      RNA regulation is central to tuning gene expression and is controlled by thousands of RNA-binding proteins (RBPs). While many RBPs require their full sequence to function, some act through modular domains that recruit larger regulatory complexes. Mapping these RNA-regulatory effector domains is important for understanding RBP function and designing compact RNA regulators. We developed a high-throughput recruitment assay (HT-RNA-Recruit) to identify RNA-downregulatory effector domains within human RBPs. By recruiting over 30,000 protein tiles from 367 RBPs to a reporter mRNA, we discovered over 100 RNA-downregulatory effector domains in 86 RBPs. Certain domains-for instance, KRABs-suppress gene expression upon recruitment to both DNA and RNA. We engineered inducible synthetic RNA regulators based on NANOS that can downregulate endogenous RNAs or maintain reporter expression at defined intermediate levels, as predicted by mathematical modeling. This work serves as a resource for understanding RNA regulators and expands the repertoire of RNA control tools. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  RNA degradation; RNA-binding proteins; gene regulation; high-throughput screening; mammalian synthetic biology
    DOI:  https://doi.org/10.1016/j.cels.2025.101450
  16. Methods Enzymol. 2025 ;pii: S0076-6879(25)00407-0. [Epub ahead of print]725 283-300
    Detecting tRNA modifications with fluorescent-labeled DNA probes.
    Kaushani Misra, Mackenzie Dillenbeck, Cailyn P Leo, Dragony Fu.
      Transfer RNAs (tRNAs) undergo extensive post-transcriptional modifications on their nucleobases and sugar moieties. Here, we describe methods to detect tRNA modifications using DNA oligonucleotide probes labeled with fluorescent dyes as an alternative to radioactive labeling. Unlike radioactive probes, fluorescent probes maintain their sensitivity after long-term storage and do not require relabeling between experiments. The first method combines Northern blotting with differential probe hybridization based upon the presence or absence of a modification in the tRNA target. In addition to detecting modifications, this method provides insight into the effects of tRNA modification on tRNA processing and abundance. The second approach uses a viral reverse transcriptase to detect primer extension blocks caused by certain tRNA modifications during cDNA synthesis of the target tRNA. The primer extension approach provides a quantitative readout of modification status in tRNAs at nucleotide resolution. Overall, the fluorescence-based approaches described in this Chapter provide a convenient and safer alternative to radioactive probes while maintaining sensitivity and resolution.
    Keywords:  Fluorescent probe; Modification; Northern blot; Primer extension; TRNA
    DOI:  https://doi.org/10.1016/bs.mie.2025.10.015
  17. Methods Enzymol. 2025 ;pii: S0076-6879(25)00396-9. [Epub ahead of print]725 177-221
    Preparation of enzymes and libraries for MapID-tRNA-seq to identify chemical modifications in human tRNAs.
    Mitchel L Tepe, Weiqi Qiu, Khalil Mimouni, Huiqing Zhou.
      Chemical modifications are abundant in tRNAs and play essential roles in tRNA biology and human diseases. We recently reported MapID-tRNA-seq that allows identification of chemical modifications in human tRNAs such as m1A and m3C. MapID-tRNA-seq utilizes an evolved reverse transcriptase (RT-1306) that reads through and generates mutation signatures at m1A and m3C modifications, which allows robust detection and semi-quantification of m1A and m3C in human tRNAs. In addition, we developed MapIDs to consolidate the sequence redundancy within the human tRNA genome, with explicit annotations of genetic variance among highly similar tRNA genes. MapIDs help resolve a critical issue of false-positive discoveries of modifications caused by reads misalignment at genetic variance sites. In this chapter, we report detailed protocols for the in-house preparation and characterization of the two enzymes used in MapID-tRNA-seq library preparation (RT-1306 and the demethylase AlkB), tRNA-seq library preparation, and MapID-assisted sequencing analysis, to facilitate application and future development of the MapID-tRNA-seq method.
    Keywords:  AlkB; Chemical modifications; Human tRNAs; RT-1306; tRNA MapID; tRNA-seq
    DOI:  https://doi.org/10.1016/bs.mie.2025.10.004
  18. Res Sq. 2025 Nov 19. pii: rs.3.rs-7933297. [Epub ahead of print]
    Age-associated G-quadruplex accumulation and DDX5 loss shape chromatin landscapes in human astrocytes.
    Andrey Tsvetkov, Vijay Kumar M J, Rocio Diaz Escarcega, Ellery Wheeler, Nitin Tandon, David Monchaud, Christopher Hartl.
      Aging disrupts genome organization and transcriptional fidelity, but the role of non-canonical DNA structures in the aging process remains unclear. G-quadruplexes (G4s), stable guanine-rich DNA and RNA structures are established regulators of gene expression and genome integrity, yet their contribution to physiological aging is unknown. Using fluorescent imaging with primary human astrocytes derived from individuals spanning early to late adulthood (22-73 years) reveals an accumulation of G4s and a reduced nuclear expression of the G4-resolving helicase DDX5 in aging cells. To investigate how these changes relate to genome architecture, we performed ATAC-seq to profile chromatin accessibility and G4 CUT&Tag to profile the G4 landscape across all astrocyte cultures. Older cells exhibited global chromatin compaction and focal G4 enrichment, with gains occurring in both accessible and closed chromatin regions, indicating locus -specific and context-dependent regulation. To determine whether DDX5 modulates these features, we overexpressed DDX5 in young astrocytes and identified transcriptional targets involved in chromatin organization and genome maintenance. Acute DDX5 knockout caused focal G4 accumulation without widespread chromatin changes, indicating that DDX5 maintains and modulates G4 dynamics at defined genomic regions. Together, these findings reveal G4s as dynamic, age-sensitive features of the genome with potential roles in epigenetic regulation and establish DDX5 as a modulator of G4 dynamics and genome integrity during human brain aging.
    DOI:  https://doi.org/10.21203/rs.3.rs-7933297/v1
  19. Nucleic Acids Res. 2025 Dec 03. pii: gkaf1204. [Epub ahead of print]
    m6AConquer: a consistently quantified and orthogonally validated database for the N6-methyladenosine (m6A) epitranscriptome.
    Xichen Zhao, Haokai Ye, Dan He, Hongxi Liu, Tenglong Li, Daniel J Rigden, Zhen Wei.
      The proper placement of N6-methyladenosine (m6A) on mRNA is essential for normal cell function, and its disruption is linked to numerous human diseases. The rapid growth of m6A data from diverse sequencing technologies presents challenges for integrative analysis due to technique-specific biases and inconsistent processing. While existing databases provide valuable catalogs, their reliance on aggregating pre-processed results can propagate inconsistencies. To overcome these limitations, we present m6AConquer, a database founded on reproducible quantification. We systematically re-processed raw data from 10 distinct profiling methods, including high-resolution GLORI and eTAM-seq, quantifying methylation at millions of consensus sites across human and mouse. Our rigorous pipeline features uniform site-calling and false-positive calibration with in vitro transcribed (IVT) controls where available. By leveraging a reproducibility-based framework across technically orthogonal methods, we identified over 135300 orthogonally validated m6A sites in human (IDR < 0.05). Beyond this validated methylome, m6AConquer provides matched multi-omics data (gene expression, splicing, variants) and identifies m6A quantitative trait loci (m6A QTLs) to link RNA modification to genetic regulation and disease. Offering intuitive query tools, interactive visualizations, and downloadable, analysis-ready data matrices, m6AConquer provides a standardized resource for rigorous exploration of the roles of m6A in biology and medicine, freely accessible at https://rnamd.org/m6aconquer/.
    DOI:  https://doi.org/10.1093/nar/gkaf1204
  20. J Mol Biol. 2025 Nov 28. pii: S0022-2836(25)00636-9. [Epub ahead of print] 169570
    RNAsolo 2.0: multimodal database to study RNAs, their structural families and intermolecular interfaces.
    Bartosz Adamczyk, Pawel Boinski, Marta Szachniuk, Maciej Antczak.
      Understanding RNA structures - essential for uncovering their biological functions, interactions, and therapeutic potential - relies on both experimental techniques and computational approaches increasingly driven by artificial intelligence. The latter are transforming RNA structural research but depend on large, reliable datasets, which remain limited, particularly for RNA-protein and RNA-DNA complexes. To address this gap, we present RNAsolo 2.0 ( https://rnasolo.cs.put.poznan.pl/), an open-access database integrating cleaned, non-redundant RNA 3D structures with detailed information on their intermolecular interactions. Building on the original RNAsolo, which has attracted over 16,000 page views from ∼5,600 users, this release adds Rfam-based family classification, >2,500 precompiled benchmark sets, and multimodal representations encompassing sequence, secondary and tertiary structure, as well as torsion angle data. RNAsolo 2.0 enables searches for RNAs that interact with specific proteins, ligands, or ions, and provides an interactive view of their binding interfaces. The tool offers a robust, user-friendly platform for RNA structural biology and next-generation AI-driven modeling.
    Keywords:  RNA 3D structure; RNA interactions; Rfam families; benchmarking; non-redundant sets; representative structures; structural database
    DOI:  https://doi.org/10.1016/j.jmb.2025.169570
  21. J Mol Biol. 2025 Dec 03. pii: S0022-2836(25)00639-4. [Epub ahead of print] 169573
    Implications of Codon Usage, tRNA Gene Redundancy and tRNA Gene Clustering in Experimental Models of Mistranslation.
    D W McDonald, L Joos, M L Duennwald.
      The genetic code converts information transcribed in messenger-RNA (mRNA) into the amino acid sequences that build proteins. Transfer-RNAs (tRNAs) are the adaptors for this conversion from nucleic acids to proteins as they discriminate mRNA codons via anticodon-codon base pairing and recruit cognate amino acids to the ribosome for faithful protein biosynthesis. Although the genetic code is identical among many common model organisms and humans, there are profound differences in genomic codon usage, tRNA gene redundancy and genomic organization of tRNA genes that may change the accuracy and efficiency by which the genetic code is translated. Furthermore, these factors may influence how organisms tolerate tRNA variants that induce translation errors. Such tRNA variants are common in human populations, yet their contribution to human disease remains mostly unclear. Thus, tRNA variants have been studied in several model organisms and induce different rates of mistranslation and toxicity. To understand why mistranslating tRNA variants affect model organisms differently, we compare codon frequency, tRNA gene abundance and the genomic organization of tRNA genes in these commonly used model organisms (yeast, roundworms, fruit flies, mice and rats) and humans. We describe unique translation biases across model systems that influence tolerance of mistranslating tRNA variants, efficiency of protein biosynthesis, and co-translational protein quality control. Our review serves as a practical resource for researchers studying tRNA biology and the regulation of protein biosynthesis in these model organisms to guide experimental design and data interpretation.
    Keywords:  codon usage; tRNA; tRNA expression; tRNA genes; translation
    DOI:  https://doi.org/10.1016/j.jmb.2025.169573
  22. bioRxiv. 2025 Nov 17. pii: 2025.11.17.688896. [Epub ahead of print]
    Capturing Translation in Action with Protein Synthesis Profiling.
    Cesar Arcasi Matta, Zhi Qi Ten, Simpson Joseph.
      Translation is a central control point of gene expression, linking nucleotide sequences to functional proteins. Dysregulated translation contributes to diverse diseases, underscoring the need for methods that can directly reveal which transcripts are actively translated. Ribosome profiling, the current gold standard, provides nucleotide-resolution maps of ribosome occupancy but requires laborious purification and sacrifices information on mRNA isoforms and mRNA modifications by restricting analysis to short ribosome-protected fragments. Here, we introduce Protein Synthesis Profiling (PSP), a proximity-labeling strategy for transcriptome-wide identification of actively translated mRNAs without ribosome isolation. PSP exploits a fusion of the enzyme APEX2 with the elongation factor eEF2, which transiently associates with ribosomes during elongation, to catalyze selective tagging of mRNAs engaged in translation. Applied in Saccharomyces cerevisiae , PSP captures condition-specific translational programs, recapitulates known stress responses, and expands the detectable repertoire of regulated genes beyond existing methods. By preserving full-length transcript features, PSP is scalable, isoform-aware, and broadly adaptable, providing a versatile platform to dissect translational regulation in health and disease.
    DOI:  https://doi.org/10.1101/2025.11.17.688896
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