bims-mirnam 2026-09-06 papers

bims-mirnam

Biomed News

on Mitochondrial RNA metabolism

Issue of 2026–09–06
twelve papers selected by
Hana Antonicka, McGill University

Guanosine 10 methylation by TRMT11-TRMT112 optimizes human mitochondrial tRNAs for efficient translation.
RBProximity-CLIP: A Method for Profiling the Interactome of an RNA-Binding Protein at Subcellular Resolution.
Ionomycin suppresses cancer cell growth by disrupting mitochondrial transcription.
Dietary effects on cytosolic and mitochondrial tRNA abundance and modification patterns across mouse tissues.
SLIRP differentially modulates RNA binding of pathogenic LRPPRC variants.
RNA Labeling for Measuring RNA Endonucleolytic Cleavage.
Analysis of tRNA Aminoacylation by Northern Blotting and Multiplex Small RNA-Seq.
Decoding the Genetic and Phenotypic Spectrum of MT-TI Mitochondrial Diseases.
Analysis of Endogenously Expressed Double-Stranded RNAs in Human Cultured Cells.
Updated guide to RNA quantification by RNA sequencing and reverse transcription-qPCR.
RNA Structure and Its Function.
RNA Structure Analysis by Chemical Probing with DMS and CMCT.

Nucleic Acids Res. 2026 Aug 24. pii: gkag859. [Epub ahead of print]54(16):

Guanosine 10 methylation by TRMT11-TRMT112 optimizes human mitochondrial tRNAs for efficient translation.

Jonny Petrosyan, Mehmet Yilmaz, Carolin P M Scheitl, Nicolas Lemus-Diaz, Angela Boshnakovska, Laurianne L E Tay, Wanwan Hu, Olga Beine-Golovchuk, David Touboul, Ricarda Richter-Dennerlein, Claudia Höbartner, Marc Graille, Peter Rehling, Katherine E Bohnsack.

Modified nucleotides are essential determinants of RNA function, and identifying the enzymes that install them is fundamental to understanding their cellular roles. Here, we show that the human RNA methyltransferase TRMT11 and its cofactor TRMT112 are imported into mitochondria via N-terminal targeting signals. Using a recently developed N2-methylguanosine (m2G)-sensitive DNAzyme, we demonstrate that TRMT11 catalytic activity and interaction with TRMT112 are required for installation of m2G at position 10 in 13 mitochondrial (mt-)tRNAs. The crosslinking profile of TRMT11 on mt-tRNAs experimentally supports a model of the TRMT11-TRMT112-mt-tRNATrp complex in which the THUMP domain contacts the 3' end of the acceptor stem, and G10 is flipped into the S-adenosylmethionine binding pocket for methylation. Transcriptome-wide mapping reveals that TRMT11 interacts with most nuclear-encoded and mt-tRNAs, but only methylates a subset. In vitro reconstitution of TRMT11-TRM112-mediated methylation defines key structural requirements for m2G10 installation across different mt-tRNAs, and reveals how pathogenic mutations influence this modification. TRMT11-TRMT112 recognizes folded mt-tRNAs, and in the degenerate mt-tRNALys, m1A9 strongly enhances m2G10 methylation efficiency. Loss of m2G10 modifications alters the conformation of numerous mt-tRNAs, perturbs mitochondrial protein synthesis, and impairs oxidative phosphorylation, highlighting an essential role of this modification in maintaining mitochondrial function.

DOI: https://doi.org/10.1093/nar/gkag859
Methods Mol Biol. 2026 ;3047 153-176

RBProximity-CLIP: A Method for Profiling the Interactome of an RNA-Binding Protein at Subcellular Resolution.

Iwona Nowak, Hang T Huynh, Mahekdeep Kaur, Orli Korchev, Veronica Moskovicz, Vivian Lobo, Michelle Fong, Aishe A Sarshad, Daniel Benhalevy.

  RNA-binding proteins (RBPs) are central players in post-transcriptional gene regulation (PTGR), and their mode of action often depends on subcellular localization. Conventional crosslinking and immunoprecipitation (CLIP) methods map the RNA targets of RBPs with nucleotide resolution but lack spatial specificity, making it impossible to distinguish how one RBP can contribute to biological processes across distinct compartments simultaneously. Here, we present RBProximity-CLIP, an extension of Proximity-CLIP that integrates APEX2-mediated proximity labeling with UV crosslinking of 4-thiouridine-labeled RNAs, followed by sequential biotin- and RBP-specific immunoprecipitations, to capture RBP-RNA complexes with both spatial and protein specificity. RBProximity-CLIP generates nucleotide-resolution maps of RBP-RNA interactions and enables proteomic characterization of complete ribonucleoprotein (RNP) compositions. Using Y-box binding protein 1 (YBX1) as a model RBP, we provide a detailed protocol for RBProximity-CLIP.

Keywords:  APEX2; Crosslinking and immunoprecipitation (CLIP); Multifunctional RBPs; Multilocalized RBPs; Post-transcriptional gene regulation; Protein–RNA interactions; Proximity-biotinylation; RNA-binding proteins (RBPs); Spatial transcriptomics; Subcellular resolution; YBX1

DOI:  https://doi.org/10.1007/978-1-0716-5352-4_13
FEBS Open Bio. 2026 Sep 01.

Ionomycin suppresses cancer cell growth by disrupting mitochondrial transcription.

Lishen Wang, Mingqiang Deng, Dongmei He, Diqin Lai, Dongjun Liu, Dengyong Zhu, Zhenhua Zhang, Peng Tang, Chuanman Zhou, Menghui Yin, Xichen Bao.

  The Warburg effect has long suggested that oxidative phosphorylation (OXPHOS) is dispensable for tumor growth. However, recent studies have shown that the mitochondrial RNA polymerase inhibitors IMT1 and IMT1b, which impair OXPHOS, are potent anticancer agents. Here, we demonstrate that ionomycin, a selective ionophore known to modulate mitochondrial homeostasis, similarly inhibits mitochondrial gene expression across cancer cell lines. Specifically, gene expression and nascent RNA profiling revealed a global downregulation of mitochondrial gene transcription in Jurkat T, THP-1, HeLa, and NCI-H441 cells. Thus, we conclude that ionomycin suppressed mitochondrial gene transcription, impaired OXPHOS, and thereby inhibited cancer cell proliferation and growth, providing a novel insight into the function of ionomycin.

Keywords:  OXPHOS; cell proliferation; ionomycin; mitochondrial gene transcription

DOI:  https://doi.org/10.1002/2211-5463.70297
Genome Res. 2026 Sep 01. 36(9): 1902-1920

Dietary effects on cytosolic and mitochondrial tRNA abundance and modification patterns across mouse tissues.

Simeiyun Liu, Andrew D Holmes, Ruoxia Zhao, Cassandra Herbert, Patrick A Limbach, Todd M Lowe, Upasna Sharma.

Transfer RNAs (tRNAs) are central to protein synthesis and are increasingly recognized as dynamic regulators of gene expression whose abundance and chemical modifications are subject to precise biological control. Here, we systematically investigate how two distinct dietary interventions, low-protein and high-fat diets, reshape the tRNA landscape across multiple mouse tissues, using RNA mass spectrometry and ordered two-template relay sequencing (OTTR-seq) to comprehensively profile cytosolic and mitochondrial tRNAs at single-nucleotide resolution. We reveal pronounced tissue-specific biases in tRNA isodecoder expression, including the unexpected presence of full-length cytosolic tRNAs in mature sperm with a distinct isotype composition. In somatic tissues such as liver and heart, dietary conditions alter both tRNA abundance and key modifications known to regulate decoding efficiency, whereas in reproductive tissues diet primarily affects the abundance of select tRNAs with comparatively limited changes in modification profiles. We further demonstrate that mitochondrial tRNAs are subject to diet-responsive changes in both abundance and modification status and that even subtle differences in dietary fat composition are sufficient to alter tRNA modification signatures. Together, these findings establish the tRNA epitranscriptome as a sensitive and tissue-specific sensor of nutritional state and provide a resource for understanding how dietary cues interface with translational regulation in somatic and reproductive tissues.

DOI: https://doi.org/10.1101/gr.281159.125
NAR Mol Med. 2026 Jul;3(3): ugag040

SLIRP differentially modulates RNA binding of pathogenic LRPPRC variants.

Louise Lambert, Urška Rovšnik, Amandine Moretton, Katarina Johansson, Mãdãlina Sãcultanu, Mathilde Chéron, Adèle Porcan, Bertil Macao, Siet van den Wildenberg, Xuefeng Zhu, Maria Falkenberg, Géraldine Farge.

The LRPPRC/SLIRP complex is a key post-transcriptional regulator of mitochondrial gene expression, stabilizing mitochondrial mRNAs and promoting their polyadenylation and translation. Mutations in LRPPRC cause mitochondrial disorders, including Leigh syndrome French-Canadian type (LSFC), primarily affecting oxidative phosphorylation. Here, we examined the RNA-binding properties of wild-type LRPPRC and three pathogenic variants (A354V, K909del, and R1276_K1300del) using electrophoretic mobility shift assays, acoustic force spectroscopy, and AlphaFold 3 modeling. All three mutations reduced intrinsic RNA binding, with R1276_K1300del showing no detectable interaction in the absence of SLIRP. Remarkably, SLIRP restored RNA binding of this mutant to near wild-type levels, likely through conformational stabilization, as supported by single-molecule and structural analyses. These findings highlight SLIRP's critical role in modulating LRPPRC function and suggest that enhancing SLIRP activity represents a potential therapeutic strategy for LRPPRC-related mitochondrial disorders.

DOI: https://doi.org/10.1093/narmme/ugag040
Methods Mol Biol. 2026 ;3047 29-46

RNA Labeling for Measuring RNA Endonucleolytic Cleavage.

Minseon Kim, Jaeil Han.

  Although endonucleolytic cleavage of RNA is an essential process for maintaining cellular RNA homeostasis, its detection and quantification are often challenging. In this chapter, we present methods for detecting and quantifying RNA endonucleolytic cleavage events using Northern blot analysis. We describe two RNA labeling strategies for probe preparation: (1) a radiolabeled probe generated by 5' end-labeling with [γ-32P]ATP and polynucleotide kinase, and (2) a nonradioactive probe labeled with digoxigenin (DIG) or biotin for chemiluminescent detection. RNA samples are resolved by gel electrophoresis and transferred to a membrane. The labeled probes are then hybridized to the blotted RNA to detect specific cleavage products. These protocols enable both qualitative assessment of cleavage patterns and quantitative measurement of cleavage efficiency. We include step-by-step procedures from probe labeling to detection of target RNAs, as well as optional modifications to improve sensitivity and specificity. The methods are broadly applicable to analyzing RNA cleavage by various nucleases, providing a reliable combination of sensitivity for different experimental needs.

Keywords:  Endonucleolytic cleavage of RNA; Northern blot

DOI:  https://doi.org/10.1007/978-1-0716-5352-4_3
Methods Mol Biol. 2026 ;3051 27-42

Analysis of tRNA Aminoacylation by Northern Blotting and Multiplex Small RNA-Seq.

Jennifer Porat, Christopher P Watkins, Mark A Bayfield.

  Measuring RNA folding is particularly challenging for tRNAs, where extensive structure and posttranscriptional modifications complicate traditional structure probing and reverse-transcriptase-based assays. Since tRNA folding is a prerequisite for aminoacylation, quantifying tRNA aminoacylation is often used as an indirect, though functionally relevant, measure of tRNA folding. Periodate oxidation and beta-elimination, resulting in the selective loss of the 3' terminal nucleotide of deacylated tRNAs, is an amino-acid-agnostic chemistry that enables differentiation of aminoacylated and deacylated tRNAs when coupled to high-resolution northern blotting or RNA sequencing. Quantification of tRNA aminoacylation has been used to demonstrate RNA chaperone activity for RNA-binding proteins that promote tRNA folding to increase the fraction of aminoacylated tRNAs, underscoring the utility of this assay in interrogating RNA chaperone-assisted tRNA folding and function.

Keywords:  Aminoacylation; Northern blotting; Small RNA sequencing; tRNA

DOI:  https://doi.org/10.1007/978-1-0716-5372-2_2
Ann Hum Genet. 2026 Aug 30.

Decoding the Genetic and Phenotypic Spectrum of MT-TI Mitochondrial Diseases.

Enchi Yuan, Ning Zhang, Haoyu He, Tao Zhang, Enze Chen, Qidi Zhang, Dongmei Ji.

   BACKGROUND: Variants in the MT-TI gene, which encodes mitochondrial transfer RNA for isoleucine, have been associated with neuromuscular, cardiac, auditory, renal, and metabolic disorders, but their clinical interpretation remains difficult.
OBJECTIVE: To integrate clinical, familial, heteroplasmy, and functional evidence across the reported MT-TI variant spectrum and clarify its implications for variant interpretation and diagnosis.
METHODS: We conducted a narrative review of reported MT-TI variants, with detailed comparison of seven representative variants and synthesis of phenotypic, familial, tissue-specific heteroplasmy, and functional findings.
RESULTS: Evidence was derived mainly from case reports and small pedigrees. Heteroplasmy differed markedly among blood, skeletal muscle, and myocardium, indicating that blood may not represent variant loads in energy-demanding tissues. Reported values generally reflected the lowest observed levels in affected individuals or family-specific boundaries rather than validated pathogenic cutoffs. Functional findings support a staged mechanism involving disturbed transfer RNA processing, structure, stability, or aminoacylation, followed by impaired mitochondrial protein synthesis and respiratory-chain dysfunction. Integrated mechanistic support was limited to a few variants, including m.4295A>G; evidence for most variants remained incomplete or indirect.
CONCLUSION: Diagnosis requires tissue-informed heteroplasmy assessment integrated with phenotype, maternal family history, and functional evidence. Current treatment is supportive, and proposed reproductive and molecular strategies lack MT-TI-specific clinical-trial evidence.

Keywords:  Ile; RNA; heteroplasmy; mitochondrial; mitochondrial diseases; oxidative phosphorylation; transfer

DOI:  https://doi.org/10.1111/ahg.70056
Methods Mol Biol. 2026 ;3047 237-255

Analysis of Endogenously Expressed Double-Stranded RNAs in Human Cultured Cells.

Moonhyeon Jeon, Yoosik Kim.

  Long double-stranded RNAs (dsRNAs) play a critical role in modulating innate immune responses. In particular, endogenously generated dsRNAs are increasingly recognized as a biomarker and effector in immune-related diseases such as cancer, viral infection, and autoimmune disorders. However, accurate detection of endogenous dsRNAs is challenging due to their transient nature and low abundance. This chapter outlines three complementary methods using the dsRNA-specific J2 antibody: J2 dot blot for semiquantitative assessment of dsRNA levels; J2 immunocytochemistry for visualization of dsRNA subcellular localization; and J2 formaldehyde-mediated crosslinking immunoprecipitation coupled with RT-qPCR and/or high-throughput sequencing for quantification and/or identification of dsRNA species. Together, these methods enable sensitive and reproducible analysis of dynamic dsRNA landscapes under pathophysiological conditions, providing a versatile workflow for studying dsRNA signaling in mammalian cell biology and human disease models.

Keywords:  Dot blot; Double-stranded RNAs; Immunocytochemistry; Innate immunity; J2 formaldehyde-mediated crosslinking immunoprecipitation

DOI:  https://doi.org/10.1007/978-1-0716-5352-4_18
Mol Cells. 2026 Sep 04. pii: S1016-8478(26)00088-9. [Epub ahead of print] 100397

Updated guide to RNA quantification by RNA sequencing and reverse transcription-qPCR.

Dajeong Bong, Kyungjin C Lee, Gee-Yoon Lee, Seung-Jae V Lee.

  RNA sequencing (RNA-seq) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) are widely used for RNA quantification. RNA species with distinct structural and biogenetic features require specific computational and experimental approaches. Here, we provide an updated MiniResource that extends our previous guides to RNA-seq analysis and RT-qPCR-based RNA quantification. We introduce available tools and key considerations for analyzing circular RNAs, double-stranded RNAs, ribosomal RNAs, and transfer RNAs. This guide will help researchers choose appropriate methods for RNA species-specific quantification.

Keywords:  Bioinformatics; RNA quantification; RNA-seq; Reverse transcription-quantitative polymerase chain reaction

DOI:  https://doi.org/10.1016/j.mocell.2026.100397
Methods Mol Biol. 2026 ;3037 185-214

RNA Structure and Its Function.

Shunsuke Sumi, Fumiya Ito, Ruiqi Xu, Hirohide Saito, Risa Karakida Kawaguchi.

  RNA molecules were once regarded merely as intermediates in the transmission of genetic information from DNA, but are now emerging as key players in complex cellular regulation. Driven by the recent discovery of diverse functions of non-coding and coding RNAs, a deep understanding of RNA structure, and ultimately their interaction has become essential for both basic and applied research in RNA biology, including drug discovery efforts targeting RNA. However, its inherent structural instability and variability have made experimental analyses, such as X-ray crystallography and NMR, challenging and often inadequate. For this reason, continuous efforts have been devoted to advancing computational structure prediction. With growing attention to the role of RNA as a signaling molecule, a vast amount of functional RNA sequence and structure data has been accumulated so that it makes it feasible to apply machine learning (ML) and artificial intelligence (AI) technologies to further enhance the power of computational structure analysis in a data-driven manner. In this section, we introduce current gold-standard RNA structure prediction approaches and cutting-edge ML methods that can contribute to inferring RNA functions. We first describe the basics of RNA structure classification and diverse non-coding functional RNAs identified so far. Next, the example use cases of several RNA analysis tools, including computational prediction with the aid of structure probing methods, are provided. Finally, we introduce software protocols for generating RNA family sequences using a novel methodology called RfamGen, enabling readers to explore how cutting-edge ML can be leveraged for not only structure prediction but also the design of artificial RNA sequences to engineer novel RNA functions.

Keywords:  Cross-linking and immunoprecipitation (CLIP); RNA binding protein (RBP); RNA family; RNA secondary structure; RNA tertiary structure; Variational autoencoder

DOI:  https://doi.org/10.1007/978-1-0716-5284-8_11
Methods Mol Biol. 2026 ;3051 217-232

RNA Structure Analysis by Chemical Probing with DMS and CMCT.

José M Andrade, Ricardo F Dos Santos, João P Sousa, Alda F Q Silva, Cecília M Arraiano.

  RNA structure is important for understanding RNA function and stability within a cell. Chemical probing is a well-established and convenient method to evaluate the structure of an RNA. Several structure-sensitive chemicals can differentiate paired and unpaired nucleotides. This chapter specifically addresses the use of DMS and CMCT. Although exhibiting different affinities, the combination of these two chemical reagents enables screening of all four nucleobases. DMS and CMCT are only reactive with exposed unpaired nucleotides. We have used this method to analyze the effect of the RNA chaperone Hfq on the conformation of the 16S rRNA. The strategy here described may be applied for the study of many other RNA-binding proteins and RNAs.

Keywords:  CMCT; Chemical probing; DMS; Primer extension; RNA fold; RNA secondary structure; rRNA

DOI:  https://doi.org/10.1007/978-1-0716-5372-2_13