bims-mirnam 2026-01-25 papers

bims-mirnam

Biomed News

on Mitochondrial RNA metabolism

Issue of 2026–01–25
nine papers selected by
Hana Antonicka, McGill University

ADAM-tRNA-seq: an optimized approach for demultiplexing and enhanced hierarchal mapping in direct tRNA sequencing.
Integrative analysis of nanopore direct RNA sequencing data reveals a global impact of pseudouridylation on m6A and m5C modifications.
Human mitochondrial helicase Twinkle has RNA binding, annealing, and strand-exchange activities.
Recent Advances in Chemical Probing Strategies for RNA Structure Determination in Vivo.
Uridine analogs prevent stress granule formation, not by blocking PKR recognition, but by inhibiting the synthesis of T7 RNA Polymerase byproducts.
Lometrexol targets MRPL2 to suppress NSCLC via dual regulation of mitochondrial ribosomal activity and nuclear PDCD11/ Ca2+ signaling.
Aminoacyl-tRNA-dependent enzymes in natural product biosynthesis: structure-function insights.
Synthesis of long and functionally active RNAs facilitated by acetal levulinic ester chemistry.
The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis.

Nucleic Acids Res. 2026 Jan 22. pii: gkag022. [Epub ahead of print]54(3):

ADAM-tRNA-seq: an optimized approach for demultiplexing and enhanced hierarchal mapping in direct tRNA sequencing.

Rodrigo Alarcon, Daniel Köster, Stine Behrmann, Zoya Ignatova.

Transfer RNAs (tRNAs) play an essential role in protein synthesis and cellular homeostasis, and their dysregulation is associated with various human pathologies. Recent advances in direct RNA sequencing by the Nanopore platform have enabled simultaneous profiling of tRNA abundance, modifications, and aminoacylation status. However, the high sequence similarity among tRNAs and the lack of robust demultiplexing strategies reduce the accuracy and limit the scalability of current approaches. Here, we developed ADAM-tRNA-seq, a framework that addresses two key limitations of the Nanopore-based direct tRNA sequencing. First, we develop an RNA-based barcode demultiplexing method, that employs a barcode embedded in the sequencing adapter, recognized by the Dorado basecaller. Second, we designed a hierarchy-based mapping strategy that mitigates read loss due to multimapping by classifying reads at the isodecoder, isoacceptor, or isotype levels, thereby enhancing quantification accuracy. We validated ADAM-tRNA-seq using both synthetic tRNAs and a complex human tRNA pool, and systematically optimized it to achieve up to 99% classification precision. Together, these developments enable more accurate, scalable, and comprehensive characterization of tRNA pools across diverse sample types.

DOI: https://doi.org/10.1093/nar/gkag022
NPJ Precis Oncol. 2026 Jan 22.

Integrative analysis of nanopore direct RNA sequencing data reveals a global impact of pseudouridylation on m6A and m5C modifications.

Mohit Bansal, Anamika Gupta, Jane Ding, Anirban Kundu, Katherine Marlow, Andrew Gibson, Zhangli Su, Sunil Sudarshan, Han-Fei Ding.

RNA modifications play a crucial role in regulating cellular functions. Among the most abundant modifications in the human transcriptome are pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C). However, the interplay between these modifications remains poorly understood due to limited integrative studies. To address the gap, we utilized nanopore direct RNA sequencing to quantify the stoichiometry of Ψ, m6A, and m5C after depleting the pseudouridine synthases PUS7 or DKC1. We used the custom tool NanoPsiPy to quantify pseudouridine by analyzing differential U-to-C base-calling errors in nanopore sequencing data. For m6A and m5C, we applied the established tool CHEUI to conduct stoichiometry differential analysis. Our investigation identified both known and novel pseudouridylation sites in tRNA, rRNA, and mRNA targeted by PUS7 or DKC1. Integrative analysis revealed that depletion of PUS7 or DKC1 reduced pseudouridylation levels while simultaneously increasing global m6A and m5C levels, with functional implications for mRNA translation regulation. These findings suggest that pseudouridylation may play an active role in repressing m6A and m5C modifications. This study demonstrates the analytical power of nanopore direct RNA sequencing for investigating co-regulation of RNA modifications.

DOI: https://doi.org/10.1038/s41698-026-01278-4
Nucleic Acids Res. 2026 Jan 14. pii: gkag008. [Epub ahead of print]54(2):

Human mitochondrial helicase Twinkle has RNA binding, annealing, and strand-exchange activities.

Anupam Singh, Laura C Johnson, Noe Baruch-Torres, Y Whitney Yin, Smita S Patel.

Twinkle is the sole replicative helicase in human mitochondria, essential for mitochondrial DNA replication. Beyond its canonical unwinding activity, Twinkle has non-canonical activities, including DNA annealing and strand-exchange. Here, we show that these non-canonical activities extend to RNA. Twinkle binds RNA and catalyzes RNA:DNA hybrid formation through annealing, strand-exchange, and toehold-mediated strand displacement. Twinkle can unwind RNA:DNA forks when loaded onto the DNA tail but not the RNA tail. Although the physiological role of these RNA-related activities remains unclear, we show that Twinkle can strand-exchange an RNA downstream of a stalled replication fork to restart replication. The annealing/strand-exchange activity can be involved in DNA replication initiation and repair, but RNA:DNA hybrids can compromise genome integrity, emphasizing the need to balance unwinding and annealing activities. Interestingly, mitochondrial SSB inhibits the RNA:DNA annealing activity of Twinkle, thus regulating the non-canonical functions of Twinkle. A disease-associated W315L variant, which is defective in DNA replication, retains annealing and strand-exchange functions with both RNA and DNA, resulting in an imbalance between replication and annealing functions that may underlie its pathogenicity. Our findings of Twinkle's RNA-binding and strand-exchange activities may have a connection to its localization within mitochondrial RNA granules.

DOI: https://doi.org/10.1093/nar/gkag008
Chemistry. 2026 Jan 20. e03291

Recent Advances in Chemical Probing Strategies for RNA Structure Determination in Vivo.

Maryana Yarshova, Jieyu Zhao, Chun Kit Kwok.

  The intricate structures of RNA molecules facilitate their diverse cellular functions. These structures are shaped by the cellular environment, a context that in silico and in vitro methods typically cannot reconstitute, making it more difficult to study the structure of RNA in cells. In response to these challenges, RNA structure probing using cell-permeable chemicals has emerged as an effective method to capture the RNA structural landscape in its native environment. The integration of these probes with advanced adduct detection techniques, particularly second- and third-generation sequencing, has propelled the field forward, facilitating a deeper understanding of the RNA structurome within its precise functional context, including the examination of RNA structure at the single-molecule and single-cell levels, within specific subcellular compartments, and across various stages of RNA biogenesis and regulation. This Review summarizes the significant advances in the field of RNA structure probing, focusing on the development of novel structural probes, strategies for RNA structure reconstruction, innovative methodologies that offer extended applicability to address unique biological questions, and concludes with an outlook on future directions in the field.

Keywords:  RNA structure; RNA‐RNA interactions; chemical probing; nucleic acids; structure determination

DOI:  https://doi.org/10.1002/chem.202503291
RNA. 2026 Jan 22. pii: rna.080481.125. [Epub ahead of print]

Uridine analogs prevent stress granule formation, not by blocking PKR recognition, but by inhibiting the synthesis of T7 RNA Polymerase byproducts.

Sean J Ihn, Emily Jiang, Nevraj S KeJiou, Yifan E Wang, Laura Farlam-Williams, Alexander F Palazzo, Hyun O Lee.

  mRNA-based therapeutics are commonly produced through T7 RNA Polymerase-mediated in vitro transcription. Introducing these exogenous RNAs into human cells activates an RNA sensor Protein Kinase R (PKR), which suppresses translation initiation and reduces their therapeutic effectiveness. Incorporating uridine analogs into these transcripts prevents PKR activation and translation shutdown, but the underlying mechanism remains unclear. Here, we demonstrate that treating T7 RNA Polymerase-produced transcripts with RNase III, which selectively degrades double-stranded RNA (dsRNA), blocks PKR activation and downstream translation-inhibition events, including eIF2α phosphorylation and stress granule formation in human cells. Interestingly, dsRNAs generated with uridine analogs robustly induce eIF2α phosphorylation and stress granules to the same extent as dsRNA containing uridine. These findings indicate that uridine analogs do not prevent PKR from detecting dsRNA. Instead, we show that uridine analogs decrease the production of T7 RNA Polymerase byproducts, including antisense RNA and dsRNA, which activate PKR and downstream stress responses. Finally, we demonstrate that higher amounts of exogenous RNA, lacking T7 RNA Polymerase byproducts, can induce stress granules independently of PKR and phospho-eIF2α, but dependent on stress granule scaffold proteins G3BP1 and G3BP2. Together, our findings show that uridine analogs mitigate PKR signaling not by blocking mRNA-PKR interactions, but by minimizing dsRNA byproducts from T7 Polymerase transcription. Furthermore, stress granule formation in response to high levels of exogenous RNA can occur through a mechanism that does not depend on PKR but relies on G3BP1 and G3BP2. These insights clarify the role of uridine analogs in PKR activation and may inform future therapeutic RNA design.

Keywords:  PKR; T7 RNA Polymerase; biomolecular condensates; stress granules; uridine analogs

DOI:  https://doi.org/10.1261/rna.080481.125
Bioorg Chem. 2026 Jan 14. pii: S0045-2068(26)00045-3. [Epub ahead of print]170 109509

Lometrexol targets MRPL2 to suppress NSCLC via dual regulation of mitochondrial ribosomal activity and nuclear PDCD11/ Ca2+ signaling.

Xuehang Jin, Lvjun Zhang, Kailing Pan, Chiqing Ying, Dan Zhu.

  Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality, necessitating novel therapeutic targets. Recent studies emphasize the significance of mitochondrial ribosomal proteins (MRPs) in cancer, showing altered expression in different cancers and their potential as prognostic biomarkers and therapeutic targets. We identified that MRPL2 was upregulated in NSCLC and decreased expression of MRPL2 diminished tumorigenicity of NSCLC. Mechanistically, USP21 directly interacts with and stabilized MRPL2 via deubiquitination process through its USP domain. Moreover, The upregulation of MRPL2 influenced mitochondrial function via its localization to mitochondria, facilitating its role in ribosomal activity. Additionally, our findings indicated that MRPL2 can also enhance the intracellular calcium signaling pathway through its nuclear localization and interaction with PDCD11, thereby playing a regulatory role in the progression of NSCLC. Furthermore, we identified lometrexol as a potential MRPL2 inhibitor that can hinder NSCLC development. Taken together, we identify a MRPL2-regulated ribosomal and non-ribosomal mechanism that involves the USP21/MRPL2/PDCD11/Ca2+ axis in NSCLC tumorigenesis, which could serve as a potential target for the treatment of NSCLC.

Keywords:  MRPL2; Non-small-cell lung cancer; USP21; Ubiquitylation

DOI:  https://doi.org/10.1016/j.bioorg.2026.109509
J Antibiot (Tokyo). 2026 Jan 22.

Aminoacyl-tRNA-dependent enzymes in natural product biosynthesis: structure-function insights.

Yu Zheng, Yanhui Zhao, Shunji Takahashi.

Aminoacyl-tRNAs, charged by aminoacyl-tRNA synthetases with cognate amino acids, are essential for protein synthesis in primary metabolism. Beyond this canonical role, increasing evidence highlights their involvement in natural product biosynthesis. In this review, we first describe the biosynthesis of the aminoacyl nucleoside sulfamate ascamycin from Streptomyces sp. 80H647, highlighting the discovery of the alanyl-tRNA synthetase-like enzyme AcmF through an AI-driven "Forecasting Biosynthesis" approach. Leveraging recent advances in AlphaFold 3, we constructed complex models of a broadened repertoire of aminoacyl-tRNA-dependent enzymes to provide preliminary structure-function insights. These include the isoleucyl-tRNA synthetase-like enzyme SbzA, Gcn5-related N-acetyltransferase-fold transferases, cyclodipeptide synthase family enzymes, and lantibiotic dehydratase-like peptide aminoacyl-tRNA ligases. The catalytic mechanisms of these aminoacyl-tRNA-dependent enzymes are summarized in detail in this review.

DOI: https://doi.org/10.1038/s41429-025-00893-w
Nucleic Acids Res. 2026 Jan 14. pii: gkaf1525. [Epub ahead of print]54(2):

Synthesis of long and functionally active RNAs facilitated by acetal levulinic ester chemistry.

Zidi Lyu, Adam Katolik, Iqra Yaseen, Adrain A Pater, Francis Robert, Sidong Huang, Keith T Gagnon, Peter J Unrau, Masad J Damha.

Recent advances in RNA-based therapeutics have created a demand for synthetic RNAs that are 100 nucleotides (nts) or longer. In this study, we present the use of 2'-acetal levulinic ester (2'-ALE) phosphoramidites for the synthesis of long RNAs that are at least 215 nts in length. We have developed protocols for rapid (2-4 min) and efficient coupling (>99%) of 2'-ALE monomers and established a rapid, on-column deprotection of RNA strands requiring short alkylamine treatments at room temperature. The results of these studies enabled the successful syntheses of sgRNAs (99 nt), sgRNA tagged with fluorogenic Mango II and Broccoli aptamers (130-170 nt), and 5'-capped minimal mRNAs (200-215 nt), each exhibiting robust functional activity in both cell-free and cellular systems. We also found that the incorporation of 2'-O-methyl-adenosine in the poly(A) tail of synthetic mRNAs markedly enhanced protein expression, highlighting the ALE platform's compatibility for systematic exploration of RNA chemical diversity. Collectively, these results establish 2'-ALE chemistry as a promising platform for the synthesis of long and functionally active RNAs.

DOI: https://doi.org/10.1093/nar/gkaf1525
Mol Cell. 2026 Jan 19. pii: S1097-2765(25)01021-4. [Epub ahead of print]

The ribosome synchronizes folding and assembly to promote oligomeric protein biogenesis.

Alžběta Roeselová, Santosh Shivakumaraswamy, Gabija Jurkeviciute, Jessica Zhiyun He, Josef Auburger, Jaro L Schmitt, Günter Kramer, Bernd Bukau, Radoslav I Enchev, David Balchin.

  Natural proteins often form intricate multidomain, oligomeric architectures. This presents a prima facie challenge to cellular homeostasis, as topologically complex proteins seldom refold efficiently in vitro. Here, we show that the efficient folding and assembly of the five-domain homotetramer β-galactosidase is obligatorily coupled to its synthesis on the ribosome, and we define the underlying mechanisms. During refolding from a denaturant, maturation of the catalytic domain is frustrated. Assembly outpaces monomer folding, and non-native oligomers accumulate. Efficient de novo folding is characterized by segmental domain folding, shaped by the binding of a nascent amphipathic helix to a cryptic pocket on uL23 on the ribosome surface. Homomer assembly also initiates cotranslationally via recruitment of a full-length subunit to the nascent polypeptide, and the failure to do so results in misassembly. Our results reveal how the ribosome can dictate the timing of folding and assembly to enable efficient biogenesis of a topologically complex protein.

Keywords:  cotranslational folding; protein assembly; protein folding; ribosome

DOI:  https://doi.org/10.1016/j.molcel.2025.12.022