RNA 2'-phosphotransferase Tpt1 catalyzes the removal of an internal RNA 2'-PO4 via a two-step mechanism in which: (i) the 2'-PO4 attacks NAD+ C1'' to form an RNA-2'-phospho-(ADP-ribose) intermediate and nicotinamide; and (ii) transesterification of the ADP-ribose O2'' to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1'',2''-cyclic phosphate. Although Tpt1 enzymes are prevalent in bacteria, archaea, and eukarya, Tpt1 is uniquely essential in fungi, where it erases the 2'-PO4 mark installed by tRNA ligases during tRNA splicing. A Tpt1 "poison" that arrests the reaction after step 1 could aid in the design of antifungals that interdict tRNA biogenesis. Whereas our previous studies of 2''OMeNAD+ established its efficacy in poisoning Runella slithyformis Tpt1 (RslTpt1, a bacterial enzyme), its application was limited insofar as Tpt1 enzymes from fungal pathogens were unable to utilize 2''OMeNAD+ for step 1 catalysis. Here we report the chemical synthesis of 2''F-NAD+, which proved to be a broad-spectrum poison against Tpt1 enzymes from five different taxa, including human pathogens Candida albicans and Candida auris. The resulting RNA-2'-phospho-(2''F-ADP-ribose) dead-end product remains stably trapped in a complex with Tpt1. A modified analog, 2''F-NAD-DTB, containing desthiobiotin (DTB) linked to adenine C2, provides an improved affinity-tag probe of RNA 2'-phosphate modification.
Adenosine-to-inosine (A-to-I) RNA editing by ADAR1 is a key post-transcriptional modification, and mutations in ADAR1 lead to Aicardi-Goutières syndrome (AGS), an autoimmune disorder. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Using a combination of biochemical approaches, inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, molecular dynamics simulations, and a cell-permeable inositol hexakisphosphate (IP6) prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. Furthermore, we identify the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly coordinates a Zn²⁺-ion. Finally, we show that the AGS-associated ADAR1 mutation N907S impairs RNA editing activity, most likely by altering the hydrogen-bond interaction network linking IP6 to the ADAR1 catalytic center. Together, these findings identify IP6 as an essential cofactor and regulator of ADAR1 activity and highlight cofactor availability and interaction networks as strategies for therapeutically modulating RNA editing.
RNA 5-methylcytidine (m⁵C) modification plays an essential role in regulating RNA metabolism and functions in cellular processes. Tools achieve temporal and transcript-specific m⁵C editing for functional studies are still limited. Furthermore, methods enabling m⁵C editing, triggered by specific cellular signals, can contribute to the understanding of m⁵C functions under specific physiological conditions but still lacking. Here, we present a temporally and conditionally controlled m⁵C writing platform engineered through integrating abscisic acid (ABA)-mediated chemically induced proximity with split-dCas13b-NSUN2/NSUN6 technology. This system enables the writing of m⁵C by reconstituting the split dCas13b-based m⁵C editing complex at the guide RNA (gRNA)-targeted RNA transcript sites under the control of the inducer ABA. The deposition of m⁵C is inducible, reversible and selective. The deposited m⁵C is biologically active and influence the stability of endogenous mRNA transcripts. Moreover, by incorporating ABA prodrugs, the m⁵C writing can be triggered by signals associated with distinct physiological or disease conditions (e.g., tumor microenvironment and senescence). This conditional m⁵C editing strategy provides a new programmable tool for studying m⁵C biology in context dependent manners and expands the repertoire of RNA modification editing technologies.
Self-amplifying mRNA (SAM) is a potential platform for protein replacement, as it enables recipients to transiently produce a therapeutic protein for 4-8 weeks. To obtain external control over the level and duration of protein expression, synthetic SAM constructs that switch OFF in the presence of a small molecule such as trimethoprim (TMP) have been developed in the past. Here, we increase the ON-state protein expression of such a TMP-responsive SAM by fusing the effector protein L7Ae to a TMP-responsive destabilizing domain (DD) on both terminals. We also lower the OFF-state by adding a duplicate of the SAM 3'UTR downstream of the DD-L7Ae subgenomic ORF. We demonstrate that fusing two DDs to L7Ae has a beneficial effect on the response rate and level of protein production after removal or addition of TMP. We further demonstrate that overexpression of DD-L7Ae results in microscopic abnormalities. These insights are implemented in a mechanistic model, and further improvements of the platform are highlighted.
Small regulatory RNAs (sRNAs) can specifically bind to their target mRNAs to inhibit their expression, either by promoting degradation or by blocking translation. However, the molecular determinants that select which repression mode an sRNA employs remain poorly understood. To investigate this, we used the model target mRNA hdeD, which is repressed by two sRNAs, CyaR and RprA, both of which use distinct modes of repression. Whereas CyaR promotes the target mRNA hdeD degradation, the sRNA RprA specifically blocks hdeD translation initiation, leaving hdeD mRNA intact. We found that mutating two nucleotides in the seed pairing region of RprA with hdeD is sufficient to switch its mode of repression from translation inhibition to degradation by recruiting RNase E. We investigated this further by using various RNase E deletion mutants of the C-terminal domain (CTD). Among these was a RNase E mutant deleted to contain a minimal C-terminal scaffold (rne 701), a form which typically fails to promote sRNA-induced mRNA degradation. Our data indicated that even with a minimal scaffold, the mutated RprA sRNA still promoted hdeD mRNA degradation, whereas the native sRNA CyaR could not. Our data suggested that the mutated RprA sRNA relies primarily on the arginine-rich RNA-binding (ARRBD) domain of RNase E to induce target mRNA hdeD degradation. This suggests that the mutated RprA sRNA requires fewer components of the CTD of RNase E and RNA degradosome to induce target mRNA degradation. .
Translation reinitiation (REI) is one of the most important gene-specific regulatory mechanisms by which eukaryotic cells influence expression of main translons, for example during highly conserved integrated stress response (ISR). In S. cerevisiae, expression of the key stress response gene, GCN4, is controlled by an intricate interplay among four short upstream translons (uTranslons, formerly uORFs), resulting in high or low levels of REI at GCN4 depending on the growth conditions. Under nutrient rich conditions, GCN4 expression is repressed, but upon amino acid starvation, it is derepressed, despite of a general translational shut down. Capitalizing on our screening reporter system, we identified three new factors influencing efficiency of REI after translation of GCN4 uTranslons: Rai1p (an RNA quality control and processing factor), and Ssz1p and Zuo1p (members of the Ribosome Associated Complex [RAC]). Importantly, we showed that depletion of these factors deregulated derepression of Gcn4p synthesis under starvation. Furthermore, we found that similar to RAC, Rai1p associates with 40S subunits and actively translating ribosomes. We also explored interactomes of these proteins. Collectively, we present three previously unknown factors that co-regulate stress response to amino acid starvation in the budding yeast by unique mechanisms.
Plasmodium falciparum, the primary cause of human malaria, relies on tightly coordinated gene-expression programs to adapt to host-derived stress despite possessing a limited repertoire of canonical transcription factors. Antisense long noncoding RNAs have emerged as important regulators of parasite biology, including virulence gene regulation and sexual commitment; however, their prevalence, origin, and broader functional significance remain poorly understood. Here, we demonstrate that antisense transcription is a widespread, reproducible, and regulated feature of the P. falciparum transcriptome rather than a byproduct of pervasive euchromatic transcription. Environmental stress, including febrile temperature exposure and artemisinin treatment, extensively remodelled antisense transcription, particularly at loci associated with virulence and stress adaptation, promoting widespread sense-antisense RNA duplex formation. Functional analyses of two stress-responsive chromatin regulators, PfGCN5 and PfHDAC1, identified as antisense-expressing loci, revealed that increased antisense expression elevated steady-state mRNA abundance while reducing cognate protein levels. Mechanistically, sense-antisense RNA duplex formation stabilized complementary transcripts but suppressed translation. Integrated transcriptomic, RNA-RNA duplex profiling, ribosome sequencing, and proteomic analyses further showed that duplex-enriched transcripts exhibit reduced ribosome occupancy and reduced protein abundance, accompanied by localized antisense enrichment near transcription end sites and altered ribosome distribution consistent with impaired translational engagement. Collectively, our findings identify an antisense RNA-ribosome regulatory axis that couples RNA duplex formation to adaptive translational control, providing a previously unrecognized mechanism underlying stress adaptation and post-transcriptional gene regulation in P. falciparum.
RNA polymerase (RNAP) is the enzyme that produces RNA for an increasingly large variety of uses. The need for efficient industrial production of high quantities of mRNA was exemplified during the SARS-CoV-2 pandemic. While the RNAP from the T7 bacteriophage has been the gold standard for some time, it has certain limitations impeding efficient manufacturing of all RNA types, including production of long transcript self-amplifying RNA (saRNA). Here we present an alternative to T7 RNAP, identified from the Klebsiella bacteriophage 32 (KP32). We show KP32 RNAP to efficiently produce RNA of varying lengths, with a wide temperature, pH and salt tolerance range. We show KP32 RNAP to be capable of efficient production of an 11,551 nt self-amplifying RNA with its critical native initiating dinucleotide, AU. This represented a 9.3-fold improvement in production levels, relative to T7 RNAP.
Antiviral defence mechanisms are typically activated upon sensing virus-derived nucleic acids. During replication, viruses generate double-stranded RNA (dsRNA) intermediates that the innate immune system can sense, triggering several defence pathways. Conversely, mammalian cells avoid accumulating their own endogenous dsRNA to prevent activating these defence mechanisms. However, we demonstrate that mammalian embryonic stem cells (ESCs) accumulate endogenous dsRNA without activating these responses, as they lack all classical dsRNA-mediated antiviral pathways. To identify these endogenous dsRNAs, we have developed a two-step purification method that includes an antibody-based immunoprecipitation followed by RNase I treatment to remove single-stranded RNA regions that do not contribute to dsRNA formation. RNase I treatment results in an enrichment of overlapping sense/antisense transcripts containing A-to-I editing sites, suggesting successful purification of cellular dsRNA. Our refined protocol reveals that transposable elements (TEs), including evolutionary young elements from the LINE and LTR classes, are the predominant source of dsRNA in ESCs. This approach will be useful for investigating the role of dsRNA in disease settings, such as autoimmunity or cancer, where endogenous dsRNA accumulation has also been observed.
The nonsense-mediated decay pathway (NMD) is an RNA quality control mechanism that regulates the stability of target RNAs. We previously identified the ER-localized SEC13 protein as a novel NMD factor in C. elegans and in HeLa cells; raising the possibility that it could be involved in regulating the stability of mRNAs translated at the ER. SEC13 is a component of several cellular complexes, including the COPII vesicle coat, the nuclear pore complex (NPC) and the nutrient sensing GATOR2 complex. Here, we show that SEC13 interacts with core NMD factors and using a newly developed dual-color fluorescent NMD sensor in U2OS cells, we assessed SEC13 NMD activity, at a single-cell level. Transcriptomic profiling revealed that unlike the previously described ER-NMD factor, NBAS, SEC13 co-regulates the stability of substrates translated both in the cytoplasm and at the ER. We also show that SEC13 function in NMD is largely independent of its function in other cellular complexes. Altogether, these results show that SEC13 is a bona fide NMD factor in mammalian cells. Finally, we utilized an ER stress-activated indicator (ERAI) in U2OS cells to demonstrate that SEC13, together with canonical NMD factors, has a role in the regulation of the unfolded protein response (UPR) at the ER. Thus, the moonlighting functions of SEC13 include a role in NMD pathway and the regulation of ER stress.
Regulation of gene silencing in large chromosomal regions is crucial for development and disease progression. One example of massive gene silencing is X chromosome inactivation (XCI), a process essential for gene dosage compensation. During XCI, most genes in the chromosome are inactivated following the transcription of long noncoding RNA XIST. Recent experiments showed that the spread of silencing is restricted in space but the mechanism of controlling the spread remains unclear. Here, we develop a continuum-based, reaction-diffusion model that elucidates chromosomal inactivation through a regulatory network for XIST-mediated gene silencing. We find that the spread of XIST can be tuned by known negative feedback loops regulating its synthesis and degradation, and that the spread of gene silencing is controlled by a wave-pinning mechanism driven by global regulation of silencing complex together with local epigenetic regulators. We use a 3D chromosome structure inferred from experimental data and our modeling framework to show the spatiotemporal regulation for spread of gene silencing. Our method enables the investigation for the inactivation dynamics of large regions of chromosomes with varying degrees of the spread of gene silencing. Our model provides mechanistic insights that quantitatively relate gene regulatory networks to tunability and stability of chromosomal inactivation.
Structural analysis of nucleic acids lags behind that of proteins, partly because most fundamental structural analysis techniques have been primarily developed for proteins. The molecular replacement (MR) method, commonly used for phase determination in protein crystallography, encounters unique challenges when applied to nucleic acids. Nucleic acids can have different three-dimensional structures even with the same sequence, which often renders database entries or predicted models unsuitable as search models for MR. To overcome the limitation, we developed a novel strategy termed 4MRNA, which stands for Massive Multi-type Model Molecular Replacement for Nucleic Acids. This method introduces a new principle for MR, which is the systematic creation of diverse search models through parameter adjustment. By identifying the parameters that critically influence MR and generating models based on their statistical analysis, 4MRNA can provide search models that closely approximate target structures and thereby improve the success rate of MR. Its effectiveness was validated across comprehensive test cases including canonical duplexes, duplexes with bulges and internal loops, the more complex structure of transfer RNA, and a previously unreported DNA structure. 4MRNA is anticipated to become an indispensable tool for nucleic acid structure determination, profoundly advancing fundamental research and extending its impact to wide-ranging applications including structure-based drug design and nucleic acid nanotechnology.
Conventional RNA extraction with acid guanidinium thiocyanate-phenol-chloroform (AGPC) reagents incompletely recovers a subset of transcripts, termed semi-extractable RNAs (seRNAs). This underrepresented RNA population includes architectural RNAs (arcRNAs), which scaffold membraneless organelles, as well as stress-induced downstream-of-gene readthrough transcripts (DoGs). Although our previously developed AGPC extraction method incorporating physical disruption, such as heating or needle shearing, enhances seRNA recovery, it also co-extracts abundant, readily extractable RNAs, resulting in mixed populations that hinder precise characterization. Here, we present SERIPH (semi-extractable RNA isolation from the interphase), a simple two-step protocol that selectively enriches seRNAs by separating them from readily extractable RNA species. In SERIPH, the aqueous phase containing extractable RNAs is first removed following conventional AGPC extraction. The remaining interphase is subsequently subjected to the extraction procedure incorporating physical disruption, yielding a fraction selectively enriched for semi-extractable transcripts. Transcriptome-wide RNA sequencing demonstrates that SERIPH robustly enriches established seRNAs, including arcRNAs and stress-induced DoGs, while efficiently depleting abundant mRNAs. Compared with the previous extraction method alone, SERIPH increases both the number and genomic extent of detectable DoG loci and enhances sensitivity for weakly semi-extractable transcripts that fall below conventional threshold-based classifications. SERIPH further reveals enrichment of intron-retaining transcript subsets within the semi-extractable RNA fraction. By expanding the detectable seRNA repertoire and enabling selective enrichment, SERIPH establishes a practical framework for focused, high-resolution analysis of seRNA populations. This methodological advance facilitates comprehensive characterization of seRNAs and supports studies of RNA processing, transcriptional regulation, and RNA-mediated nuclear organization in stress responses and disease.
Pseudouridine is an abundant posttranscriptional modification important to RNA structure and function. The isomerization of uridine (U) to pseudouridine (Ψ) is catalyzed by members of the pseudouridine synthase (Pus) family throughout all domains of life. All Pus enzymes modify noncoding RNAs, and a subset also pseudouridylate protein-coding messenger RNAs (mRNAs). Although the precise role of Ψ in mRNAs remains to be established, emerging evidence suggests that Ψ might contribute to the posttranscriptional control of gene expression. However, the mechanisms driving mRNA target selection by individual Pus enzymes still need to be defined. The bacterial Pus enzyme TruB has been well characterized and modifies tRNA at position U55 within T-loops. In addition to catalyzing Ψ55 in most tRNAs, eukaryotic TruB orthologs also pseudouridylate mRNAs. While it has been proposed that eukaryotic TruB orthologs modify mRNA at sites that mimic their tRNA targets in sequence and secondary structure, only a fraction of such sites are pseudouridylated in cells. Here, we demonstrate that the Saccharomyces cerevisiae TruB ortholog Pus4 binds and modifies RNAs that differ in secondary structure from its established tRNA substrates in vitro. Comparison of Pus4 and TruB activities on structurally diverse substrates reveals that while both enzymes can modify a variety of substrates, TruB does so less robustly than Pus4. We also find that the bacterial-specific PUA domain modulates TruB substrate selection. These findings are consistent with reports demonstrating that other mRNA-modifying Pus enzymes are more promiscuous in vitro than in cells. Our results suggest that Pus4 substrate selection might rely on additional factors beyond protein-RNA recognition in cells.
Multisystem proteinopathy (MSP) is a pleiotropic degenerative disorder which affects the nervous system, muscles, and bones. The identification of risk factors and their molecular contribution to MSP expands our understanding of disease mechanisms. Here, we describe a family with dominantly inherited MSP, in which a mutation in the serine/arginine repetitive matrix protein 2 gene (SRRM2) that co-segregates with disease, is identified. SRRM2 is essential for nuclear speckle formation and a constitutive member of the RNA splicing machinery. To investigate how the mutation in SRRM2 might contribute to MSP pathogenesis, we examined its effect on a model cell line, where the point mutation was introduced in the endogenous gene. Surprisingly, we found that the resulting single amino acid exchange led to the loss of protein-protein interaction between SRRM2 and the splicing factor ACIN1. Transcriptome studies further revealed widespread differential gene expression, which converged on the dysregulation of synapse-associated pathways. Together, our findings identify SRRM2 as a novel MSP risk factor and provide mechanistic insights into how its mutation can be linked to MSP pathology.
Genetic code reprogramming has enabled the ribosomal incorporation of diverse nonproteinogenic amino acids (npAAs) into nascent peptide chains. While backbone-altering npAAs—such as β-amino, γ-amino, d -amino, and N -methylamino acids—are typically poor substrates, the development of engineered tRNAs, notably tRNA Pro1E2 , has facilitated their efficient incorporation. tRNA Pro1E2 possesses a distinct D-arm motif recognized by the translation factor EF-P, which enhances peptide bond formation with these npAAs. α-Hydroxy acids can also be ribosomally incorporated to form ester bonds, serving as valuable building blocks for novel bioactive depsipeptides. In this study, α-hydroxy acid incorporation was unexpectedly found to be significantly inhibited by EF-P when charged onto EF-P-responsive tRNAs, such as tRNA Pro1 and tRNA Pro1E2 . This inhibition was effectively disrupted by a single mutation in the D-arm motif. To enable the efficient co-incorporation of α-hydroxy acids and other backbone-altering npAAs (e.g., β- and d -amino acids) in a single translation, a dual-tRNA strategy was employed: α-hydroxy acids were charged onto an EF-P-insensitive tRNA (tRNA AsnE2 ), while β-/ d -amino acids were charged onto tRNA Pro1E2 . Using this approach, a model depsipeptide containing two α-hydroxy acids, one β-amino acid, and one d -amino acid was successfully synthesized in an EF-P-dependent manner.
Mutations in BRCA1 are key drivers of breast cancer by impairing homologous recombination. While these tumors are often sensitive to PARP inhibitors, resistance frequently emerges, highlighting the need to identify additional molecular vulnerabilities. HORMAD1 is frequently overexpressed in triple-negative breast cancer and associated with genomic instability, yet its role in therapy response in BRCA1-deficient tumors remains unclear. Here, transcriptomic profiling of BRCA1-mutant breast cancer identified HORMAD1 as one of the most upregulated and alternatively spliced genes. The splicing inhibitor Isoginkgetin globally altered alternative splicing patterns in BRCA1-mutant cells, promoting HORMAD1 exon 4 inclusion. We found that RNA-binding protein RBM38 is correlated with exon 4 inclusion, and RBM38 knockdown further sensitized BRCA1-mutant cells to MEK1 inhibition. Together, these findings define an RBM38-HORMAD1 signaling as a potential therapeutic vulnerability in BRCA1-mutant breast cancer and suggest that targeting splicing regulation may represent a promising strategy to enhance treatment efficacy.
Human papillomavirus (HPV)-driven cancers remain a major global health burden, and understanding how posttranscriptional regulation shapes viral gene expression may inform new therapeutic strategies. Here, we reanalyzed independently generated public HeLa data sets to identify candidate RNA modification sites on integrated HPV18 transcripts expressed from the HeLa genome. Using Oxford Nanopore direct RNA sequencing of native RNAs and in vitro transcribed controls, together with GLORI, eTAM-seq and staged 4sU-GLORI data sets, we identified a set of candidate m 6 A sites on HPV18 early transcripts. m 6 A levels at a subset of these sites were reduced following perturbation of the m 6 A pathway, most clearly after METTL3 inhibition, WTAP knockdown, or FTO overexpression. We further show that the E6*I -proximal m 6 A site at position 224 is enriched on unspliced transcripts in direct RNA sequencing, as supported by several of the analyzed data sets. In contrast, we find no convincing evidence for m 5 C or pseudouridine within the HPV18 regions covered by these data sets. Together, our analyses provide a candidate map of RNA modifications on HeLa-expressed HPV18 early transcripts.
RNAmed - Future Leaders in RNA-based Medicine is a unique graduate training program in the area of RNA-based medicine. Financed by the Free State of Bavaria, Germany, it is jointly run by several universities and research institutes from Würzburg, Regensburg, and Munich. It aspires to equip doctoral students with a comprehensive idea of RNA therapeutics, spanning fundamental biology, translational research, clinical application, regulation, ethics, and societal implications. This integrative approach is meant to cultivate exceptional qualifications for careers across academia, industry, and policy. The RNAmed program held its 2025 annual retreat on San Servolo (Venice, Italy), with a total of 40 doctoral researchers, principal investigators (PIs), and invited guests from academia and pharmaceutical industry attending. The three-day program combined keynotes, student presentations, flash talks, a career panel, and topic-table discussions of advances and challenges across RNA therapeutics to catalyze exchange and collaboration. Presentations highlighted emerging directions for antisense strategies, circular RNAs, delivery technologies, and AI-enabled molecular design, reflecting the program's cross-disciplinary nature. Discussions emphasized shared priorities such as clear experimental standards, robust delivery solutions, and stronger academia-industry ties to accelerate safe, effective RNA medicines. Updates on RNAmed's growth and extended funding underscored its mission to develop talent through integrated scientific and professional training. Overall, the retreat strengthened a network of early-career scientists and mentors committed to advancing RNA-based modalities from concept to clinic.
Processing bodies (P-bodies) are cytoplasmic granules that regulate mRNA storage, repression, and decay, yet how their internal organization supports selective mRNA regulation remains poorly understood. Here, we show that the conserved LSm protein Trailer Hitch (Tral) is a key organizer of P-body architecture and function in the Drosophila melanogaster female germline. Using quantitative confocal imaging, super-resolution microscopy, and chemical perturbation of intermolecular interactions, we demonstrate that Tral coordinates the incorporation and spatial organization of the core P-body proteins Me31B and Cup. Loss of Tral alters their partitioning into P-bodies, promotes demixing into distinct subdomains, and shifts condensates toward a less dynamic, structurally heterogeneous state. These organizational changes have functional consequences for mRNA storage: Tral depletion selectively releases maternal mRNA bicoid , while nanos mRNA remains P-body associated and stable. We further identify twinstar mRNA, encoding the homolog of the actin regulator Cofilin, as a Tral-dependent P-body client whose localization and organization within P-bodies requires Tral:RNA interactions and electrostatic forces. Reduced twinstar mRNA levels in the absence of Tral are associated with decreased nuclear G-actin and altered transcription of me31B and cup , revealing a potential feedback mechanism that links cytoplasmic P-body organization to nuclear gene expression. Together, these findings establish Tral as a central regulator of P-body architecture that couples condensate organization to selective mRNA regulation and transcriptional homeostasis.
