O20
Prediction of RNA localization elements with a conserved secondary structure

Presenting Author: Annika Ladewig

University of Wuerzburg, Bioinformatics II, Eichendorffstraße 16, 97072 Würzburg, DE, annika.ladwig@uni-wuerzburg.de

Author(s)
Annika Ladewig¹

RNA localization enables the spatial control of gene expression through the subcellular transport of RNAs. In Saccharomyces cerevisiae, this process is mediated by the RNA-binding protein She2p, which recognizes its target transcripts via so-called localization elements (LEs). These short RNA elements are characterized by specific sequence and structural features and are recognized largely based on their structure. However, only very few LEs have been experimentally characterized to date.
Here, we developed a computational strategy to predict candidate She2p LEs based on a conserved sequence-structure motif found in validated examples. A structure-based sequence alignment generated with LocARNA was used to build a covariance model for motif searches with Infernal. Candidate regions were then prioritized by suboptimal folding and structure-based filtering. The workflow identified several candidate LEs within She2p target transcripts. A transcriptome-wide search further identified candidate LEs in transcripts not previously associated with She2p, including two transcripts encoding proteins with interesting functionality.  In summary, our prediction strategy provides a solid basis for the systematic identification and prioritization of candidate LEs for experimental validation.

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D1
Pseudouridine increases ribosome stability in a thermophilic eukaryote

Presenting Author: Klemens Wild

Heidelberg University, Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, DE, klemens.wild@bzh.uni-heidelberg.de

Author(s)
Dr. Klemens Wild¹, Dr. Marius Klein¹, Alicia Burkard², Stefan Pastore², Tamer Butto², Mark Helm², Irmgard Sinning¹

Ribosomal RNA (rRNA) modifications stabilize the structure of ribosomes and are critical for their function. They are known to cluster around functionally important sites such as the peptidyl transferase center, ribosomal subunit bridges and the polypeptide tunnel. Here, we investigate the rRNA modifications of the thermophilic fungus Chaetomium thermophilum (ct), a model system for eukaryotic structural stability and thermophily¹. Using LC-MS/MS, orthogonal second and third generation RNA-sequencing and high-resolution cryo-electron microscopy (cryo-EM), we describe a cross-correlating method to assign and quantify all ct rRNA modifications. The 2.4 Å structure of the idle ct60S ribosome, retaining nascent chains and including metal ions, polyamines and water molecules, allows for a comprehensive and mechanistic structure-function study highlighting the strong increase of pseudouridines (PSIs) compared with mesophilic ribosomes from Chaetomium globosum, yeast and human. Overall, a doubling of modified rRNA nucleotides to 4% explains ribosomal stability with an unusual density and extension towards peripheral functional sites. The number of pseudouridines lineally correlates with growth temperature suggesting statistical modification. A ct-specific PSI substitution at the polypeptide tunnel exit (PTE), forming a tight 5'-turn we delineate as 'PSI-turn', exemplifies mechanistic adaptations of this eukaryotic ribosome at elevated temperatures up to 55 °C.

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D2
Life in Flux: Dynamic RNA:Protein Complex Assembly Shapes Biomolecular Function

Presenting Author: Nils Walter

University of Michigan - Ann Arbor, Chemistry, 930 North University Avenue, 48109-1055 Ann Arbor, US, nwalter@umich.edu

Author(s)
Prof. Dr. Nils Walter¹

The explosion of cryo-EM structures in recent years has underscored the stepwise assembly of stable biomolecular machines with defined, fixed compositions.  In contrast, advances in single-molecule imaging—both in vitro and in live cells—are revealing a very different picture:  Many biological complexes are not static but highly dynamic and transient.  Instead of persisting as stable entities, their functions emerge from short-lived, fluid assemblies, whose lifetimes and outputs are governed by the kinetics of their components.  This chemistry-driven paradigm shift—from rigid machines to kinetically controlled assemblies—offers a powerful framework for understanding gene regulation, proofreading, checkpoint control, and cellular adaptability.

This talk will illustrate this evolving view with two case studies: the kinetically programmed exchange behavior of RNA-guided gene expression machines, and the dynamic assembly of phase-separated RNA-protein structures (or RNP granules).  I will also explore broader implications of this model, including how regulatory signals can fine-tune molecular function by modulating kinetic parameters, rather than altering structure or affinity per se.  By reframing molecular cell biology through the lens of RNA-mediated kinetic control and spatiotemporal organization, this presentation aims to offer a unifying conceptual foundation across diverse areas of biomolecular science.

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O1
Order from Disorder: A Coiled-Coil Domain Drives Rbfox1 Stress Responses

Presenting Author: Nils Schumann

Medizinische Hochschule Hannover (MHH), Institute for Cell Biochemistry, Carl-Neuberg-Straße 1, 30625 Hannover, DE, Schumann.Nils@mh-hannover.de

Author(s)
Nils Schumann¹, Prof. Halyna Shcherbata¹

Intrinsically disordered regions (IDRs) make up a large percentage of the human proteome and have been increasingly linked to various diseases. RBFOX proteins are a family of RNA-binding proteins implicated in autism spectrum disorder and schizophrenia. While most known for their role in alternative splicing via their structured RNA recognition motif (RRM), each family member is mostly intrinsically disordered, containing multiple low-complexity domains (LCDs). These LCDs contribute to RBFOX multimerization during splicing, but might also mediate non-splicing and RNA-binding-independent functions. Nevertheless, the specific roles of individual LCDs remain poorly understood.

We use Drosophila, encoding a single RBFOX ortholog, to study LCD function in vivo. One of its LCDs was predicted to form coiled-coil structures with binding partners, and we generated CRISPR/Cas9 mutants lacking this domain (Rbfox1-ΔLCD^CC). Given RBFOX's stress-responsiveness, we analyzed mutants under different stress conditions, revealing opposing phenotypes: increased thermotolerance but reduced survival under metabolic stress. RNP granule assembly is a key cellular response to metabolic stress, and Rbfox1 associates with these granules upon starvation; this localization was strongly reduced in Rbfox1-ΔLCD^CC mutants. This identifies the coiled-coil LCD as a previously uncharacterized but critical determinant of Rbfox1-dependent stress responses. 

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D3
Dynamic RNA conformational ensembles as drug targets: an integrated NMR and chemical probing approach

Presenting Author: Rosalie Koros

Uppsala universitet, Department of Medical Biochemistry and Microbiology, IMBIM, Husargatan 3, 75237 Uppsala, SE, rosalie.koros@imbim.uu.se

Author(s)
Rosalie Koros¹, Dr. Christian Steinmetzger¹, Dr. Rubin Dasgupta¹, Dr. Joanna Sajkowska¹, Prof. Katja Petzold¹

RNA is an emerging therapeutic target, but rational drug discovery is constrained by incomplete knowledge of conformational dynamics. RNA exists as dynamic conformational ensembles, sampling transient states on μs–ms timescales that may define ligand-binding opportunities invisible to X-ray crystallography or cryo-EM. R1ρ relaxation dispersion NMR spectroscopy uniquely accesses these states at atomic resolution, detecting and quantifying conformational exchange directly.

This project investigates the conformational dynamics of the ribosomal A-site within helix 44 of the bacterial small ribosomal subunit, a clinically validated drug target bound by aminoglycoside antibiotics. NMR measurements on unlabelled A-site constructs have confirmed correct sample folding, establishing the foundation for relaxation dispersion experiments and antibiotic titrations. In parallel, in vivo chemical probing of bacterial rRNA in E. coli using SHAPE (2A3) and DMS reagents followed by targeted Oxford Nanopore sequencing has yielded reproducible nucleotide-resolution reactivity profiles. These data will be compared to NMR-informed ground and excited state information, to determine whether dynamic signatures detected by NMR are reflected in probing reactivities.

 

The broader goal is to develop a quantitative framework linking probing signatures to NMR-derived exchange parameters, enabling identification of druggable RNA motifs across biologically relevant systems.

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O2
One Fold, Many Rates

Presenting Author: Antonia Bangnowski

Universität Zürich, Department of Chemistry, Winterthurerstrasse 190, 8057 Zürich, CH, antonia.bangnowski@chem.uzh.ch

Author(s)
Antonia Bangnowski¹, Dr. Kasimir Dahlby-Kienbeck¹, Dr. Silke Johannsen¹, Prof. Dr. Roland K.O. Sigel¹, Dr. Susann Zelger-Paulus¹

HDV-like self-cleaving ribozymes are among the most widespread catalytic RNAs in nature.¹ Despite sharing a highly conserved tertiary fold and catalytic mechanism, members of this family exhibit cleavage rates spanning several orders of magnitude, suggesting that subtle differences in sequence and structural context can profoundly influence activity.

We recently discovered over 1,700 HDV-like theta ribozymes in genomic and metagenomic datasets, providing an unprecedented resource for investigating structure–function relationships in this ribozyme family.² Using biochemical analyses, we explore how substrate sequence, local secondary structure and cleavage-site architecture influence self-cleavage activity. We additionally assess how these structural features interact with metal ion conditions to modulate catalysis.

By systematically probing the structural context of the cleavage site, we aim to identify the RNA features that contribute to the broad range of activities observed among HDV-like ribozymes and to better understand how conserved catalytic folds accommodate functional diversity.

 [1] Sharmeen et al. (1988) J Virol 62:2674

[2] Kienbeck et al. (2024) Nat Commun 15:1559

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O3
The structural basis of SRSF6-mediated splicing decisions governed by tandem-domain RNA recognition and RS-domain tuning

Presenting Author: Julian von Ehr

Greifswald University , Institut für Biochemie, Felix-Hausdorff-Straße 4, 17489 Greifswald, DE, vonehr@bio.uni-frankfurt.de

Author(s)
Julian von Ehr¹

Why and how cells exploit redundant versus complementary specificities of the twelve splice-regulatory SRSF (1–12) proteins has remained enigmatic and is particularly evident for SRSF6, for which no structures are available to explain its pre-mRNA splicing specificity. Here, we combine RNA Bind-n-Seq with integrated structural biology to determine the first atom-resolved structures of SRSF6 with RNA. Together with iCLIP, splicing analyses and biochemical analysis of the full-length phosphorylated protein, our data suggest that SRSF6 engages RNA through cooperative interactions of its two RRMs: ΨRRM2 recognizes GGA-elements while RRM1 binds C-rich RNAs, enabling high-affinity binding to composite CGGACU-like motifs, present in SRSF6-regulated exons. In contrast, GAA repeats, often found in constitutive exons, are bound with lower affinity and require repeats. The differential association of SRSF6 with alternative and constitutive exons resolves previous inconsistencies in motif analyses. Splicing analyses confirm that RRM1 contributes to exon selection. The RS domain globally reduces RNA-binding and likely acts as a selectivity filter. Comparative binding studies with SRSF1 reveal cross-regulated splicing, providing a blueprint for an integrated understanding of SR protein networks. Our findings establish a structural and mechanistic framework, in which multidomain RNA recognition, RS-domain-mediated tuning and binding competition define SRSF6’ cellular functional specificity.

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D4
DDX47 organizes perichromosomal RNA in mitosis

Presenting Author: Ramona Jühlen

RWTH Aachen University, Institute of Biochemistry and Molecular Cell Biology, Pauwelsstraße 30, 52074 Aachen, DE, rjuehlen@ukaachen.de

Author(s)
Ramona Jühlen¹, Karina Mertens¹, Uliana Budzinskaya¹, Xenia Rosenkranz¹, Sabine Wiesmann¹, Prof. Wolfram Antonin¹

During mitosis, chromatin first condenses in order to distribute the chromatin correctly to the daughter cells, and later decondenses so that the interphase chromatin structure can be re-established. The mechanisms underlying condensation are well studied, whereas those governing decondensation are not.

We have shown that the DEAD-box RNA helicase eIF4A1/2 plays a role in chromatin decondensation and, as an RNA chaperone, regulates the composition of the perichromatin, a layer of RNA and proteins coating mitotic chromatin. When eIF4A1/2 is reduced, perichromosomal components mislocalize to the cytosol. This previously unknown function of eIF4A1/2 is independent of its role in translation initiation. It is unclear whether additional helicases are involved. Therefore, we tested other RNA helicases for a possible function in chromatin decondensation and identified DDX47 as potential factor.

DDX47 localizes to perichromatin, and a reduction of DDX47 leads to perichromosomal aggregates directly on the chromatin, clearly different from the phenotype observed after eIF4A1/2 depletion. Simultaneous knockdown of eIF4A1/2 and DDX47 mislocalizes perichromosomal components to the mitotic cytosol and produces the same phenotype as single eIF4A1/2 knockdown. Our findings imply that eIF4A1/2 and DDX47 execute successive steps in perichromatin organization: eIF4A1/2 may transport structured RNA to the perichromatin surface, whereas DDX47 may distribute RNA uniformly over the chromatin.

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O4
Structure-based Design of RNA targeting Peptides

Presenting Author: Marvin Albers

Vrije Univ. Amsterdam, Chemistry and Pharmaceutical Sciences, De Boelelaan 1108, 1081 HZ Amsterdam, NL, m.a.albers@vu.nl

Author(s)
Marvin Albers¹, Prof. Dr. Tom Grossmann²

RNA is increasingly recognized as an attractive therapeutic target due to its central role in disease-associated pathways. However, designing high-affinity ligands remains challenging because RNA lacks well-defined binding pockets and adopts dynamic conformations. RNA-binding proteins, which have co-evolved with RNA, provide ideal templates for structure-guided ligand design for these challenging targets.

In this work, we explore structure-based design approaches to design high-affinity RNA-targeting peptides. Inspired by RNA-protein complexes, such as the Tomato Aspermy Virus protein 2b (TAV2b), we apply rational design principles to develop peptides that selectively bind RNA structures. To enhance their properties, we employ chemical engineering strategies such as hydrocarbon stapling, which improves protease stability and promotes cellular uptake. These optimized peptides ultimately yielded bioactive inhibitors with improved selectivity.

Furthermore, TAV2b served as a template for designing dimeric stapled peptides that bind and stabilize dsRNA. By promoting the cellular uptake of siRNA, these RNA-targeting peptides tackle a major bottleneck for the therapeutic application of RNA-based technologies.

 

Together, these approaches demonstrate how structure-guided approaches combined with chemical engineering can yield potent RNA-targeting ligands. Expanding such strategies will help accelerate the exploitation of RNA in drug discovery and diagnostics.

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D5
Compact genome, clever regulation: a multi-layered riboswitch that bind 2’-deoxyguanosine

Presenting Author: Matteo Lisibach

Universität Zürich, Department of Chemistry, Winterthurerstrasse 190, 8057 Zürich, CH, matteo.lisibach@chem.uzh.ch

Author(s)
Matteo Lisibach¹, Prof. Dr. Roland K. O. Sigel¹, Dr. Susann Zelger-Paulus¹

Riboswitches are structured non-coding RNAs that regulate gene expression by coupling metabolite recognition to conformational changes in RNA structure. We studied a 2’-deoxyguanosine riboswitch from Mesoplasma florum (mfl-2dG) that regulates transcription of the ribonucleotide reductase beta-subunit, a key enzyme for deoxynucleotide biosynthesis. Unlike canonical purine riboswitches, mfl-2dG contains a unique 5’ flanking stem-loop that competes with conserved structural elements. Previous studies using ensemble-averaged methods (SHAPE, NMR) captured static conformational snapshots but could not resolve the dynamic interplay between distinct states. To directly monitor these dynamics, we developed an enzymatic RNA labeling strategy that enables site-specifically dual-labeled RNA suitable for single-molecule investigations.

Our measurements reveal that the mfl-2dG samples multiple, ligand-modulated conformations rather than operating as a simple binary switch. Strikingly, the 5’ flanking motif expands accessible conformations and leads to an inversion of the expected hierarchy of RNA folding events. These results demonstrate that small structural elements can dictate the order and outcome of riboswitch folding and uncover an unexpected RNA-mediated regulatory mechanism.

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D6
RNAs untangled: Shedding light onto complex structures of long RNAs

Presenting Author: Lukas Pekarek

TU Dresden, B CUBE, Tatzberg 41, 01307 Dresden, DE, lukas.pekarek1@tu-dresden.de

Author(s)
Lukas Pekarek¹, Dr. Andreas Hartmann¹, Fiona Anilkumar¹, Simon Doll¹, Leo König¹, Cesar Augusto Quintana Catano¹, Dr. Marcus Jahnel, Prof. Dr. Michael Schlierf¹

RNA is an intriguing molecule. Despite its relatively simple composition, RNA's functional versatility underscores the crucial role of RNA structure. Proper folding enables distant segments of the RNA molecule to come into close proximity, facilitating essential biological functions. This is particularly critical for long RNAs such as mRNAs, rRNAs, and lncRNAs, which can span over 1000 nucleotides. These RNAs play diverse roles in gene expression, regulation, chromatin organization, and post-transcriptional control. The function of these RNAs often depends on their structure and ability to cooperatively interact with RNA-binding proteins, which often contain intrinsically disordered regions prone to condensation. When the RNA structure is compromised, the consequent lack of function can be severe for the cell.

This raises a fundamental challenge: how do living organisms ensure the robust and accurate folding of long regulatory RNAs? What is the hierarchy of RNA folding? And how are the RNA structures affected by the presence of RNA-binding proteins?

 

In this project, we aim to understand how long RNA molecules fold into their complex structures. We took lncRNA HOTAIR as a case study to shed some light on this folding enigma. By employing methods such as single-molecule optical tweezers and fluorescence correlation spectroscopy, we aim to understand the key aspects of the dynamic folding of complex RNAs. 

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O5
Eukaryotic tRNA ligases mediate RNA break repair

Presenting Author: Jirka Peschek

Heidelberg University, Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, DE, jirka.peschek@bzh.uni-heidelberg.de

Author(s)
Alexander N. Wirth, Dr. Isabel Naarmann-De Vries, Anne Pinnen, Aiswarya Gopal, Anna Righetti, Prof. Dr. Kathrin Leppek, Prof. Dr. Christoph Dieterich, Dr. Jirka Peschek¹

RNA is continuously exposed to damage during physiological metabolism and stress, yet cellular responses to RNA damage remain less understood than DNA repair pathways. RNA strand breaks are particularly deleterious because they generate chemically incompatible RNA ends. Eukaryotic tRNA ligases have been implicated in RNA processing and repair, but whether they function as general RNA repair enzymes remains unresolved. Here, we show that the evolutionarily divergent tRNA ligases, human RTCB and fungal Trl1, mediate RNA break repair (RBR) targeting ribosomes and other ribonucleoprotein (RNP) complexes. Using direct RNA nanopore sequencing, we map these repair events at nucleotide resolution, demonstrating that tRNA ligases repair breaks in ribosomal RNA and restore translational activity of repaired ribosomes. We further identify repair across additional structured cellular RNAs. We show that loss of RBR activity leads to RNA fragmentation in human cells and impairs cell viability upon oxidative stress. Together, these findings uncover a broader role for eukaryotic tRNA ligases in repairing RNA breaks and maintaining transcriptome integrity.

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D7
High-Resolution NMR Analysis of the Dynamic NmPAL-RNA Interaction Network that enables optoribogenetics

Presenting Author: Luis Gardon

Heinrich Heine Universität Düsseldorf, Institut für Physikalische Biologie, Universitätsstraße, 1, 40225 Düsseldorf, DE, lugar102@hhu.de

Author(s)
Luis Gardon¹, Dr. Jessica Schmuck¹, Dr. Mohanraj Gopalswamy¹, Dr. Manuel Etzkorn¹

Artificial chimeric RNAs have emerged as powerful tools for controlling cellular processes and probing signaling pathways. In optoribogenetics, these RNAs form light-dependent complexes with photoreceptors, enabling precise regulation of biological functions. In previous work, optoribogenetic systems had been developed based on the photoreceptor NmPAL and a range of functionally diverse chimeric RNAs. Three RNA aptamer motifs were identified that bind with high affinity to the light-adapted state of NmPAL.

To gain structural and mechanistic insight into this optoribogenetic system, we employ nuclear magnetic resonance (NMR) spectroscopy, as alternative structural biology approaches proved insufficient. NMR provides a detailed view of both structural and dynamic properties of the RNA aptamers, allowing investigation of NmPAL–RNA interactions, contacts between individual RNA modules, and potential modulation by metal ions or other cellular factors. Preliminary data already reveal clear differences among the investigated RNA aptamers, suggesting distinct molecular properties and conformational behaviors of the respective aptamer motifs.

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V1
Interferon-inducible PARPs ADP-ribosylate RNA ends and RNA nucleobases

Presenting Author: Karla Feijs-Žaja

RWTH Aachen University, Institute of Biochemistry and Molecular Biology, Pauwelsstraße 30, 52074 Aachen, DE, kfeijs@ukaachen.de

Author(s)
Dr. Roko Žaja, Jonas Siefert, Victoria Ngangbam, Wiwik Bauten, Dr. Karla Feijs-Žaja¹

ADP-ribosylation has been studied as protein posttranslational modification for decades. Only recently, it has become clear that mammalian PARPs can also attach ADP-ribose to RNA substrates.

In 2019, an in vitro study suggested that PARPs can attach ADP-ribose to the RNA 5’-phosphate, which we were able to detect in human cells in 2022. The ADPr cap does not allow canonical translation, but it protects the RNA from degradation. The biological function of this non-canonical cap remains elusive. In addition to serving as cap, we recently observed that incubation of specific RNA oligonucleotides with PARP10 or PARP15 leads to incorporation of several ADP-ribose moieties. Using complementary strategies, we were able to identify both uracil as well as guanine bases as ADP-ribose acceptor. After developing suitable methods, we were able to confirm the existence of ADP-ribosylated RNA nucleobases in human cells. ADP-ribose can thus serve both as RNA cap, as well as RNA internal modification, thereby expanding the spectrum of RNA modifications.

One of the outstanding questions concerns the function of RNA ADP-ribosylation. As most of the PARPs which modify RNA are upregulated in response to viral infection and play a poorly understood role in the antiviral defense, it is possible that ADP-ribosylation of RNA is involved in the antiviral response. Our current work focuses on deciphering the unknown functions of ADPr-RNA in human cells.

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D8
Structural and thermodynamic effects of pseudouridine positioning in miR159b/miR159b* RNA duplexes

Presenting Author: Alena Dolińska

Adam Mickiewicz University, Poznań, ul. Janusza Meissnera 4B/8, 60-408 Poznań, PL, aledol3@st.amu.edu.pl

Author(s)
Alena Dolińska¹, Prof. Agnieszka Kiliszek²

Pseudouridine (Ψ), the most abundant post-transcriptional RNA modification, is known to influence RNA structure, stability, and function. Although its effects have been extensively characterized in tRNA or rRNA, its structural and thermodynamic consequences in microRNAs remain poorly understood. Recent evidence suggests the presence of naturally occurring pseudouridine residues in the plant microRNA miR159b, indicating a potential role in modulating duplex properties and biological activity.

This study investigates how pseudouridine positioning affects the thermodynamic stability and crystallization potential of RNA duplexes derived from the miR159b/miR159b* sequence. A set of synthetic RNA duplexes containing pseudouridine at defined positions was designed and chemically synthesized. Thermodynamic stability was quantified by UV melting experiments, and melting profiles were analyzed using MeltWin 3.5 to derive thermodynamic parameters.

In parallel, crystallization screening was performed, followed by X-ray diffraction analysis of obtained crystals where applicable. This combined approach enables correlation of pseudouridine localization with global RNA duplex stability and crystallization propensity.

The results are expected to provide mechanistic insight into how site-specific RNA modifications influence structural properties of microRNA duplexes, contributing to a better understanding of RNA modification-driven modulation of RNA structure and function.

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O6
Targeting RBM3 Exon 3a via Antisense Oligonucleotides as a Neuroprotective Strategy in Parkinson's Disease

Presenting Author: Bo Zhao

Freie Universität Berlin, Department of Biology, Chemistry, Pharmacy, Takustr.6, 14195 Berlin, DE, bozhao95@hotmail.com

Author(s)
Dr. Bo Zhao¹, Prof. Dr. Florian Heyd

Parkinson's disease (PD) is the most common movement disorder worldwide, with its incidence rising steadily. In this study, transcriptomic analysis of PD patients and cellular PD models revealed downregulation of the neuroprotective RNA-binding motif protein 3 (RBM3), suggesting it as a potential therapeutic target. Further experiments in a PD cell culture model confirmed that MPP⁺ treatment reduced RBM3 expression, while the inclusion of exon 3a, identified as a poison exon, was increased upon MPP⁺ treatment, likely serving as a mechanism underlying RBM3 reduction. To elucidate the upstream regulatory mechanism of this splicing event, we analyzed splicing regulators of RBM3 in a bioinformatics approach. We identified SRSF2 as the most prominent negative regulator and HNRNPH1 as a positive regulator (previously reported), and confirmed experimentally that knockdown of SRSF2 increased RBM3 expression. Given that the effect of SRSF2 is likely associated with its binding to an exonic splicing enhancer (ESE), we treated cells with an ASO designed against the ESE region of RBM3 exon 3a. The results showed that ASO pretreatment effectively blocked MPP⁺-induced RBM3 downregulation. Collectively, this study suggests that exon 3a alternative splicing regulates RBM3 expression in PD and validates ASO targeting this exon as a potential intervention strategy. Future experiments in MPTP-treated mice will be conducted to assess whether this ASO can prevent or alleviate PD symptoms in vivo.

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O7
Structure of the tobramycin riboswitch solved by NMR

Presenting Author: Elke Duchardt-Ferner

Goethe - Universität Frankfurt a. M., Molekulare Biowissenschaften, Max-von-Laue Str. 9, 60438 Frankfurt, DE, duchardt@bio.uni-frankfurt.de

Author(s)
Elke Duchardt-Ferner¹, Dr. Leon Kraus², Anahita Limouchi¹, Prof. Dr. Beatrix Suess², Prof. Dr. Wöhnert Jens¹

 

Recently, a novel tobramycin-responsive riboswitch was developed by a combination of Capture-SELEX and in vivo screening exhibiting best of class regulatory activity [1]. This riboswitch is functional in S. cerevisiae on the translational level. To provide a structural basis for the remarkable regulatory efficiency and ligand selectivity of this riboswitch, we investigated its structure in complex with its cognate ligand tobramycin by high-resolution solution NMR spectroscopy. The structure reveals a novel aminoglycoside binding motif with a unique pattern of intermolecular hydrogen bonds and electrostatic interactions between the RNA and functional groups of all three rings of the ligand. The structure of the ligand-RNA complex also allows to rationalize the around 35fold discrimination against the closely related aminoglycoside kanamycin A. Comparison with the free form of the RNA shows that the latter is much less compact, lacking many RNA-RNA interactions in particular in the bulge regions, thereby immediately providing a rationale for the exceptional switching efficiency of this synthetic riboswitch.

 

[1] Kraus, L., Duchardt-Ferner, E., Bräuchle, E., Fürbacher, S., Kelvin, D., Marx, H., Boussebayle, A., Maurer, L.-M., Bofill-Bosch, C. and Wöhnert, J. et al. (2023) Development of a novel tobramycin dependent riboswitch. Nucleic Acids Res., 51, 11375–11385.

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O8
Stimulus-Responsive Protein and RNA Networks of the ASCC Complex

Presenting Author: Aruna Arumugam

Freie Uinversität Berlin, Biology, Chemistry and Pharmacy, Takustrasse 6, 14195 Berlin, DE, arua96@zedat.fu-berlin.de

Author(s)
Aruna Arumugam¹, Benno Kuropka¹, Dr. Nicolás Lemus-Díaz², Dr. Petia Adarska¹, Nicole Holton¹, Prof. Dr. Francesca Bottanelli¹, Dr. Katherine E. Bohnsack³, Prof. Dr. Markus T. Bohnsack⁴, Prof. Dr. Lydia Herzel¹, Prof. Markus C. Wahl⁵

 

The human activating signal co-integrator complex (ASCC) consists of the core subunits ASCC1, ASCC2, ASCC3, and TRIP4 and associates with additional proteins to regulate genome maintenance and gene expression processes. However, it remains unclear how the core ASCC interacts with auxiliary factors, how these interactions relate to nucleic acid binding, and how the complex responds to cellular stimuli. We therefore mapped TRIP4-associated proteins and RNAs using proximity labeling and cross-linking and analysis of cDNA (CRAC) analysis. We generated CRISPR/Cas9-engineered HeLa cells producing TurboID-tagged TRIP4 and used established Flp-In™ T-REx™ 293 cells producing Flag-tagged TRIP4 for interactome analyses by proximity labeling and co-immunoprecipitation coupled to mass spectrometry, in the presence or absence of 9-cis retinoic acid (RA). These analyses revealed that TRIP4 participates in interconnected networks involved in transcription, translation, RNA metabolism, and cytoskeletal organization, and that these interactions are remodeled following RA treatment. Integration of TRIP4 CRAC data with the protein interactome identified extensive overlap at RNA regulatory hubs, particularly within the eukaryotic translation initiation factor 4F complex. Collectively, these findings provide a framework for understanding how ASCC and its associated factors coordinate multiple cellular processes.

 

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O9
GTP binding by a topologically complex G-quadruplex in a GTP aptamer

Presenting Author: Helena Stafflinger

Goethe - Universität Frankfurt, Molekulare Biowissenschaften, Max-von-Laue Str. 9, 60438 Frankfurt, DE, stafflinger@bio.uni-frankfurt.de

Author(s)
Helena Stafflinger¹, Dr. Elke Duchardt-Ferner¹, Prof. Dr. Ronald Micura², Prof. Dr. Hermann Schindelin³, Prof. Dr. Jens Wöhnert¹

RNA molecules are capable of forming complex secondary and tertiary structures which can serve as binding sites for a variety of small molecule ligands, proteins or other RNAs. The structural complexity of these motifs is often the basis for a high affinity and specificity of RNA-ligand interactions. However, it is still very difficult to accurately predict RNA three-dimensional structures due to the still small number of experimentally determined RNA structures available as training data.
For GTP as the ligand a large number of in vitro selected RNA aptamers are known which differ widely in sequence, secondary structure and ligand binding affinity and specificity. Here we report a 1.6 Å crystal structure of the class V-GTP aptamer bound to GTP, revealing a topologically complex three-layered G-quadruplex. The GTP ligand is directly incorporated into one G-quadruplex layer. The quadruplex is only formed upon ligand binding and is further stabilized by a variety of non-canonical interactions. Importantly, the structure does not correspond to typical G-quadruplex topologies with G-tracts that are separated by short single-stranded loops. Instead, in our structure the G-tracts are separated by an A-form double helix.
This complex tertiary folding could not be predicted from the sequence and the previously determined secondary structure and therefore raises the question, if this or similar RNA folds occur in other functional RNAs in biological systems.

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O10
The Effect of tRNA Modifications Studied on mascRNA as a Model System

Presenting Author: Lucija Sunko

Goethe - Universität Frankfurt, Molekulare Biowissenschaften, Max-von-Laue Str. 9, 60438 Frankfurt, DE, s1011235@stud.uni-frankfurt.de

Author(s)
Lucija Sunko¹, Dr. Elke Duchardt-Ferner¹, Katja Hollnagel¹, Prof. Dr. Jens Wöhnert¹

The tRNA-like structure of MALAT1-associated small cytoplasmic RNA (mascRNA) is able to recruit tRNA-processing enzymes. Its compact L-shaped fold, which closely mimics the highly conserved elbow region of tRNAs, also enables its recognition by tRNA-modifying enzymes whose substrate recognition elements are contained within this region. Together with its smaller size compared to canonical tRNAs, this makes mascRNA an excellent model system to investigate structural and dynamic consequences of tRNA modifications using NMR-spectroscopy. This is particularly relevant because tRNAs are the RNA family with the greatest diversity of modifications and the highest number of modifications per molecule. Here, we used the Escherichia coli (E. coli) TruB enzyme to introduce pseudouridine (Ψ) at position 40 of mascRNA, corresponding to pseudouridine 55, the most abundant modification in tRNAs. Given that in vitro-transcribed mascRNA does not contain other modifications, this enabled us to investigate the impact of this single pseudouridine on RNA structure and stability, independently of the complex modification patterns found in tRNAs. Although pseudouridine is generally considered to stabilize RNA structure, and Ψ55 has also been associated with enhanced tRNA stability, our results show the opposite. Our findings suggest that pseudouridine should not be viewed as a generally stabilizing modification, but that its effects depend on the overall structural context of the modification site.

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O11
Protonated adenine nucleotides as a central role for high affinity GTP binding in an RNA aptamer

Presenting Author: Helena Stafflinger

Goethe - Universität Frankfurt, Molekulare Biowissenschaften, Max-von-Laue Str. 9, 60438 Frankfurt, DE, stafflinger@bio.uni-frankfurt.de

Author(s)
Helena Stafflinger¹, Dr. Elke Duchardt-Ferner¹, Prof. Dr. Christoph Kreutz², Prof. Dr. Hermann Schindelin³, Prof. Dr. Jens Wöhnert¹

RNA can fold into intricate tertiary structures with a unique set of hydrogen bonds. This enables, besides the canonical base pairing, the formation of base triplets and quartets or hydrogen bonding interactions including the ribose or the phosphodiester backbone of RNAs. Also, modifications, such as base protonations, can increase the structural diversity. However, based on pK_a values, which are far from the physiological pH, (~3.7 for A and ~4.5 for C), protonated nucleotides are normally not considered as suitable building blocks. 
Here, we report the 2.5 Å crystal structure of a GTP-binding RNA aptamer, called 10-10 in complex with its ligand. Upon high affinity ligand binding the RNA adopts a compact helical shape with numerous non-canonical base pairing interactions including several base triplets and a quartet. Furthermore, there are three adenine residues, which are protonated at the N1 to enable an even larger set of hydrogen bonds, but also high affinity binding to the negatively charged GTP ligand. Nuclear magnetic resonance spectra and isothermal titration calorimetry measurements at different pH values demonstrate the stability of these protonation sites even at pH values of up to 9.0, highlighting an enormous shift of the pK_a values in this structured context. Therefore, protonated nucleobases need to be considered more routinely as RNA building blocks for further structural analysis and structure predictions.

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D9
Exploring regulatory efficiency of the tobramycin riboswitch via mutational and dynamic studies

Presenting Author: Anahita Limouchi

Goethe - Universität Frankfurt , Molekulare Biowissenschaften, Max-von-Laue Str. 9, 60438 Frankfurt, DE, limouchi@bio.uni-frankfurt.de

Author(s)
Anahita Limouchi¹, Dr. Elke Duchardt-Ferner¹, Dr. Leon Kraus², Prof. Dr. Jens Wöhnert¹

The tobramycin riboswitch exhibits the highest switching efficiency observed so far for synthetic small-molecule driven riboswitches in its class. One of its key structural features in the ligand-bound state is a non-canonical A11-C24 base pair containing a protonated adenine, which is essential for regulatory activity despite not directly being part of the ligand-binding site. To investigate which structural features are important for the exceptional regulatory efficiency of this riboswitch, we generated RNA variants where the A:C base pair was replaced with Watson-Crick base pairs or other mismatches. Using imino proton solvent exchange measurements combined with chemical shift analysis, we investigated the dynamic behavior of these RNAs in both free and ligand-bound states. Our study provides mechanistic insights into how local base-pair properties influence global RNA dynamics and illustrates that the riboswitch functions as a mechanically coupled system rather than a collection of structurally independent helical stems. These findings contribute to a better understanding of the structural determinants of high-efficiency RNA switches and offer guidance for the rational design of synthetic riboswitches.

 

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P1
Unraveling the (un-)folding of complex RNA structures using a correlative coarse-grain force-spectroscopy approach

Presenting Author: Simon Doll

TU Dresden, B CUBE, Tatzberg 41, 01307 Dresden, DE, simon.doll@tu-dresden.de

Author(s)
Simon Doll¹, Lukáš Pekárek¹, Andreas Hartmann¹, César Augusto Quintana-Cataño¹, Leo König¹, Michael Schlierf¹

The function of many non-coding RNAs depends on their structure. Yet, determining the structural ensembles remains challenging. Recently single-molecule approaches based on force spectroscopy have become more popular to resolve RNA structures and structural ensembles. While force-spectroscopy yields mechanical and nanometer-resolved structural information, the reconstruction of a full secondary or tertiary structural map remains challenging especially for RNAs with multiple structural elements. Here, we present a coarse-grained framework to predict and interpret the unfolding of complex structured RNAs. By combining secondary structure stabilities and polymer elasticity we model force-induced unfolding, allowing us to predict pathways, the probability of observing intermediate states, and energetic contributions governing the process. The framework enables the interpretation of force spectroscopy data by connecting experimental observables to the underlying structural landscape. In addition, it facilitates the identification of rare intermediates, structural bottlenecks, and mechanically resistant states, providing insight into the unfolding mechanisms of complex RNAs. We anticipate, that the combination of force spectroscopy experiments and the coarse grained pathway search will allow to gather further insights into the structurally reach landscape of non-coding RNAs.

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P2
Integrated NMR/MD investigation reveals differences after reweighting in conformational ensembles of the GAAG and GCAA tetraloops

Presenting Author: David Leopold

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, leopold@nmr.uni-frankfurt.de

Author(s)
David Leopold¹, Dr. Andreas Oxenfarth¹, Dr. Emil Thomasen², Dr. Felix Kümmerer², Dr. Christian Richter¹, Dr. Anna Wacker¹, Prof. Kresten Lindorff-Larsen², Prof. Harald Schwalbe¹

In recent years, the concept of thinking about even highly structured RNA as an ensemble of conformations gained significant foothold within the RNA research community. However it proves difficult to elucidate these conformational ensembles, as individual experimental methods either display the averaged data or an energy minimized state. MD simulations provide a detailed insight into short framed dynamical behaviour. However, the force fields in use are still in progress of optimization and often fail to reproduce the experimental data on longer simulations.

In our work we aim to gain a complete picture of the conformational ensembles of tetraloops in solution as a resource for future force field optimization and to improve the understanding of dynamic RNA behaviour in solution. We therefore use a Bayesian/Maximum Entropy reweighting approach combined with extensive experimental data, obtained by nuclear magnetic resonance measurements, to examine the structural motifs of the MD and determine which are underrepresented by the initial MD. A comparison with the structure prediction tools FarFar2 and Alphafold3 and NOE based structure calculation shows, that the reweighted MD ensemble explains the experimental data better by occupying a larger set of structural conformations. With the GAAG and GCAA tetraloops we applied this procedure, that was already published on the UUCG and CUUG tetraloop, on more dynamic tetraloops.

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V2
Solving the NMR solution structures of two elements 5_SL5a and 5_SL5b as part of the translational start site of SARS-CoV-2

Presenting Author: Andreas Oxenfarth

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, oxenfarth@nmr.uni-frankfurt.de

Author(s)
Dr. Andreas Oxenfarth¹, Dr. Klara R. Mertinkus¹, Dr. Christian Richter¹, Dr. Anna Wacker¹, Dr. Julia Wirmer-Bartoschek¹, Prof. Dr. Harald Schwalbe¹

The Covid-19-pandemic showed the need for understanding the function of RNA viruses. Since structure and function are often interlinked in nature, the need for RNA structures arose. Before the pandemic, the structures of viral RNAs were lacking since crystallization for X-ray is often difficult and solution NMR structures are time consuming. Previously the secondary structures were confirmed based on chemical shift assignments.

Here we show the structures and structural analysis of the two sub constructs 5_SL5a and 5_SL5b of the 5_SL5 construct, which is found in the 5’-UTR. In addition, we analyzed the most frequently occurring two mutations found in VoCs, namely 5SL5a_G210U and 5SL5b_C241U. The overall 4-way-junction RNA is of great interest since it is the largest RNA in the 5’-UTR and contains the AUG start codon of ORF1a/b.

To achieve this, we combined a classical approach of measuring NOESYs as well as analyzing the underlying dynamics using hetNOE, T1-relaxation and T1rho. Additionally, we used carbon and nitrogen detected experiments to gain further insight into the base pairing of the U rich bulge of 5_SL5a and loops of the two constructs, which would otherwise be difficult. Lastly, we used residual dipolar couplings to analyze the orientation of the RNAs in solution.

With this work, we were able to provide a 3D model for 5_SL5a and 5_SL5b of the 5_SL5 RNA and additional can give insight into how two mutations in VoCs of these elements change their respective behavior.

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D10
Binding mode investigation of a photoswitchable azo compound binding to the SMN2 exon 7 5’-ss:U1 RNA

Presenting Author: Alexander Herr

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, herr@nmr.uni-frankfurt.de

Author(s)
Alexander Herr¹, Dr. Frank Abendroth², Dr. Henry Jonker¹, Dr. Julia Wirmer-Bartoschek¹, Dr. Christian Richter¹, Dr. Anna Wacker³, Prof. Dr. Olalla Vázquez², Prof. Dr. Harald Schwalbe¹

SMA is a neuromuscular disorder caused by insufficient levels of SMN protein, which in part results from skipping of exon 7 during splicing of the SMN2 pre-mRNA. Small molecules, i.e. risdiplam, can restore sufficient SMN levels by stabilizing a transient dsRNA between exon 7 and the U1 snRNA (5’-ss:U1). From this, a photoswitchable azo analog has been developed (ZZZ1135) with E-isomer specific binding properties to this dsRNA. We aim to understand the action mode of this molecule by determining the apo and holo 3D NMR solution structures of the RNA and utilize the compound’s photoswitchability for investigation of binding kinetics and ligand-induced conformational changes. So far, we could confirm E-isomer specific binding of ZZZ1135 to the A-bulge of 5’-ss:U1 by using NMR spectroscopy with in situ photo-isomerization of the ligand. Using pseudouridine (Ψ) to uridine substitutions, a negligible effect of Ψ nucleotides on ligand binding could be detected. This allowed RNA construct optimization for biochemical accessibility including isotopic labelling, resulting in a model hairpin RNA for comprehensive NMR structure analysis. Light-dependent binding site mapping and ligand-based titrations showed high specificity of ZZZ1135 binding to our model RNA with a K_D of ~60-70 μM. By probing the conformational dynamics of the RNA by {¹H}-¹³C hetNOEs, a reduction of the A-bulge dynamics upon ligand binding could be shown, indicating a stabilization of the RNA around the binding site.

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P3
Detailed structural characterization of novel antivirals bound to stem 3 of the SARS-CoV-2 pseudoknot RNA

Presenting Author: Jennifer Adam

Goethe - Universität Frankfurt a. M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, adam@nmr.uni-frankfurt.de

Author(s)
Jennifer Adam¹, Dr. Frank Kaiser, Julian Schoth, Dr. Christian Richter, Prof. Dr. Julia Weigand, Prof. Dr. Harald Schwalbe

The COVID-19 pandemic highlighted the need for novel antiviral strategies. While current therapies mainly target viral proteins, the clinical success of RNA-targeting small molecules, exemplified by the FDA approval of the splicing modifier risdiplam, has underscored the potential of structured RNA elements as promising drug targets. Among these, the highly conserved SARS-CoV-2 frameshift element (FSE) is of particular interest. The FSE comprises a three-stemmed pseudoknot RNA (PK) that stalls and backtracks the ribosome, together with a heptameric slippery sequence that enables tRNA realignment into the -1 frame.

Here, we describe the development of small molecule antivirals targeting the PK RNA using ligand- and RNA-observed NMR approaches. A multi-stage NMR-based screening campaign identified HSJA12 as a low-micromolar binder. NMR resonance assignment of the 69-nt RNA enabled mapping of the binding site and mutational analysis guided the design of a 21-nt model RNA. Using 29 intermolecular NOEs, we determined the three-dimensional structure of the RNA-ligand complex, which was validated by RDC analysis and supported by structure-activity relationship studies.

Reporter gene assays showed a concentration-dependent reduction in frameshifting efficiency that was largely abolished upon mutation of the binding site. Together, these results establish the SARS-CoV-2 FSE as a tractable RNA target and provide a structural basis for the rational design of antiviral agents.

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O12
Biophysical studies of the intramolecular triplex lncRNA MALAT1

Presenting Author: Maximilian Braun

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, braun@nmr.uni-frankfurt.de

Author(s)
Maximilian Braun¹, Prof. Dr. Harald Schwalbe², Dr. Julia Wirmer-Bartochek

Long non-coding RNA (lncRNA) transcripts are gradually elucidated in their roles in human diseases. Increasing findings suggest their important roles in cancer development and proliferation.¹ LncRNA have the ability to form triple helices (triplexes) either with double stranded DNA or intramolecular resulting in a RNA:RNA•RNA triplex. The formation of these unusual structural elements has potential roles in vivo for cellular functions, like transcriptional regulation, post transcriptional RNA processing or chromatin modification. Therefore, lncRNA and their respective structures gained more and more attention over the past years due to their potential of being drug targets of interest.²

This was further underlined by the findings of certain proteins with the ability of recognizing triplex structures. We set out to investigate the highly stable intramolecular triplex MALAT1 employing biophysical methods of structural elucidation. We utilized electrophoretic mobility shift assays (EMSA), circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. We are hereby showing first results of the highly stable parallel triplex MALAT1 by investigating potential protonation states in different buffer conditions as well as comparing stabilities of different utilized constructs in an effort to gain a better understanding. Furthermore, first looks into the Interaction between MALAT1 and METTL16 are also shown.

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V3
Biophysical Characterization of Dengue Virus Serotype 2 Stem-Loop A RNA Dimerization and Its Ligand-Binding Properties

Presenting Author: Chenxi Sun

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, sun@nmr.uni-frankfurt.de

Author(s)
Chenxi Sun¹, Dr. Anna Wacker, Dr. Christian Richter, Prof. Dr. Harald Schwalbe, Dr. Sridhar Sreeramulu

 

Dengue virus serotype 2 stem-loop A RNA (DENV2-SLA) is an essential promoter for viral RNA replication^[1] and has been proposed to dimerize through intermolecular RNA–RNA interactions.^[2] To identify the structural determinants of this process, we compared a GC-stabilized construct, DENV2-SLA_GC, containing additional GC base pairs for enhanced stability, with DENV2-SLA_GAAA, in which the native UAA-containing side loop was replaced by a stable GAAA tetraloop.

Native gel electrophoresis, size-exclusion chromatography, and SAXS showed that DENV2-SLA_GC partially forms a dimeric RNA species. In contrast, DENV2-SLA_GAAA remained predominantly monomeric, indicating that the native UAA side loop drives SLA dimerization. Solution NMR spectroscopy did not reveal concentration-dependent spectral changes under the tested conditions but showed that the GAAA side loop is better structured and less flexible than the corresponding UAA-containing region in DENV2-SLA_GC, while the overall SLA fold is retained.

Together, our data identify the UAA side-loop motif as a key determinant of DENV2-SLA dimer formation. Replacing this motif by GAAA stabilizes the local loop but disrupts UAA-mediated dimerization. Future work will investigate how the monomer–dimer equilibrium affects ligand-binding affinity and binding-site recognition, providing insight into ligand targeting of the flaviviral SLA promoter element.

 

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D11
Resolving the conformational landscape of a large ribozyme by multi-trajectory smFRET

Presenting Author: Susann Zelger-Paulus

Universität Zürich, Department of Chemistry, Winterthurerstrasse 190, 8057 Zürich, CH, susann.paulus@chem.uzh.ch

Author(s)
Matteo Lisibach¹, Abdul Rahman Sadiq¹, Prof. Roland K.O. Sigel¹, Dr. Susann Zelger-Paulus¹

Single-molecule FRET (smFRET) is widely used to study RNA dynamics and is often applied using a single distance constraint. For large, multidomain RNAs such as our model system, this is not sufficient, as their underlying structural rearrangements are inherently complex. Here, we resolve the conformational landscape of a catalytic group II intron ribozyme undergoing multiple structural transitions during self-cleavage by integrating multiple FRET trajectories.

We introduced four labeling positions, yielding six pairwise FRET trajectories recorded sequentially, each reporting on distinct interdomain distances. While each dataset revealed a complex conformational landscape, correlating states across labeling schemes remained a major challenge. To overcome this, we disrupted a key tertiary interaction, reducing activity and redistributing conformational populations. This perturbation enabled mapping of corresponding states across all datasets. By integrating the trajectories, we identified three major conformational states and defined their domain organization.

Our results show that the ribozyme does not simply adopt a compact active structure but instead coordinates its domain arrangements in a functional manner. This work demonstrates that multi-trajectory smFRET, combined with targeted perturbations, can resolve the dynamic landscapes of large RNAs.

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V4
Viral inhibition through RNA structure targeting the frameshifting element of SARS-CoV-2

Presenting Author: Fatima Zahra Lissane Eddine

Avenue Collonges 5, 1004 Lausanne, CH, fatima.lissane-eddine@chuv.ch

Author(s)
Fatima Zahra Lissane Eddine, Dr. Marianna Bufano, Prof. Andrea Brancale, Prof. Valeria Cagno

The SARS-CoV-2 programmed −1 ribosomal frameshifting element is essential for viral replication and a promising antiviral target. However, all validated inhibitors have been tested only against a minimal 84-nucleotide RNA fragment derived from in vitro studies. Recent cellular probing revealed alternative conformations, including a 1.4 kb long-range interaction that appears to predominate in infected cells, while the minimal structure is likely only transiently formed near the translating ribosome. Targeting these cellular conformations could inhibit the predominant viral RNA structures and prevent the conformational switch required for frameshifting. However, these conformations remain unexplored as drug targets.

 

My PhD research addresses this gap through experimental and computational approaches. I screened an RNA-targeting compound library using a dual-luciferase reporter assay with a 527-nucleotide construct containing the 5′ region of the long-range interaction. Hits were counterscreened to remove false positives, while the same library was screened in parallel by RNA-ligand docking. Validated compounds were then tested in SARS-CoV-2-infected cells. Eleven molecules inhibited viral replication, and four were identified by both approaches. Computational analysis revealed binding pockets that will guide screening of larger libraries and optimization of lead compounds, providing a framework for RNA-targeting antivirals against SARS-CoV-2 and related RNA viruses.

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P4
Investigations on different stem loop II motifs of various viruses

Presenting Author: Quentin Glatz

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, glatz@nmr.uni-frankfurt.de

Author(s)
Quentin Glatz¹, Dr. Tobias Matzel¹, Dr. Inge Schwedt², Dr. Anna Wacker¹, Dr. Christian Richter¹, Prof. Julia Weigand², Prof. Harald Schwalbe¹

The stem-loop II motif (s2m) is a highly conserved mobile genetic element (MGE) located in the 3’-UTR of various positive-sense single-stranded RNA (+ssRNA) viruses. Its irregular distribution across several viral families, including Astroviridae, Caliciviridae and Coronaviridae, suggests a possible horizontal transfer mechanism in addition to its vertical transfer. Despite its high degree of sequence conservation, the biological function of s2m remains elusive. Recent studies indicate a role in viral replication and virus-host interactions. However, only a limited number of s2m structures have been experimentally characterized to date.

The aim of this work was to investigate the similarities, differences and conformational stability of s2m variants from different viruses and to compare them with previously characterized SARS-CoV-2 s2m variants. The secondary structures were analysed using NMR spectroscopy, SHAPE-MaP, SAXS, CD spectroscopy, and computational approaches including structure prediction and sequence alignment.

Preliminary results revealed that all investigated s2m variants exhibit pronounced structural dynamics, while the presence of Mg²⁺ Ions enhances structural stability. By integrating newly acquired experimental and computational data with published structural information, this study aims to identify conserved structural features across diverse s2m variants and to establish a general structure that captures the defining characteristics of the s2-motif.

 

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V5
Targeting Pathogenic Repeat Expansion RNAs with Small Synthetic Molecules: Structural Rearrangements and Therapeutic Implications

Presenting Author: Agnieszka Kiliszek

Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, PL, kiliszek@ibch.poznan.pl

Author(s)
Prof. Agnieszka Kiliszek¹, Dr. Leszek Błaszczyk

The presented work is part of our crystallographic studies focused on the structural analysis of interactions between disease-related RNAs and synthetic molecules. Recent progress in high-throughput screening of small-molecule libraries has led to the discovery of numerous drug-like compounds. However, their further optimization requires the determination of three-dimensional structures revealing the molecular details of RNA–ligand interactions.
    The abnormal expansion of repetitive sequences within specific genes gives rise to mutant RNAs that acquire pathogenic properties. Specific binding of small molecules to these mutant RNAs can interfere with pathological pathways and potentially prevent disease progression.
    Using X-ray crystallography and biophysical methods, we analysed a series of small molecules that recognize unique patterns of nucleobases engaged in non-canonical base pairing or exposed within single-stranded regions of repeat expansion RNAs. The structures of the complexes enabled a detailed characterization of RNA–ligand interactions and revealed how small molecules can induce structural rearrangements of RNA molecules. Moreover, these structural insights lay the foundation for the rational design of therapeutics targeting pathogenic repeat expansion RNAs and for improving computational predictions of RNA–ligand recognition.

 

Acknowledgment: NCN UMO-2022/45/B/NZ7/03543 and UMO-2017/26/E/NZ1/00950; PLGrid PLG/2025/018816 and PLG/2026/019240.

 

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D12
Structural interplay between the ribosomal RNA anti-termination complex (rrnTAC) and the nascent pre-16S rRNA RNase IIII target duplex

Presenting Author: Rachel L Redler

Freie Universiät Berlin, Departments of Biology, Chemistry, Pharmacy, Takustr. 6, 14195 Berlin, DE, r.redler@fu-berlin.de

Author(s)
Rachel L Redler¹, Dr. Zhennan Tian¹, Jörg Bürger¹, Dr. Tarek Hilal¹, Prof. Markus C Wahl¹

Ribosomal RNA (rRNA) is the limiting component whose synthesis and maturation sets the pace at which functional ribosomes can be assembled. The ability of bacterial RNA polymerase (RNAP) to resist termination and support co-transcriptional maturation of nascent rRNAs from the precursor transcript has been attributed to a dedicated anti-termination complex (rrnTAC). However, the precise mechanisms by which rrnTACs support rapid rRNA synthesis and maturation are not yet understood. We utilize cryoEM to study reconstituted rrnTACs containing the nascent pre-16S RNA duplex targeted by RNase III. As the individual strands of this duplex are separated by the long intervening 16S rRNA sequence, their ability to meet during ongoing rapid transcription represents a particular challenge to efficient 16S maturation. To assemble rrnTACs more closely recapitulating this in vivo context, we employ a strategy in which the RNA signal for rrnTAC assembly is separated from the RNA encoding the downstream arm of the RNase III target duplex. By varying the length of this downstream complementary region, we structurally characterize complexes representing potential intermediate states in RNase III target duplex formation. Analysis of such complexes in the presence and absence of RNase III enables us to probe the structural basis by which rrnTACs facilitate the initial steps required for 16S rRNA maturation: long-range formation and presentation of the substrate duplex to RNase III.

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V6
NMR-based secondary structure characterization of the West Nile Virus Frameshift Element RNA and investigation of small molecule binding

Presenting Author: Kathrin Skorodumov

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, skorodumov@nmr.uni-frankfurt.de

Author(s)
Kathrin Skorodumov¹, Ioanna Maria Andreadou¹, Dr. Anna Wacker¹, Dr. Christian Richter², Dr. Sridhar Sreeramulu¹, Prof. Dr. Harald Schwalbe¹

Programmed ribosomal frameshift is a regulatory mechanism for protein translation that occurs in many positive-stranded RNA viruses.^[1][2] It enables the expression of different proteins form the same mRNA sequence and is primarily controlled by cis-acting elements in the mRNA. In the West Nile Virus (WNV) the ‑1 PRF induces the translation of the NS1' protein, which plays a role in viral replication.^[3]

The structural characterization and understanding of the function of the frameshift element (FSE) in WNV and the investigation of frameshift-inhibiting ligands can contribute to the development of new antiviral pharmaceuticals.^[4][5]

Solution-state NMR spectroscopy was used to investigate the secondary structure of the FSE RNA. We confirmed the predicted structure^[6] of the RNA as a pseudoknot, forming two base pairing stems and possible additional base pairs in the loop regions. The addition of Mg²⁺ showed a stabilizing effect on the pseudoknot formation.

A fragment library NMR screening was carried out to identify small molecule fragments as binders. Using structural information from the chemical shift assignment, we identified possible binding sites of these fragments in the FSE RNA.

To investigate the activity of fragment binders in ribosomal translation, we are establishing a cell-free frameshift assay in mammalian cell lysates. The use of dual reporter constructs will allow us to determine the ratio of produced proteins and detect changes in the frameshift efficiency.

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E1
Expanding the crystallographic toolbox: FAB-assisted crystallisation of DNAzymes

Presenting Author: Zoé Kürsteiner

ETHZ, Vladimir-Prelog-Weg 1-5 / 10, 8093 Zürich, CH, zoe.kuersteiner@pharma.ethz.ch

Author(s)
Zoé Kürsteiner¹, Dr. Artur Łaski¹, Dr. Kenny Jungfer², Dr. Pascal Röthlisberger¹, Dr. Andreas Gloger¹, Dr. Jacqueline Mock¹, Prof. Dr. Jörg Scheuermann¹, Prof. Dr. Martin Jinek², Dr. Stefanie Jonas¹, Prof. Dr. Jonathan Hall¹

The growing interest in nucleic acids as functional molecules has increased the need for structural information to assist the engineering of nucleic acids for therapeutic or biosensing applications. However, obtaining well-diffracting crystals remains a major bottleneck in nucleic acid structure research. Nucleic acid crystallisation is inherently challenging: the uniformly charged flexible backbone and the four natural bases offer limited tertiary contacts and disfavour crystal packing by electrostatic repulsion. Consequently, only about 8% of structures in the Protein Data Bank contain nucleic acids.

To overcome these challenges, protein co-crystallisation has emerged as a powerful strategy, although only few proteins serve as truly robust crystallisation chaperones. A proven crystallisation chaperone is the antigen binding fragment (FAB) BL3-6 developed by the Piccirilli lab. Its RNA binding motif can be engrafted onto diverse nucleic acid sequences and enabled the solving of several RNAs, but so far has limited broader adoption.

In our work, we improved on the production of FAB BL3-6 and applied it for the first time to crystallise catalytically active DNA sequences (DNAzymes). We synthesized the DNAzymes as RNA-DNA chimeras to contain the FAB-binding RNA pentaloop. The method yielded reproducible, well-diffracting crystals. This demonstrated the robustness of the system and extends the use of FAB BL3-6 to elusive DNA structures, expanding the crystallographic toolbox.

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P5
Deciphering the structural landscape of human p53 mRNA: from secondary structure mapping to cryo-EM analysis

Presenting Author: Leszek Błaszczyk

Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań, PL, blaszcz@ibch.poznan.pl

Author(s)
Dr. Monika Jóźwiak¹, Dr. Angelika Andrzejewska-Romanowska¹, Dr. Paweł Joachimiak¹, Adam Ciesiołka², Prof. Agnieszka Kiliszek¹, Prof. Katarzyna Pachulska-Wieczorek¹, Prof. Agnieszka Fiszer¹, Prof. Chantal Pichon³, Dr Leszek Błaszczyk¹

The tumor suppressor p53 is a central regulator of cellular homeostasis. Dysfunction of the p53 pathway is a common event in human cancers, making p53 one of the most intensively studied proteins. Although extensive research has focused on the p53 protein and its regulatory network, increasing evidence indicates that structural elements within p53 mRNA actively participate in the regulation of p53 expression. However, the global structural organization of p53 mRNA and the functional significance of its structural motifs remain poorly understood.

Here, we present a strategy aimed at building a structural atlas of human p53 mRNA. Using in vitro and in vivo high-throughput RNA structure probing, we investigate the global secondary structure of p53 mRNA and identify conserved motifs with potential functional importance. We further examine the influence of different cellular stress conditions on p53 mRNA folding and assess the structural dynamics of the transcript by probing its thermal stability. The obtained structural information is used to select RNA motifs for functional characterization and cryo-EM analysis. This approach is expected to reveal previously uncharacterized structural determinants governing p53 mRNA function, providing insights into the role of RNA architecture in the regulation of p53 expression.

This research was funded by National Science Centre, Poland, UMO-2024/53/B/NZ5/01942 and PLGrid PLG/2025/018816 and PLG/2026/019240

 

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O13
Deciphering the RNA binding preferences and mRNA targets of Roquin proteins

Presenting Author: Lasse Oberstrass

Universität Marburg, Fachbereich Pharmazie | Institut für Pharmazeutische Chemie, Marbacher Weg 6, 35037 Marburg, DE, oberstrass@uni-marburg.de

Author(s)
Dr. Lasse Oberstrass¹, Dr. Jan-Niklas Tants², Prof. Dr. Andreas Schlundt³, Prof. Dr. Julia Weigand¹

Cis-regulatory elements in the untranslated regions of mRNAs are recognized by RNA-binding proteins (RBPs) to control posttranscriptional regulation. Most studied RBP‑RNA contacts rely on short, single‑stranded sequence motifs. However, many proteins can recognize RNA 3D structure. So, both the sequence and folding must be considered.

Roquin proteins are key posttranscriptional regulators of immune responses and serve as a model for studying the recognition of structured RNA motifs. Upon binding a target mRNA, Roquin triggers its decay. Roquin contains two RNA-binding domains: 1) The unique ROQ domain, which recognizes the shape of stem-loops, and 2) a CCCH-type zinc finger, whose RNA‑binding preference had been unclear.

We applied RNA Bind-n-Seq (RBNS) to screen for binding motifs in vitro using completely randomized or pre-structured RNA pools. Specifically, we introduced sRBNS (structured RBNS) to characterize the preferred size and nucleotide composition of stem-loops recognized by the ROQ domain. We used these motifs as a blueprint to bioinformatically predict binding sites in the human transcriptome and verify novel target mRNAs in cells. We also found that the Zinc finger contributes to target recognition by binding of two adenines spaced by at least one uracil next to Roquin stem-loop motifs. 

Our study provides a platform for detailed analysis of RBP‑RNA interactions, enabling discovery of non‑canonical RBP binding preferences, involving complex binding events.

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P6
Targeting c-myc IRES structures with antisense oligonucleotides

Presenting Author: Melanie Flohr

Goehte-Universität Frankfurt a.M., Institute of Biochemistry I, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, DE, me.flohr@biochem.uni-frankfurt.de

Author(s)
Melanie Flohr¹, Dr. Giulia Cardamone¹, Nina Kaltenschnee², Prof. Dr. Alexander Heckel², Dr. Tobias Schmid²

Translation is a highly regulated process that is mainly controlled at its initiation step. Under physiological conditions, translation is predominantly initiated by a canonical cap-dependent mechanism which is inhibited during tumor-associated stress, such as hypoxia or nutrient limitation. Under these circumstances, tumor relevant proteins are translated by cap-independent mRNA specific mechanisms, such as internal ribosome entry site (IRES)-mediated translation, to ensure tumor growth. In the present study, we focused on the transcription factor c-myc known to regulate critical functions during tumorigenesis. Regarding c-myc translation, an IRES-mediated mechanism within the c-myc 5’ untranslated region has been identified, which was reported to be highly activated during tumorigenesis. Therefore, this study aimed at the characterization of the c-myc IRES structure and activity. We validated the c-myc IRES and identified two regions that are potentially responsible for the IRES activity. We further modulated the c-myc IRES activity by targeting the identified structures with antisense oligonucleotides that are designed to interfere with the RNA structure. Therefore, our study provides insights into whether targeting the c-myc IRES could be a strategy to regulate tumor-associated c-myc expression.

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P7
Fragment-based NMR screening and NMR structure determination to support development of high-affinity binders to CAG repeat RNA

Presenting Author: Julia Schäfer

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, j.schaefer@nmr.uni-frankfurt.de

Author(s)
Julia Schäfer¹, Dr. Henry Jonker¹, Dr. Sridhar Sreeramulu¹, Christian Richter¹, Dr. Anna Wacker², Prof. Dr. Harald Schwalbe¹

The expansion of CAG repeats beyond a gene-specific threshold leads to nine neurodegenerative diseases, including Huntington's disease. The disease pathogenesis is caused by the elongated stretches of polyglutamine polypeptide chains and by a CAG-only hairpin in the mRNA, which is only present in the pathological mRNA. Previous studies have shown that targeting the A-A mismatches within this hairpin can alleviate the toxicity in infected cells.

Using the iNEXT DSI-poised library  containing 607 fragments for NMR ligand observed fragment based screening, 81 initial hits were found. Single-compound screens of the best 42 of these hits confirmed 32 of them as binder to r(gG(CAG)₈Cc). RNA-observed binding site mapping was conducted for the twelve highest-priority hits, all of which induced CSPs on the adenosine H8, H2 and guanosine H1′ resonances. For three of the compounds, intermolecular NOEs to the RNA were observed, with one showing 19 intermolecular NOEs and a K_D of 60 µM. This molecule is currently being used with the intermolecular NOE contacts as restraints for docking simulations using HADDOCK.

The project aims to target the A-A mismatch of CAG repeat RNA using small molecules identified through NMR detected screening of fragments, alongside an NMR-based hit-to-lead strategy within RNA constructs with single or several A-A mismatches.  Such studies allow to exploit neighbouring A-A mismatches to increase binding specificity for RNA binders.

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E2
New Tools and Workflows for the NMR-Based Structural Analysis of RNA

Presenting Author: Jennifer Adam

Goethe - Universität Frankfurt a. M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, adam@nmr.uni-frankfurt.de

Author(s)
Dr. Christian Richter, Dr. Philip Wurm, Matthias Becker, David Leopold, Dr. Henry Jonker, Dr. Anna Wacker, Dr. Daniel Mathieu, Dr. Rüdiger Weisemann, Harald Schwalbe

Given the growing importance of RNA NMR spectroscopy, the research group of Prof. Harald Schwalbe at the Goethe University Frankfurt has collaborated with Bruker Biospin to compile a comprehensive collection of established and newly developed experiments for characterizing unlabeled, ¹⁵N-labeled, and ¹³C/¹⁵N-labeled RNA¹. To facilitate implementation, these experiments are integrated into Bruker’s “BioTop” tool, which enables a straightforward setup (even for inexperienced researchers). Moreover, the procedure and approaches are supported by a web-based tutorial covering RNA sample preparation, experiment selection, and step-by-step workflows for data analysis.

Initiatives such as Instruct-ERIC² play a crucial role in facilitating access to such advanced methods in structural biology. This infrastructure provides researchers with funded, peer-reviewed access to specialized technologies that are essential for understanding human physiology and for drug discovery. Following Germany’s accession to Instruct-ERIC, researchers will receive free access to state-of-the-art high-field NMR instruments (600 MHz to 1.2 GHz) housed at Goethe University Frankfurt, as well as full coverage of travel and accommodation expenses.

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O14
Human tRNA splicing in health and disease: structural insights into the TSEN–CLP1 complex

Presenting Author: David Schumacher

Goethe - Universität Frankfurt, Biochemie, Max-von-Laue Str. 9, 60438 Frankfurt am Main, DE, da.schumacher@em.uni-frankfurt.de

Author(s)
David Schumacher¹, Charlotte Wiegand¹, Dr. Samoil Sekulovski¹, Dr. Simon Trowitzsch¹

Pre-tRNA splicing is essential for tRNA maturation and accurate protein synthesis. In humans, intron excision is catalyzed by the tRNA splicing endonuclease (TSEN), which associates with the RNA kinase CLP1. Mutations in TSEN and CLP1 cause pontocerebellar hypoplasia (PCH), but the molecular consequences remain poorly understood. Using single-particle cryo-EM, we determined two high-resolution structures of human TSEN bound to a chimeric pre-tRNA substrate containing an archaeal bulge–helix–bulge motif and a human tRNA body. The structures capture distinct stages of substrate processing and reveal splice-site organization during catalysis. We further mapped the CLP1–TSEN54 interaction by pull-down assays and peptide spot arrays, identifying three interaction sites within the intrinsically disordered region of TSEN54. The PCH-associated mutations TSEN54 A307S and CLP1 R140H significantly weakened CLP1–TSEN binding, while phosphomimetic substitutions at TSEN54 A307 abolished binding, suggesting a role for post-translational modifications in disease. Finally, the cryo-EM structure of apo CLP1 revealed a previously uncharacterized dimeric assembly, with R140 located at the dimer interface, indicating that the PCH mutation destabilizes CLP1 oligomerization. Together, our findings provide a structural framework for TSEN–CLP1 function and support convergent disease mechanisms in which impaired protein interactions and defective complex assembly contribute to PCH pathogenesis.

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O15
Systematic identification of destabilizing 3’-UTRs in human mRNAs

Presenting Author: Friederike Kollmeier

Philipps-Universität Marburg , AG Weigand, Marbacher Weg 6 , 35037 Marburg, DE, kollmei4@staff.uni-marburg.de

Author(s)
Friederike Kollmeier¹, Dr. Chiara Lichtenthäler, Dr. Lasse Oberstraß, Dr. Sara Ali, Prof. Dr. David Mathews, Prof. Dr. Julia Weigand

 

Considering untranslated regions (UTRs) make up a large percentage of mammalian mRNAs and play a pivotal role in gene expression, for example controlling mRNA localization, degradation and translation efficiency, a lot is still unknown about them. mRNA regulation occurs by cis-regulatory elements located in UTRs that are recognized by RNA-binding proteins (RBPs), which manipulate the mRNA, for example inducing mRNA degradation. While sequence-specific RBP binding is well understood, RBPs are also able to bind specifically to RNA structures. However, this mode of RNA recognition and its role in gene regulation is hardly understood so far. Therefore, we searched for previously unknown structured cis-regulatory elements in human 3'-UTRs by bioinformatic prediction of evolutionarily conserved RNA structures across five mammals. Via this approach we discovered 3700 candidate elements and determined their structure in vitro using SHAPE-MaP. Further, we tested whether these candidates have an effect on mRNA degradation by stably integrating a reporter plasmid pool into HeLa cells and performing a massively parallel reporter assay, with Actinomycin D treatment followed by time course sampling and NGS sequencing. Analyzing the NGS data, we were able to identify ~180 destabilizing 3'-UTR structures. Currently, we analyze how mRNA folding affects mRNA degradation rates and perform nascent-chain pulldowns to discover mRNA structures that affect translation efficiency.

 

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P8
SPHERE: SELEX and SHAPE-MaP for High-Resolution Exploration of Riboswitch Elements

Presenting Author: Inge Schwedt

Marburg University, FB16 Pharmacy, Marbacher Weg 6, 35037 Marburg, DE, schwedt@uni-marburg.de

Author(s)
Dr. Inge Schwedt¹, Vincent Gunawan², Dr. Marcus Lechner¹, Prof. Dr. Beatrix Suess², Prof. Dr. Julia Erika Weigand¹

 

Riboswitches are regulatory RNA-elements, containing an aptamer domain, which binds a ligand with high affinity, and an expression platform, allowing ligand dependent gene regulation. Natural riboswitches are mostly present in bacteria and bind to metabolites, limiting their suitability for synthetic biology. Synthetic riboswitches to any ligand are generated by systematic evolution of ligands by exponential enrichment (SELEX), which results in high affinity aptamers, but requires subsequent mutational studies to identify and optimize the ligand binding pocket. We utilized selective 2ˈ-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to structurally probe a SELEX-pool, enabling the identification of the ligand binding pocket at nucleotide resolution. We developed MultiShaper, a sorting algorithm fitted for SELEX-SHAPE-MaP data to reliably calculate SHAPE-reactivity profiles for each aptamer and could show, that a change in SHAPE-reactivity within an internal loop upon ligand binding is a strong indicator for riboswitch potency. This way, we managed to identify a new riboswitch showing a base dynamic range of regulation comparable to unoptimized riboswitches, speeding up riboswitch discovery. SPHERE aids with riboswitch discovery and is therefore a valuable tool for synthetic biology approaches, like selective gene expression control, biosensors or genetic circuits.

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O16
Exploring CsrA Binding Motifs with RNA Bind-n-Seq

Presenting Author: Louisa Koch

Marburg University, Pharmacy, Marbacher Weg 6, 35037 Marburg, DE, louisa.koch@pharmazie.uni-marburg.de

Author(s)
Louisa Koch¹, Dr. Lasse Oberstraß¹, Prof. Dr. Julia E. Weigand¹

The RNA-binding protein (RBP) CsrA is a global post-transcriptional regulator in E. coli, controlling carbon metabolism, motility, and virulence pathways by binding to target mRNAs and interfering with translation initiation. CsrA typically interacts with short sequence motifs in the 5' UTR of transcripts, thereby modulating ribosome accessibility. A canonical binding consensus sequence (ANGGA) was originally identified by SELEX experiments. Strikingly,  not all verified CsrA targets match this motif, suggesting that additional sequence or structural features could influence RNA recognition.

Therefore, we applied RNA Bind-n-Seq (RBNS), a high-throughput technique for systematically identifying the RNA-binding motifs of an RBP of interest within randomized RNA libraries thus providing a quantitative view on motif enrichment across diverse sequence contexts. 

Our data confirms the canonical ANGGA element; however, the enrichment of sequence variants suggests that binding preferences may extend the strict consensus sequence. These findings motivate further exploration of contextual features that may modulate recognition. In order to achieve this, we are implementing structured RBNS (sRBNS) with partially constrained RNA pools to directly interrogate potential contributions of RNA secondary structure to binding specificity. This strategy refines the CsrA binding landscape beyond the canonical consensus, improving the characterization of sequence and structural determinants.

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E3
Mechanical Folding of mRNAs Revealed by Single-Molecule Force Spectroscopy

Presenting Author: Borja Ibarra

FUNDACIÓN IMDEA NANOCIENCIA , Campus Cantoblanco, Faraday 9, 28049 Madrid, ES, borja.ibarra@imdea.org

Author(s)
Dr. Borja Ibarra¹, Dr. Rebeca Bocanegra¹, Dr. Xavier Viader-Godoy², Dr. Maria Manosas², Prof. Dr. Felix Ritort²

Unlike highly structured non-coding RNAs, messenger RNA (mRNA) does not generally adopt a single native fold; instead, their global organization emerges from a dynamic balance between local base pairing, base stacking, and electrostatic interactions. How these competing interactions determine the mechanical behavior and folding of mRNAs remains poorly understood.

Here, we used optical tweezers to measure force–extension curves of individual mRNAs, spanning different sequences and lengths. All molecules exhibited largely reversible force–extension behavior without signatures of large stable structural transitions. Nevertheless, each RNA displayed a characteristic mechanical fingerprint, with sequence- and context-dependent mechanical properties. Increasing ionic strength reduced molecular extension, consistent with salt-dependent stabilization of transient intramolecular interactions, although the RNAs remained resistant to complete globular collapse.

We developed a cooperative three-state model in which nucleotides populate compact, stacked, or unstacked domains. The model reproduces force–extension curves across RNA sequences and ionic conditions, resolving the energetic interplay between compaction, stacking, and electrostatic screening. Our results provide a physical framework to understand how mRNAs maintain structured yet translation-compatible conformational ensembles.

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P9
smART: specific mRNA tARgeTing

Presenting Author: Julia Wirmer-Bartoschek

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, wirmer@nmr.uni-frankfurt.de

Author(s)
Dr. Julia Wirmer-Bartoschek¹, Prof. Dr. Julia Weigand², Prof. Dr. Harald Schwalbe¹

 

Ribonucleic acids (RNAs) are among the most essential building blocks of cells. They are part of the information transfer that converts genetic information into cellular function. Along this information path, pre-messenger RNA (pre-mRNA) and messenger RNA (mRNA) are functionally highly relevant in governing genome diversity and protein synthesis. In addition, they play key roles in regulating subcellular architecture and localization. mRNAs adopt 3D structures that are targeted by a variety of ways: by metabolites, nucleic acids and proteins. mRNA targeting has evolved in Nature, and medicinal chemistry and synthetic biology follow Nature’s principles for targeted manipulation of mRNAs.

Our overall scientific approach in this project is to exploit a key property of pre-mRNA and mRNA to modulate cellular function: the formation of functional three-dimensional structures. Our long-term vision is to create a versatile toolbox to target any mRNA at any location within eukaryotic cells.

We will modulate mRNA function in three essential cellular processes: splicing, translation, and mRNA abundance. In Focus Area A – Splicing, we will design, synthesize and evaluate small molecules that target pre-mRNA. In Focus Area B – Translation, we will investigate how mRNA structure and modifications modulate protein output. Focus Area C – Abundance aims at understanding how proteins decorate and protect mRNA to advance packaging technologies for delivery and protection of fragile mRNAs.

 

 

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P10
Structure or Sequence dependent binding of RBPs on 3'-UTR

Presenting Author: Anna Schlauersbach

Philipps-Universität Marburg, AG Weigand, Marbacher Weg 6, 35037 Marburg, DE, schlauea@staff.uni-marburg.de

Author(s)
Anna Schlauersbach¹

3'-untranslated regions (3'-UTRs) control mRNA levels by encoding cis-regulatory elements. Such 3'-UTR elements can be evolutionarily conserved across different species indicating a crucial role in gene regulating processes such as mRNA stability. The decay of mRNAs is controlled by RNA-binding proteins (RPBs) recognizing the cis-regulatory elements by sequence, structure or both. An example for RBPs inducing the degradation of mRNAs are the Roquin-1 and -2 proteins. Both are major immunoregulators suppressing inflammatory reactions. Roquin recognizes stem-loop structures in the 3'-UTRs of their targets known as ADEs and CDEs – alternative and constitutive decay elements – via their ROQ domain [Braun et al. 2018; Oberstrass et al. 2024]. Interestingly, Roquin proteins recognize AU-pure stem-loops, which can be recognized in their linear form by AU-rich binding proteins, such as AUF1/HNRNPD [Binas et al. 2020]. Using RBNS (RNA Bind-N-Seq), we unravel RNA-binding motifs of Roquin for which it has to compete with other mRNA degradation inducing RBPs like AUF1. Further, we want to investigate the sequence and folding dynamics of shared RNA motifs recognized by Roquin and AUF1 and if there is a competitive dynamic between these two RBPs to fine-tune mRNA levels.

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E4
Development of pseudo-4D experiment for sequential RNA backbone assignment

Presenting Author: Florian Amend

Goethe-Universität Frankfurt a.M., Institut für Organische Chemie und Chemische Biologie, Max-von-Laue-Str. 7, 60438 Frankfurt, DE, amend@nmr.uni-frankfurt.de

Author(s)
Florian Amend¹, Jennifer Adam¹, Dr. Jihyun Kim², Dr. Christian Richter¹, Prof. Dr. Lucio Frydman², Prof. Dr. Harald Schwalbe¹

 

In the recent years, numerous RNA functions have been discovered enabled by a high diversity of flexible structural elements. In these regions, imino resonances are often undetectable due to solvent exchange. An alternative is the NOE-based sequential backbone assignment, using correlations of aromatic (H6/H8) and ribose protons. However, even 3D experiments are limited by low chemical shift dispersion, making assignments challenging even for medium-sized RNAs. A 4D HMQC-NOESY-HMQC experiment method helps to overcome this limitation but requires significantly longer measurement times.

Previously, we showed that J-driven cross-polarization selectively inverts ¹H resonances, reducing a 3D NOESY-HMQC experiment to a series of selective 1D spectra. Here, we adapted this approach to the aromatic ¹³C-¹H spin pairs of a 24 nt long RNA and added a NOESY mixing period. HSQC detection targeted either C1′H1′ or all ribose resonances. Low power pulses under Hartmann-Hahn matching conditions selectively inverted individual ¹H resonances. Transfer times were optimized and the effect of field strength was studied. Purines showed high selectivity at 1.2 GHz, whereas pyrimidines exhibited reduced selectivity due to additional C–C couplings. NOE signals were enhanced by looping the selective inversion and mixing time.

The method reduces measurement time from 7 days for the 4D experiment to 1 day (C1’H1’) or 2 days (all riboses) while enabling targeted analysis of selected resonances.

 

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O17
Circular RNA aptamers to modulate MAPK signaling

Presenting Author: Simon Marpert

Universität Bonn, LIMES Institute, Gerhard-Domagk-Straße 1, 53121 Bonn, DE, smarper1@uni-bonn.de

Author(s)
Simon Marpert¹, Noushin Zahedi¹, Friederike Schlotmann¹, Thea Schinkel², Philipp Simon³, Katrin Paeschke³, Julian Válero Moreno², Günter Mayer¹

 

Signalling pathways coordinate diverse biological processes and are frequently dysregulated in disease, making them attractive targets for mechanistic study and therapeutic intervention. Intracellular aptamers (intramers) offer a powerful tool for the selective modulation of protein function, however achieving sufficient intracellular concentrations for effective target inhibition remains challenging. Here, we employ the Tornado expression system to express the Erk2-targeting aptamer C5 as a circular RNA. The C5 aptamer is embedded alongside fluorogenic light-up aptamers (Broccoli, Okra, Pepper or RhoBAST), enabling real-time visualization of inhibitory RNA expression. Tornado-Broccoli-C5 suppresses MAPK pathway activity, resulting in diminished SRE-dependent reporter signal in HEK293T, HeLa and HCT116 cells. Expression of circular RNA does not induce detectable cellular stress levels in HEK293T cells as assessed by eIF2α phosphorylation and G3BP1-dependent stress granulae formation. A preliminary cryo-EM structure of the Tornado-Broccoli-C5 in complex with Erk2 underlines the modularity of the bifunctional RNA and provides initial insights into the structural basis of target recognition for future aptamer optimization. Together, our findings highlight the Tornado platform as a versatile strategy for the intracellular expression of functional RNA aptamers and underscore the potential of intramers as inhibitory modulators of intracellular signalling networks.

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V7
ApeA cleaves genomic RNA to defend against RNA phage infection

Presenting Author: Jens Hör

Helmholtz Institute for RNA-based Infection Research (HIRI), Josef-Schneider-Str. 2 / D15, 97080 Würzburg, DE, jens.hoer@helmholtz-hiri.de

Author(s)
Arina Drobysheva¹, Dr. Manuel Velasco Gomariz², Dr. Shazeb Ahmad¹, Sarah Reichardt², Prof. Dr. Jens Hör²

To protect themselves against viral infection, bacteria encode a vast diversity of antiphage defense systems. However, the mechanisms of action of most of these systems have exclusively been studied using phages with DNA genomes as the models, while phages with RNA genomes remain understudied. Here, we investigated how the defense system ApeA confers resistance against RNA phage infection. We show that two ApeA homologs, Ec1ApeA and Ps2ApeA, protect against a variety of single-stranded RNA phages. Focusing on Ec1ApeA, we find that it senses infection through a conserved pocket that binds an RNA structure in the phage genome. This activates the HEPN (higher eukaryotes and prokaryotes nucleotide-binding) RNase domain of Ec1ApeA which consequently cleaves the phage genomic RNA to restrict replication. In contrast to many other described defense systems, Ec1ApeA activity directly stops viral replication without inducing cell death, establishing ApeA as a non-abortive defense system that protects against RNA phages. Our results add to the increasingly diverse targets of antiviral HEPN RNases and provide insights into the understudied field of RNA phage defense.

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D13
The histone mRNA stem-loop structure is modulated during replication-dependent histone mRNA decay

Presenting Author: Sarah Lewandowski

Freie Universiät Berlin, BCP, Luitpoldstr. 28, 10777 Berlin, DE, sarah.lewandowski@fu-berlin.de

Author(s)
Sarah Lewandowski¹, Dr. Alexandrina Machado de Amorim¹, Emma A. Kautz², Dr. Mario Schubert³, Prof. Dr. Florian Heyd², Prof. Sutapa Chakrabarti¹

Metazoan replication-dependent (RD) histone mRNAs are produced and expressed during S-phase of the cell cycle. These mRNAs are unusual in that they have a unique stem-loop (SL) structure at the 3’-end instead of a poly(A)-tail. This stringently conserved SL is bound by the stem-loop binding protein (SLBP) and the 3’-5’ exoribonuclease (3’hExo). Previous studies on RD histone mRNA suggest that SLBP protects the histone SL from 3’-end degradation during the S-phase while 3’hExo initiates decay of the SL at the end of the S-phase. Our recent work indicates that while SLBP does marginally stabilize the histone SL, the intact SL consisting of a G:C-rich stem is resistant to degradation by 3’hExo. Initiation of 3’-end decay requires the concerted action of SLBP, 3’hExo and the RNA helicase UPF1. Stable SL intermediates generated by 3’hExo are oligouridylated and rapidly degraded in cells by poly(A) mRNA-decay factors. We propose that SL-unwinding by UPF1 and oligouridylation of the SL-3’-end collectively destabilize the structure of the intact histone SL and SL-degradation intermediates to make them amenable to degradation. Using a combination of biochemical and structural methods, we compare the structural stability of the intact and the oligouridylated SL-degradation intermediates and correlate it to their propensity for decay. Our work suggests that the SL is the major determinant of histone mRNA stability and modulation of SL-structure is a key step in RD histone mRNA decay.

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D14
Translation regulation in immune cell differentiation - a case study on dFmr1

Presenting Author: Jonathan Lenz

Marburg University, Institute of Molecular Biology and Tumor Research, Hans-Meerwein-Str. 2, 35039 Marburg, DE, jonathan.lenz@imt.uni-marburg.de

Author(s)
Dr. Jonathan Lenz¹, Marco Amend¹, Marie Unverzagt¹, Ole Pielhoop¹, Dr. Witold Szymanski¹, Dr. Andrea Nist¹, Prof. Dr. Johannes Graumann¹, Prof. Dr. Thorsten Stiewe¹, Prof. Dr. Sven Bogdan¹, Prof. Dr. Alexander Brehm¹

Generation and translation of lineage-specific mRNA is central to cellular differentiation. While the field has focussed on the transcriptional regulation, post-transcriptional mechanisms of differentiation control remain insufficiently understood. We use Drosophila melanogaster hematopoiesis as a paradigm to study molecular processes during differentiation.

We have identified a novel pathway that suppresses immune cell differentiation involving Casein kinase 2 (CK2) and Fragile X Messenger Ribonucleoprotein 1 (dFmr1), a regulator of translation. Mutation of human  FMR1 causes Fragile X syndrome (FXS), a neurodevelopmental disorder that impacts both the central nervous system as well as immune responses. The role of FMRP in the immune system has not been systematically investigated. We propose that dFmr1 phosphorylation by CK2 is required to block translation of lineage-specific mRNAs, thereby restricting inappropriate differentiation.

To define molecular mechanisms of translation inhibition by CK2 and dFmr1 we aim to
1) identify the phosphorylation-dependent RNA interactome of dFmr1,

2) investigate effects of dFmr1 and its phosphorylation on the translatome,

3) study the influence of dFmr1 phosphorylation and its targets on hematopoiesis in vivo.

This will contribute to a better understanding of post-transcriptional differentiation control and its implications in FXS.

 

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O18
A minimal regulatory module controls chloroplast transcription in the red alga Cyanidioschyzon merolae

Presenting Author: nelly said

Freie Universitat Berlin, Structural biochemistry, Takustrasse 6, 14195 Berlin, DE, nellysaid@zedat.fu-berlin.de

Author(s)
Dr. nelly said¹, Dr. Tarek Hilal², Prof. Dr. Daniel Schubert¹, Prof. Martha Stark³, Prof. Stephen Rader⁴, Prof. Dr. Dennis Nürnberg⁵

Chloroplasts originated from an ancient cyanobacterial endosymbiont and retain a bacterial-type, plastid-encoded RNA polymerase (PEP) essential for chloroplast gene expression. While land-plant PEP forms a ~1 MDa complex with numerous PEP-associated proteins (PAPs) of eukaryotic origin, algal PEP has been proposed to resemble a simple bacterial enzyme. However, its molecular organization remains unknown. Here, we combined native purification, single-particle cryo-electron microscopy and mass spectrometry to determine structures of the chloroplast RNA polymerase from the red alga Cyanidioschyzon merolae. We identify three previously uncharacterized nucleus-encoded proteins, CmPAP5, CmPAP15 and CmPAPα, that are stably integrated into the polymerase and engage conserved surfaces occupied by multiple PAPs in land plants. CmPAP5 binds the dynamic β′-Si3 insertion, suggesting a role in regulating nucleotide addition, whereas CmPAPα stabilizes the complex by bridging the α- and ω-subunits. Elongation complex structures show that these proteins remain associated during active transcription while stabilizing the nucleic acid scaffold and dynamic regions of the enzyme. Our findings reveal that C. merolae PEP is not a bacterial-like enzyme but a structurally elaborated transcription complex employing a minimal regulatory module, providing an evolutionary link between cyanobacterial RNA polymerase and the highly elaborate plant PEP machinery.

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O19
Disruption of the Pro-Survival AR/AP-1/CD44-DT Axis by a Novel PROTAC lncRNA Suppresses Prostate Cancer Growth and Induces Senescence

Presenting Author: Forough Hakiminia

Universitatsklinikum Jena, Am Klinikum 1,, 07747 Jena, DE, forough.hakiminia@uni-jena.de

Author(s)
Forough Hakiminia¹, Julia Kallenbach², Katrin Schindler², Golnaz Atri Roozbahani², Aria Baniahmad², Dr. Malek Hossein Asadi³

Aberrant androgen receptor (AR) signaling driven by androgens plays a critical role in prostate cancer (PCa) progression. Interestingly, bipolar androgen therapy (BAT), a novel therapeutic strategy with cycles between near-castrate and supraphysiological androgen levels (SAL), suppresses PCa growth. SAL induces cellular senescence in various PCa models. However, the regulatory function of oncogenic long non-coding RNAs (lncRNAs) in SAL-induced senescence remains poorly understood. Here, we investigated the role of CD44-DT in SAL- induced cellular senescence in PCa . TCGA data analysis revealed that CD44-DT is upregulated in low-grade PCa and shows coordinated expression with CD44, a stemness marker, across all analysed cancer types, likely due to co-regulation of transcription from a bidirectional promoter. SAL induces CD44-DT upregulation in PCa cells and in a castration- resistant xenograft mouse model. We further found that the divergent transcript lncRNA CD44- DT suppresses SAL-induced senescence in both PCa 2D cell culture and 3D tumor spheroids. Knockdown and overexpression experiments suggest that CD44-DT acts via AKT-p70S6K and E2F1-pRb pathways to inhibit cellular senescence. Transcriptome, ATAC-seq, and ChIP-seq analyses of SAL-treated PCa cells reveal AR recruitment to genomic region at the first intron of CD44 gene locus. The data also suggest that AP-1 is an upstream activator to upregulate CD44-DT expression in AR-positive PCa cells. Inhibition of the AP-1 complex or degradation of AR result in downregulation of CD44-DT expression. These findings suggest that CD44- DT acts as a novel onco-lncRNA that promotes PCa growth and suppresses SAL-induced cellular senescence. Building on this mechanism, we engineered a chimeric lncRNA capable of ubiquitinating and degrading the AR. This lncRNA-degrader downregulates CD44-DT and dramatically restricts PCa growth as a novel type of PROTAC. Thus, the data suggest the identification of a novel direct AR target gene that acts as an oncogenic lncRNA in the context of SAL and may serve as a potential therapeutic target to enhance the efficacy of BAT.

Keywords: Prostate cancer, Androgen receptor, Supraphysiological androgen level, Cellular
senescence, CD44-DT, PROTAC lncRNA

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