Research at NUCLEATE

RNA-binding proteins (RBPs) of the STAR family play key roles in mammalian development, yet their contributions to lineage specification remain incompletely understood. Here, using CRISPR-Cas9 knockout models combined with multi-omics approaches, we investigate the functions of two STAR proteins, SAM68 and QKI, in mouse embryonic stem cells (mESCs). Both RBPs support mESC proliferation, self-renewal, and efficient differentiation into cardiomyocytes. Although SAM68 and QKI belong to the same protein family, they control largely distinct regulatory programs during differentiation. We uncover an unexpected role for SAM68 in cardiomyocyte specification through multiple post-transcriptional mechanisms. SAM68 modulates alternative splicing and promotes the biogenesis of a subset of cardiac-enriched circular RNAs, through binding to intronic regions flanking back-splice junctions and potentially through association with NF90/110. In addition, SAM68 binds untranslated regions of key differentiation-related transcripts, including Gata4 mRNA, and functions in ribonucleoprotein complexes to regulate their translation. Together, these findings identify SAM68 as a multifunctional regulator coordinating multiple layers of RNA metabolism-including splicing, circRNA biogenesis, and translation-during cardiomyocyte differentiation and provide insight into how STAR proteins shape post-transcriptional gene regulatory networks during early development.

Ribozymes for site-specific RNA modification provide an elegant approach for the installation of diverse functional groups, fluorophores, affinity tags, or crosslinkers at defined positions within an RNA of interest. There is increasing interest in expanding the ribozyme toolbox, since recently reported in vitro selected ribozymes have been mostly limited to labeling at adenosine sites, either by alkylation of the nucleobase or phosphodiester formation at the 2'-OH group. Here we report a cytidine-specific alkyltransferase ribozyme (CSAR) that uses O-benzylguanines as alkyl group donors. CSAR is the first ribozyme that catalyzes direct alkylation of the exocyclic amino group of a nucleobase and generates N-alkylated cytidine in a defined sequence context of a short RNA hairpin loop. In combination with tuning the electronic parameters of the transferred benzyl group, CSAR enables highly efficient cytidine alkylation for the installation of bioorthogonal functional groups.

Respiratory syncytial virus (RSV) causes severe lower respiratory disease, yet how it reshapes airway epithelial cells and evades innate immunity remains incompletely understood. We infected adult primary human airway epithelial cultures with RSV and analyzed infected and bystander cells over time using single-cell RNA sequencing and imaging. RSV mainly infected ciliated cells, triggering a virus load-dependent shutdown of genes involved in ciliogenesis, antigen presentation, and innate sensing, including key interferon (IFN) and pattern recognition pathways. Only a subset of infected cells produced type I and III IFNs, while bystander cells exhibited strong IFN-stimulated gene (ISG) signatures. Neither IFN treatment nor ISG induction eliminated infection, but IRF1, an antiviral transcription factor not suppressed by RSV, remained robustly expressed. Ectopic IRF1 expression in vitro reduced viral replication. These findings reveal how RSV evades antiviral defenses and highlight IRF1 as a potential target for therapeutic intervention.

Alternative splicing (AS) is a key layer of regulation in eukaryotic gene expression that is investigated in all areas of life sciences. Differences in AS between conditions can be quantified from transcriptome-wide short-read RNA sequencing (RNA-seq) data with designated computational tools. However, not all short-read RNA-seq data are equally suited for AS analysis. Here, we perform an exemplary AS analysis to showcase the impact of the RNA-seq library characteristics on the obtained results. Using two standard ENCODE data sets with widespread AS changes, we modulate read length and read depth and compare their influence on the detection, quantification, and classification of AS events with the state-of-the-art AS algorithm MAJIQ. We find that both longer reads and higher read depth are effective measures to improve the sensitivity and precision of the AS analysis. Our results provide valuable insights to help researchers make informed decisions when choosing the short-read RNA-seq library specifications for AS analysis.

Deoxyribonucleic acid (DNA) enables the precise arrangement and positioning of chromophores in order to study their interactions, leading, for example, to through-space energy transfer processes. BNB- and NBN-doped phenalenyls are electronically complementary fluorophores that are neutral BN/CC isosteres of the phenalenyl cation and anion, respectively. Herein, we present a pair of BNB- and NBN-doped phenalenyl-extended nucleosides, which we introduced into DNA via phosphoramidite chemistry. The two chromophores act as a donor-acceptor pair in a Förster resonance energy transfer (FRET) process, which results in the quenching of the BNB-phenalenyl fluorescence due to the nonradiative decay of the charge transfer (CT) state of the NBN-phenalenyl acceptor in an aqueous environment. The DNA duplex serves as a supramolecular scaffold to control the arrangement of the interacting BNB- and NBN-doped chromophores. The performance of the fluorophore-quencher pair was evaluated in a toehold-mediated strand displacement (TMSD) experiment, demonstrating its potential for DNA-based applications.

Synthetic nucleic acid catalysts serve as powerful tools to interrogate and manipulate biological processes with high specificity. Advances in in vitro selection, high-throughput sequencing, and computational design have yielded increasingly efficient and chemically diverse ribozymes and deoxyribozymes. Beyond applications in nucleic acid research and diagnostics, the study of nucleic acid catalysts offers fundamental insights into the origin of life and the RNA world hypothesis. This review covers both fundamental and applied perspectives and summarizes recent advancements in the field of in vitro evolution and the development of synthetic ribozymes and DNAzymes. The topics include the latest research on RNA-ligases and polymerase ribozymes as well as ribozymes and DNAzymes for targeted modification of RNA and peptides including novel catalysts for site-specific methylation, alkylation, and acylation. Finally, the versatility of RNA-cleaving DNAzymes as sensors and for the detection of RNA modifications, as well as the ability of DNAzymes to catalyze light-activating reactions are presented.

Macrophages are essential for both, to clear pathogens and preserve tissue homeostasis, yet the molecular regulators of this equilibrium remain incompletely defined. Here, we identify SAILR (survival associated immune-regulatory RNA), a primate-specific long noncoding RNA (lncRNA), as a critical modulator of macrophage viability under infection conditions. SAILR is induced during monocyte-to-macrophage differentiation, but rapidly downregulated upon bacterial challenge in a nuclear factor kappa B (NF-κB) dependent manner. In both naïve and immune-activated macrophages, SAILR dampens the expression of adhesion, phagocytosis, and invasion factors, which include SIGLEC1 and MMP7. During infection with Typhimurium, depletion of SAILR sensitizes macrophages to apoptosis, resulting in loss of intracellular replication niches and reduced bacterial recovery. Conversely, enforced SAILR expression promotes macrophage survival and increases intracellular pathogen burden. Mechanistically, SAILR interacts with the antiapoptotic adaptor protein 14-3-3β to support macrophage survival. Notably, downregulation of SAILR is mirrored in circulating immune cells from patients with severe COVID-19 and sepsis. Together, our findings position SAILR as a central regulator in linking macrophage survival to host-pathogen interaction and disease pathophysiology.

RNA structure is central to the function of every RNA class yet the gap between annotated sequences and experimentally determined structures remains large. Computational methods to fill this gap have evolved from thermodynamic free energy minimization through supervised deep learning to self-supervised RNA language models trained on millions of sequences, progressively improving structure prediction. Here we review the state of the art in RNA structure prediction, covering key training datasets, community benchmarks, and the performance of current models. We further discuss perspectives on integrating other data modalities, such as chemical probing signals and RNA modifications, as well as the emerging role of generative models. Challenges in generalization, handling of noncanonical interactions, and contextual structure prediction remain open frontiers for the field.

The chemical richness of RNAs is greatly enhanced by post-transcriptional modifications with RNA methylation as the most prominent type. RNA modifications modulate the stability, folding and interaction pattern of RNA molecules. Furthermore, emerging data suggests RNA modifications also directly regulate the activity of catalytic RNA molecules, i.e., ribozymes. Here, we employ classical and hybrid quantum-classical (QM/MM) molecular dynamics (MD) simulations to investigate the reaction mechanism of an artificial methyltransferase ribozyme MTR1. Importantly, we pinpoint how 2'-O-methylations of active site nucleotides synergistically enhance ribozyme activity by reducing the conformational flexibility of the ribose rings and rigidifying the active site. Finally, the herein reported crystal structure of the modified MTR1, solved at 2.6 Å resolution, validates the results of our simulations. Taken together, our work supports the purported central role of modified RNA for early RNA catalysis and may guide rational design of more efficient ribozymes.

Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) enables transcriptome-wide mapping of RNA-binding protein (RBP)-RNA interactions. Here, we present iCLIP version 3 (iCLIP3), a streamlined protocol optimized for generating high-quality iCLIP libraries from low-input material. We describe steps for near-infrared visualization of RBP-RNA complexes, silica column-based RNA isolation, and unique dual indexing using TruSeq adapters for cost-effective multiplexing and sequencing. We detail a complete bioinformatics workflow to identify crosslinking events and define RBP binding sites from raw sequencing data.

RNAanalyzer3 ("RNA analyzer cubic"; https://rnaanalyzer.bioapps.biozentrum.uni-wuerzburg.de) substitutes the frequently consulted current RNAanalyzer webserver (https://rnaanalyzer-old.bioapps.biozentrum.uni-wuerzburg.de). RNAanalyzer3 is free/open via the secure HTTPS protocol, with example data, help and tutorial, web-link to results, and rich data output. We combine a general detailed structure analysis with motif analyses. It accepts either a single plain-text nucleotide sequence or batch submission in FASTA format, which can be pasted or uploaded as a FASTA file. Our tool (i) has up-to-date software and operating systems, (ii) combines diverse RNA motif analyses with RNA structure prediction, (iii) puts found motifs into structural context, and (iv) offers dedicated tools for probing RNA-protein binding interactions. RNAanalyzer3 links motif searches to Rfam and miRNA search to miRbase. It focuses on structural features first, looks for stem-loops, hairpins, and specific enrichment regions such as stem-GG pairs, plus AU-rich regions with their locations for easier identification, while providing structural context and interactive RNA structure visualization. A tabulated overview shows all RNA features including structure details, coding potential, untranslated regions (UTRs, including Shine-Dalgarno sequences, Kozak sequences, and polyadenylation signals), transfer RNA (tRNA), microRNA (miRNA), long noncoding RNA (lncRNA), trans-splicing motifs, iron response elements (IRE), riboswitches, small nuclear ribonucleoprotein (snRNP) motifs, and spliceosomal Sm-sites.

Only ∼1%-2% of the human genome directly codes for proteins. The remainder consists of non-coding DNA, often referred to as the 'dark genome'. This includes regulatory elements, transposable and repetitive sequences, structural genomic features, pseudogenes, intronic and intergenic regions, and non-coding RNA (ncRNA) genes. These components are increasingly recognized as major regulators of gene expression, cell identity, and disease susceptibility. Currently, dark genome elements, particularly ncRNAs are increasingly recognized as important regulators of cardiovascular health and disease. Advances in genome analysis technologies have greatly improved our understanding of these non-coding regions and revealed clearer connections between the dark genome and cardiovascular traits. This review highlights major parts of the dark genome involved in cardiovascular disease, with emphasis on those for which mechanistic understanding and translational relevance are beginning to emerge. As mechanistic insight into individual and collective components of the dark genome advances, it increasingly enables the development of new opportunities for targeted therapeutics for cardiovascular prevention and disease management.

We report a fatal case of parkinsonism following treatment with ciltacabtagene autoleucel (cilta-cel). To investigate underlying mechanisms, we performed a multipronged longitudinal analysis using single-cell RNA (scRNA)/T-cell receptor (TCR) sequencing, flow cytometry, and cytokine measurements including cerebrospinal fluid (CSF) and peripheral blood (PB) samples, spanning more than 6 months after chimeric antigen receptor (CAR) T-cell therapy. Combined clinical and molecular findings revealed a biphasic immunologic process in the CSF. The early phase was characterized by a selective influx of predominantly CD4+ CAR T cells, accompanied by the evidence of endothelial dysfunction, prior to the clinical manifestation of parkinsonism. A second phase was preceded by a locally restricted inflammatory process in the CSF. Subsequently, an increase in the CSF to serum albumin ratio indicated disruption of the blood-brain barrier, coinciding with a pronounced influx of T cells-primarily CAR T cells but also clonally expanded, cytotoxic CD8+ non-CAR T cells-which was associated with neuronal injury and clinical decline.

Single-cell RNA sequencing is increasingly applied to bacterial model species, but a dedicated technique for anaerobic microbiota members of the Bacteroidota phylum has been lacking. Here, working with Bacteroides thetaiotaomicron, we describe experimental steps for transcriptome stabilization, fluorescence-activated cell sorting (FACS) collection, and optimized lysis of single cells from this group of organisms. We detail procedures for reverse transcription of RNAs via the multiple annealing and dC-tailing-based quantitative single-cell RNA sequencing (MATQ-seq) protocol, sensitive Cas9-based depletion of ribosomal sequences, and cDNA library generation. For complete details on the use and execution of this protocol, please refer to Bornet et al..

Chimeric antigen receptor (CAR) T cells have shown great potential in hematological cancers, but lack efficacy in solid tumors, highlighting the need for novel strategies. Stimulator of interferon genes (STING) activation was shown to inflame the tumor microenvironment, but combination of STING agonists and CAR-T cells might be limited by detrimental outcomes of T cell-intrinsic STING activation. In this study, we evaluated the potential of combining STING agonists and CAR-T cells in the context of pancreatic cancer.

The ATP-dependent INO80 chromatin remodeller slides and repositions nucleosomes to shape and maintain chromatin around gene regulatory elements and replication origins. Recent work uncovered capabilities of yeast and fungal INO80 to bind and slide hexasomes, but whether this is a universal feature is unknown. Here, we show that human INO80 also slides hexasomes as efficiently as H2A and H2A.Z nucleosomes. By determining a variety of structures of human INO80 bound to canonical and H2A.Z nucleosomes as well as hexasomes, we reveal a predominantly topological sensing of nucleosomal species with at least three positions depending on entry DNA unwrapping. INO80 spin-rotates around the nucleosomal core particle as a function of entry DNA unwrapping. Different degrees of unwrapped entry DNA lead to two different nucleosomal and one hexasomal locations of INO80, determined by binding of the Snf2 ATPase to entry point of extranucleosomal DNA at the nucleosome/hexasome core. Acidic patch binding by the INO80 subunit IES2 can differentiate between (sub)nucleosomal species, is important for nucleosome but not hexasome sliding, and may sense unwrapped exit DNA. These findings provide structural and mechanistic insights into how human INO80 remodels diverse chromatin substrates in a topology driven manner.

Excessive RNA damage activates cellular stress responses, triggering cell death. However, pathways that negatively regulate RNA damage responses are largely uncharacterized. Using genetic screens, we find that the ubiquitin ligase RNF25 provides tolerance to RNA damage caused by the nucleoside analogue azacytidine, a chemotherapeutic agent used to treat acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Mechanistically, we show that azacytidine is incorporated into mRNA, where it causes lesions that stall elongating ribosomes, leading to cytotoxic activation of the GCN2-dependent integrated stress response (ISR). Furthermore, we establish that RNF25 prevents ISR hyperactivation by ubiquitylation of ribosomal protein eS31, thereby suppressing cell death upon azacytidine treatment. Our study reveals an mRNA damage tolerance mechanism that determines cellular survival in response to azacytidine, highlighting RNA damage-induced stress response as a potentially critical component of chemosensitivity in AML and MDS.