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.

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.

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.

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.

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.