Andreas Schlundt

Haploinsufficiency is a common cause of developmental disorders, yet the mechanisms regulating protein abundance of dosage-sensitive genes remain incompletely understood. SMARCAD syndrome comprises inherited ectodermal dysplasias caused by mutations affecting SMARCAD1-s, the skin-specific short isoform of the ATP-dependent chromatin remodeller SMARCAD1. Here, we show that independent patient-derived mutations impair splicing of this isoform, resulting in intron retention. Although total mRNA abundance is reduced, spliced transcripts remain translationally competent and yield variably reduced levels of wild-type protein, providing evidence for a threshold-dependent haploinsufficiency model of disease. Under physiological conditions, the isoform-specific non-coding exon 1 dampens protein production without altering transcript levels, revealing previously unrecognized post- transcriptional control of SMARCAD1-s dosage. Our findings implicate upstream open reading frames as candidate mediators of translational control. Unexpectedly, SMARCAD1-s exhibits regulated nucleocytoplasmic distribution. Functional analyses identify two nuclear localization signals that differentially contribute to localization of the SMARCAD1 isoforms. Together, our findings clarify the molecular basis of SMARCAD syndrome and identify post- transcriptional and spatial mechanisms controlling the abundance and localization of a dosage- sensitive chromatin remodeller, providing insight into protein dosage control.

Why and how cells exploit redundant versus complementary specificities of the 12 splice-regulatory SRSF(1–12) proteins remains enigmatic, but points at subtle details in their RNA binding modules. The resulting need in high-resolution information is especially evident for SRSF6 with no existing structures explaining its pre-mRNA selectivity. Using iCLIP, global splicing analyzes, RNA-Bind-and-Seq, integrated structural biology, molecular simulations, and systematic interaction studies including purified full-length eukaryotic protein, we provide the first atom-resolved basis of RNA-recognition by SRSF6 through RRM-, linker-, and RS-domains. SRSF6 uses its two differently typed RRMs in distinct interactions with constitutive (GAA-rich) or alternative exons (GGA) modulated by the RS domain, explaining previous inconsistencies within SRSF6-targeted motifs. Our findings suggest a mode of redundant and mutually exclusive splicing decisions between the closely related SRSF1 and SRSF6 as a blueprint for integrated splicing regulation by all 12 SRSF proteins.

The human serine-arginine rich splicing factor 6 (SRSF6) is part of the SR-protein family consisting of 12 members and is involved in (alternative-) splicing regulation. It is composed of an N-terminal RRM domain, followed by a pseudo-RRM and a C-terminal serine/arginine-rich disordered domain. Despite extensive studies on the functions of SRSF6, its determinants for specific RNA-recognition have remained unresolved due to a lack of high-resolution structures based on unambiguous boundaries of individual domains and the involved disordered regions. We here provide the first near-complete NMR backbone and sidechain assignments of human SRFS6 RRM1 and backbone assignments of the pseudo-RRM2, as well as the tandem RRM. The derivable carbon secondary chemical shifts were used to define secondary structure elements as well as exact domain boundaries, showcasing a canonical β1α1β2β3α2β4-fold for either RRM. Consequently, we defined the inter-domain linker with a length of 37 residues. Furthermore, 15N-relaxation data measured for the single and tandem RRMs indicate that the two RRMs tumble as one entity. However, the minor chemical shift differences between the respective constructs suggest a merely transient interaction with no defined interface between the two RRMs. Lastly, we show that AlphaFold3 models are majorly supported by our solution NMR data, but there are subtle inconsistencies with regard to the formation of the β4-strand. In sum, our initial work underlines the role of NMR for visualizing crucial functional details of biology and sets the critical basis for future high-resolution structures of and residue-resolved determinants of specific RNA-recognition by the splicing regulator SRSF6.

Proline is a paradox in biology—integral to protein structure and functionality, yet exceptionally challenging during translation. This review examines what sets proline apart from the other proteinogenic amino acids, detailing its unique chemical and structural characteristics that hinder efficient peptide bond formation. Although proline’s impact is evident across all domains of life, our focus is primarily on bacteria, where the evolutionary pressure to overcome proline-induced ribosomal stalling has led to a sophisticated suite of specialized translation factors. Ultimately, we discuss how the interplay between proline and its translational rescue systems not only ensures protein synthesis but also drives the evolution of bacterial proteomes.

The interaction of mRNA and regulatory proteins is critical for posttranscriptional control. For proper function, these interactions, as well as the involved protein and RNA structures, are highly dynamic, and thus, mechanistic insights from structural biology are challenging to obtain. In this study, we employ a multifaceted approach combining single-molecule force spectroscopy (SMFS) with NMR spectroscopy to analyze the concerted interaction of the two RNA-binding interfaces (A-site and B-site) of the immunoregulatory protein Roquin’s ROQ domain with the 3’ untranslated region (UTR) of the Ox40 mRNA. This 3’UTR contains two specific hairpin structures termed constitutive and alternative decay elements (CDE, ADE), which mediate mRNA degradation through Roquin binding. Our single-molecule experiments reveal that the CDE folds cooperatively, while ADE folding involves at least three on-pathway and three off-pathway intermediates. Using an integrated microfluidics setup, we extract binding kinetics to Roquin in real time. Supported by NMR data, we find opposing effects of the two Roquin subdomains on distinct regions of the ADE: While the A-site interacts strongly with the folded apical stem-loop, we find that the B-site has a distinct destabilizing effect on the central stem of the ADE owed to single-strand RNA binding. We propose that RNA-motif nature and Roquin A- and B-sites jointly steer mRNA decay with context-encoded specificity, and we suggest plasticity of stem structures as key determinant for Roquin–RNA complex formation. The unique combination of NMR and SMFS uncovers a mechanism of a dual-function RNA-binding domain, offering a model for target RNA recognition by Roquin.

RNA cis elements play pivotal roles in regulatory processes, e.g. in transcriptional and translational regulation. Two stem-looped cis elements, the constitutive and alternative decay elements (CDE and ADE, respectively) are shape-specifically recognized in mRNA 3' untranslated regions (UTRs) by the immune-regulatory protein Roquin. Roquin initiates mRNA decay and contributes to balanced transcript levels required for immune homoeostasis. While the interaction of Roquin with several CDEs is described, our knowledge about ADE complex formation is limited to the mRNA of Ox40, a gene encoding a T-cell costimulatory receptor. The Ox40 3'UTR comprises both a CDE and ADE, each sufficient for Roquin-mediated control. Opposed to highly conserved and abundant CDE structures, ADEs are rarer, but predicted to exhibit a greater structural heterogeneity. This raises the question of how and when two structurally distinct cis elements evolved as equal target motifs for Roquin. Using an interdisciplinary approach, we here monitor the evolution of sequence and structure features of the Ox40 ADE across species. We designed RNA variants to probe en-detail determinants steering Roquin-RNA complex formation. Specifically, those reveal the contribution of a second RNA-binding interface of Roquin for recognition of the ADE basal stem region. In sum, our study sheds light on how the conserved Roquin protein selected ADE-specific structural features to evolve a second high-affinity mRNA target cis element relevant for adaptive immune regulation. As our findings also allow expanding the RNA target spectrum of Roquin, the approach can serve a paradigm for understanding RNA-protein specificity through back-tracing the evolution of the RNA element.

The SARS-CoV-2 nucleocapsid protein is indispensable for viral RNA genome processing. Although the N-terminal domain (NTD) is suggested to mediate specific RNA-interactions, high-resolution structures with viral RNA are still lacking. Available hybrid structures of the NTD with ssRNA and dsRNA provide valuable insights; however, the precise mechanism of complex formation remains elusive. Similarly, the molecular impact of nucleocapsid NTD mutations that have emerged since 2019 has not yet been fully explored. Using crystallography and solution NMR, we investigate how NTD mutations influence structural integrity and RNA-binding. We find that both features rely on a core network of residues conserved in Betacoronaviruses, crucial for protein stability and communication among flexible loop-regions that facilitate RNA-recognition. Our comprehensive structural analysis demonstrates that contacts within this network guide selective RNA-interactions. We propose that the core network renders the NTD evolutionarily robust in stability and plasticity for its versatile RNA processing roles.

Polyproline sequences are deleterious to cells because they stall ribosomes. In bacteria, EF-P plays an important role in overcoming such polyproline sequence-induced ribosome stalling. Additionally, numerous bacteria possess an EF-P paralog called EfpL (also known as YeiP) of unknown function. Here, we functionally and structurally characterize EfpL from Escherichia coli and demonstrate its role in the translational stress response. Through ribosome profiling, we analyze the EfpL arrest motif spectrum and find additional sequences beyond the canonical polyproline motifs that both EF-P and EfpL can resolve. Notably, the two factors can also induce pauses. We further report that EfpL can sense the metabolic state of the cell via lysine acylation. Overall, our work characterizes the role of EfpL in ribosome rescue at proline-containing sequences, and provides evidence that co-occurrence of EF-P and EfpL is an evolutionary driver for higher bacterial growth rates.

The interaction of mRNA and regulatory proteins is critical for post-transcriptional control. For proper function, these interactions as well as the involved protein and RNA structures are highly dynamic and thus, mechanistic insights from structural biology are challenging to obtain. In this study, we employ a multifaceted approach combining single-molecule force spectroscopy with NMR spectroscopy to analyze the concerted interaction of the two RNA-binding interfaces (A-site, B-site) of the immunoregulatory protein Roquin’s ROQ domain with the 3’ untranslated region (UTR) of the Ox40 mRNA. This 3’UTR contains two specific hairpin structures termed constitutive and alternative decay elements (CDE, ADE), which mediate mRNA degradation via binding of Roquin. Our single-molecule experiments reveal the CDE folds cooperatively, while ADE folding involves at least 3 on-pathway and 3 off-pathway intermediates. Utilizing an integrated microfluidics setup allows to extract binding kinetics to Roquin in real time. Supported by NMR, we find opposing effects of the two Roquin sub-domains on distinct regions of the ADE: while the A-site interacts strongly with the folded apical stem-loop, we find that the B-site has a distinct destabilizing effect on the central stem of the ADE owed to single-strand RNA binding. We propose that RNA-motif nature and Roquin A- and B-sites jointly steer mRNA decay with context-encoded specificity, and we suggest plasticity of stem structures as key determinant for Roquin-RNA complex formation. The unique methodological combination of NMR and single-molecule force spectroscopy reveals an unknown mechanism of a dual-function RNA-binding domain suggesting a new model for target RNA recognition. SIGNIFICANCE STATEMENT: Local RNA structure is decisive for specific engagement with gene-regulatory proteins and, as a consequence, correct cellular function. However, its existence often appears dynamic and thus, challenging to study. This study shows how NMR and single-molecule force spectroscopy efficiently complement each other to provide high-resolution, time-resolved data on RNA folding intermediates during dynamic complex formation with the immune-regulating protein Roquin, which exploits multiple RNA-binding sites. Our data reveal a dual-mode binding of Roquin to RNA by firmly attaching to the stem-loop and, at the same time, destabilizing other regions making them accessible to downstream interaction partners.