Biology subjects

Tants, J.-N.

Publications and source records attributed to Tants, J.-N..

5 recordsLinked to original sources

bioRxiv2024

Roquin exhibits opposing effects on RNA stem-loop stability through its two ROQ domain binding sites

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 Roquins ROQ domain with the 3 untranslated region (UTR) of the Ox40 mRNA. This 3UTR 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 STATEMENTLocal 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.

biophysics↗

bioRxiv2024

Evolution of the RNA alternative decay cis element into a high-affinity target for the immunomodulatory protein 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 homeostasis. 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 for a T-cell co-receptor. The Ox40 3UTR 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 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.

biochemistry↗

bioRxiv2024

Structure and RNA-binding of the helically extended Roquin CCCH-type zinc finger

Zinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich elements (ARE). We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence specificity through the ROQ-ZnF tandem.

biochemistry↗

bioRxiv2022

Insight into the structural basis for dual nucleic acid - recognition by the Scaffold Attachment Factor B2 protein

The family of scaffold attachment factor B (SAFB) proteins comprises three members and was first identified as binders of the nuclear matrix/scaffold. Over the past two decades, SAFBs were shown to act in DNA repair, mRNA/(l)ncRNA processing, and as part of protein complexes with chromatin-modifying enzymes. SAFB proteins are approximately-100-kDa-sized dual nucleic acid-binding proteins with dedicated domains in an otherwise largely unstructured context, but whether and how they discriminate DNA- and RNA-binding has remained enigmatic. We here provide the SAFB2 DNA- and RNA-binding SAP and RRM domains in their functional boundaries and use solution NMR spectroscopy to ascribe DNA- and RNA-binding functions. We give insight into their target nucleic acid preferences and map the interfaces with respective nucleic acids on sparse data-derived SAP and RRM domain structures. Further, we provide evidence that the SAP domain exhibits intra-domain dynamics and a potential tendency to dimerise, which may expand its specifically targeted DNA sequence range. Our data provide a first molecular basis of and a starting point towards deciphering DNA- and RNA-binding functions of SAFB2 on the molecular level and serve a basis for understanding its localization to specific regions of chromatin and its involvement in the processing of specific RNA species.

biochemistry↗

bioRxiv2018

All four double-stranded RNA binding domains of Staufen2 contribute to efficient mRNA recognition and transcript localization

Throughout metazoans, Staufen (Stau) proteins are core factors of mRNA localization particles. They consist of three to four double-stranded RNA binding domains (dsRBDs) and a C-terminal dsRBD-like domain. Mouse Staufen2 (mStau2) like Drosophila Stau (dmStau) contains four dsRBDs. Existing data suggest that only dsRBDs 3-4 are necessary and sufficient for mRNA binding. Here, we show that dsRBDs 1 and 2 of mStau2 bind RNA with similar affinities and kinetics as dsRBDs 3 and 4. While RNA binding by these tandem domains is transient, all four dsRBDs recognize their target RNAs with high stability. Rescue experiments in Drosophila oocytes demonstrate that mStau2 partially rescues dmStau-dependent mRNA localization. In contrast, a rescue with mStau2 bearing RNA-binding mutations in dsRBD1-2 fails, confirming the physiological relevance of our findings. In summary, our data show that the dsRBDs 1-2 play essential roles in the mRNA recognition and function of Stau- family proteins of different species.

biochemistry↗