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Walbrun, A.

Publications and source records attributed to Walbrun, A..

2 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

Single-Molecule Force Spectroscopy of Toehold-Mediated Strand Displacement

Toehold-mediated strand displacement (TMSD) is extensively utilized in dynamic DNA nanotechnology and is the foundation for a wide range of DNA or RNA-based reaction circuits. Previous studies have typically relied on bulk fluorescence measurements to investigate the kinetics of TMSD, which only provide effective, bulk-averaged reaction rates and do not resolve the process on the level of individual molecules or even base pairs. In this work, we addressed this limitation by exploring the dynamics of strand displacement processes at the single-molecule level using single-molecule force spectroscopy (SMFS) with an optical trap supported by state-of-the-art coarse-grained simulations. By probing the ends of the hairpin of a toehold structure, we can trigger and observe TMSD in real time with microsecond and nanometer resolution. Using a microfluidic assay where we expose the hairpin to a solution of trigger strands, we find that under load, TMSD proceeds very rapidly, with single step times of 1 s. Introducing mismatches into the invader sequence allows us to tune stability such that invasion and re-invasion occur in equilibrium, even under load. This allows us to study thousands of invasion/re-invasion events on a single molecule and analyze the kinetics of the invasion process. Extrapolating our findings to zero load, we find single step times for DNA invading DNA four times faster than for RNA invading RNA. Moreover, we used force to study the kinetics of DNA invading RNA, a process that in the absence of force would rarely occur. Our results reveal the importance of sequence effects for the TMSD process and have importance for a wide range of applications in nucleic acid nanotechnology and synthetic biology.

biophysics↗