Biology subjects

Kaiser, W.

Publications and source records attributed to Kaiser, W..

4 recordsLinked to original sources

bioRxiv2024

Deciphering the RNA recognition by Musashi-1 to design protein and RNA mutants for in vitro and in vivo applications

RNA Recognition Motifs (RRMs) are essential post-transcriptional regulators of gene expression in eukaryotic cells. The Human Musashi-1 (MSI-1) is an RNA-binding protein that recognizes (G/A)U1-3AGU and UAG sequences in diverse RNAs through two RRMs and regulates the fate of target RNA. Here, we combined structural biology and computational approaches to analyse the binding of the RRM domains of human MSI-1 with single-stranded and structured RNAs ligands. We used our recently developed computational tool RRMScorer to design a set of mutants of the MSI-1 protein to bind novel RNA sequences to alter the binding selectivity. The in-silico predictions of the designed protein-RNA interactions are assessed by NMR and SPR. These experiments also are used to study the competition of the two RRM domains of MSI-1 for the same binding site within linear and harpin RNA. Our experimental results confirm the in-silico designed interactions, thus opening the way for the development of new biomolecules for in vitro and in vivo studies and downstream applications.

biophysics↗

bioRxiv2022

Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, to implement combinatorial regulation, and to reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.

synthetic biology↗

bioRxiv2018

Utilisation of staphylococcal immune evasion protein Sbi as a novel vaccine adjuvant

Co-ligation of the B cell antigen receptor with complement receptor 2 on B-cells via a C3d-opsonised antigen complex significantly lowers the threshold required for B cell activation. Consequently, fusions of antigens with C3d polymers have shown great potential in vaccine design. However, these linear arrays of C3d multimers do not mimic the natural opsonisation of antigens with C3d. Here we investigate the potential of using the unique complement activating characteristics of Staphylococcal immune-evasion protein Sbi to develop a pro-vaccine approach that spontaneously coats antigens with C3 degradation products in a natural way. We show that Sbi rapidly triggers the alternative complement pathway through recruitment of complement regulators, forming a tripartite complex that acts as a competitive antagonist of factor H, resulting in enhanced complement consumption. These functional results are corroborated by the structure of this complement activating Sbi-III-IV:C3d:FHR-1 complex. Finally, we demonstrate that Sbi, fused with Mycobacterium tuberculosis antigen Ag85b, causes efficient opsonisation with C3 fragments, thereby enhancing the immune response significantly beyond that of Ag85b alone, providing proof of concept for our pro-vaccine approach.

biochemistry↗

bioRxiv2018

TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) is powerful technique to study transcriptional regulation. However, the requirement of millions of cells to generate results with high signal-to-noise ratio precludes it in the study of small cell populations. Here, we present a Tagmentation-Assisted Fragmentation ChIP (TAF-ChIP) and sequencing method to generate high-quality results from low cell numbers. The data obtained from the TAF-ChIP approach is amenable to standard tools for ChIP-Seq analysis, owing to its high signal-to-noise ratio. The epigenetic profiles from TAF-ChIP approach showed high agreement with conventional ChIP-Seq datasets, thereby underlining the utility of this approach.

genomics↗