Biology subjects

Perez-Rafols, A.

Publications and source records attributed to Perez-Rafols, A..

3 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↗

bioRxiv2023

HuR modulation with tanshinone mimics impairs LPS response in murine macrophages

Lipopolysaccharide exposure to macrophages induces an inflammatory response that is heavily regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA binding protein that binds and regulates the maturation and half-life of AU/U rich elements (ARE) containing cytokines and chemokines transcripts, mediating the LPS-induced response. Here we investigated how and to what extent small molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. We show TMs exist in solution in keto-enolic tautomerism and that, by molecular dynamic calculations, the orto quinone form is the bioactive species interacting with HuR and inhibiting its binding mode vs mRNA targets. A chemical blockage of the diphenolic, reduced form as a diacetate caused the loss of activity of TMs in vitro but resulted to prodrug-like activity in vivo. The murine macrophage cell line RAW264.7 was treated with LPS and TMs, and the modulation of cellular LPS-induced response was monitored by RNA and Ribonucleoprotein immunoprecipitation sequencing. Correlation analyses indicated that LPS induced a strong coupling between differentially expressed genes and HuR-bound genes, and that TMs reduced such interactions. Functional annotation addressed a specific set of genes involved in chemotaxis and immune response, such as Cxcl10, Il1b, Cd40, and Fas, with a decreased association with HuR, a reduction of their expression and protein secretion. The same effect was observed in primary murine bone marrow-derived macrophages, and in vivo in an LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing TMs such as TM7nox with remarkable anti-inflammatory properties in vivo.

molecular biology↗

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↗