A02: Structural basis and mechanisms of strand- specific microRNA trafficking and function in cardiac disease

Research Details

  • Project Leaders  
    Prof. Dr. Christian Weber
    Institute for Cardiovascular Prevention (IPEK)
    LMU Munich
    chweber@med.lmu.de

    Prof. Dr. Michael Sattler
    Department of Chemistry
    Technical University of Munich (TUM)
    sattler@helmholtz-muenchen.de
  • Research Staff  
    Prof. Dr. Donato Santovito (Postdoc)
    donato.santovito@med.uni-muenchen.de

    Dr. James Henderson (Postdoc)
    james.henderson@med.uni-muenchen.de

    Agathe Marcelot (Postdoc)
    agathe.marcelot@ibpc.fr

    Philipp Mayer (PhD Student)
    philipp.h.o.mayer@tum.de

    Laure Gauthé (PhD Student)
    laure.gauthe@tum.de

The RNA-binding protein Mex3a is a component of the processing bodies (P-bodies) and engages in RNAdependent interactions with Argonaute (AGO) proteins. Its sequence contains two KH-domains conferring RNA-binding capability and a RING-domain with E3-ubiquitin ligase activity, but its biological function remains largely unknown. In the previous funding period, we identified the role of Mex3a in selecting the passenger strand of miR-126 (miR-126-5p), promoting its nuclear transfer during autophagy to preserve endothelial cell viability upon autophagy. While the interaction of miR-126-5p with KH-domains is required for the assembly of a complex with AGO2, our preliminary data show that the C-terminal region of Mex3a can stabilize the complex, likely by protein-protein interactions, and that the N- and C-terminal regions further contribute to RNA-binding. Notably, other cell types express Mex3a but not miR-126, and Mex3a expression and nuclear localization are increased in response to cardiac-relevant pathological stimuli, such as ischemia and pressure overload. We hypothesize that Mex3a interacts with other miRNAs (and RNA species) and instruct their localization and function, possibly affecting cardiac pathophysiology. In this project, we aim (a) to uncover Mex3a-interacting miRNAs, investigate potential motifs that mediate sequence specificity, and explore their functional relevance in nucleus and P-bodies; (b) to map protein-protein and protein-RNA binding epitopes and to determine the structural basis of the ternary complex involving Mex3a, AGO2, and miRNA, and to screen for small molecules for stabilization of the interaction; (c) to assess the role of Mex3a in cardiac response to pathological stimuli in vivo. To address these issues, we will combine biophysical and biochemical approaches (i.e., surface plasmon resonance, nuclear magnetic resonance spectroscopy, mutational scanning of proteins and miRNAs), experiments in cells involving cross-linking and immunoprecipitation (CLIP) followed by RNA sequencing, gain- and loss-of-function approaches by RNA-interference and shRNAs/CRISPR-Cas9, with in vivo experiments of cardiac injury (i.e., coronary artery ligation, transverse aortic constriction) in transgenic mouse models with global or specific Mex3a deletion in relevant cell types (i.e., endothelial cells, cardiomyocytes, fibroblasts). Our results will reveal the role of Mex3a-miRNA interactions in different cells contributing to cardiac adaptation following pathological stimuli and may provide translational insights for molecular targeting with the aim of improving cardiac function following ischemic and non-ischemic damage.

Team A02