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Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
Published on: November 9, 2019
Human antigen R transfers miRNA to Syntaxin 5 to synergize miRNA export from activated macrophages
Sourav Hom Choudhury1, Shreya Bhattacharjee2, Kamalika Mukherjee3
1RNA Biology Research Laboratory, Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
Abstract:
Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from the endoplasmic reticulum to endosomes is a rate-determining step in the miRNA export process and plays an important role in controlling cellular miRNA levels and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin 5 (STX5) to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. STX5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how STX5 acts as an immunostimulant, we have identified the de novo RNA-binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAVL1 protein, Human antigen R. This activity ensures the export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with the plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.
Insights
Syntaxin 5 (STX5) protein facilitates macrophage immune responses by binding microRNAs (miRNAs) and regulating their export. This process controls inflammatory cytokine expression and pathogen clearance.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Intercellular microRNA (miRNA) exchange regulates gene expression post-transcriptionally.
- miRNA export from macrophages controls inflammatory cytokine expression.
- Intracellular miRNA trafficking is crucial for cellular miRNA levels and inflammation.
Purpose of the Study:
- To investigate the role of Syntaxin 5 (STX5) in macrophage immune function.
- To elucidate the mechanism by which STX5 influences miRNA export and cytokine regulation.
- To explore STX5's role in combating intracellular pathogens.
Main Methods:
- Identified STX5 expression patterns in activated macrophages.
- Assessed STX5's necessity and sufficiency in macrophage activation and pathogen clearance.
- Investigated STX5's RNA-binding properties and interaction with ELAVL1.
- Analyzed STX5's role in endosome trafficking and extracellular vesicle release.
Main Results:
- STX5 expression synchronizes with miRNA activity loss in activated macrophages.
- STX5 is essential and sufficient for macrophage activation and clearance of Leishmania donovani.
- STX5 binds specific miRNAs, facilitating their endosomal accumulation with ELAVL1.
- STX5 promotes miRNA export by preventing lysosomal targeting and enhancing extracellular vesicle release.
Conclusions:
- STX5 acts as an immunostimulant by binding and facilitating miRNA export.
- STX5 regulates inflammatory cytokine expression through miRNA-mediated gene silencing.
- STX5 enhances macrophage defense against intracellular pathogens via miRNA regulation and enhanced vesicle release.
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