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Final Report Summary - NRNPS & INFLAMMATION (Nuclear HuR-hnRNP interactions in post-transcriptional regulation of inflammatory gene expression)


The inflammatory response is a complex physiological process that requires the coordinated induction of cytokines, chemokines, angiogenic factors, effector-enzymes, and proteases. However, excessive inflammation can itself exacerbate tissue damage and result in chronic inflammatory diseases, a massive burden on our society. The expression of crucial inflammatory mediators can be tightly regulated at the post-transcriptional level via changes in mRNA maturation, localisation, translation and destruction. It is usually driven by the assembly of ribonucleoprotein (RNP) complexes, in which RNAs interact with trans-acting RNA-binding proteins (RBPs). Most current studies focus on cytoplasmic events of RNA regulation in immune cells-however a large majority of RBPs relating to inflammation are expressed in the nucleus. This is also the case for the RBP Elavl1/HuR which has been linked to the cytoplasmic regulation of inflammatory mRNAs in macrophage and whose dysfunction supports the development of chronic inflammatory syndromes. Work undertaken by the recruited researcher aimed to identify nuclear HuR:RNPs of relevance to inflammatory reactions and their relationship to HuR:mediated recognition of its targets. First, the researcher set up a compartmentalized proteomics approach and identified several hnRNP:HuR nuclear interactions involved in mRNA maturation in macrophages that could be altered in response to an inflammatory stimulus or by the transgenic loss of HuR. Additional, novel interactors of HuR were identified in the course of these experiments connecting the functions of HuR:RNPs to inflammatory signalling cascades.

Having identified the existence of discrete nuclear HuR-containing RNP entities involved in inflammatory responses, the researcher proceeded to the identification of the RNA components of these RNPs. To that end, the researcher set up -for the first time in Greece- a genome wide approach technique, namely the Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation methodology (PAR-CLIP), which allows for identification of the RNA entities that are bound by the HuR:RNPs via next generation sequencing and determines the site on the RNA where the protein is bound. The researcher also proceeded to apply this technique under the kinetic conditions of a modelled inflammatory response of primary macrophages and in many biological replicas; by doing so the researcher identified RNA targets of HuR that are differentially altered upon inflammation and highlighted the involvement of co-existing levels of mRNA maturation and mRNA regulation. Most importantly the differential binding of HuR:RNPs in exonic, intronic and UTR sequences of mRNA populations related to macrophage polarization implicating their involvement in macrophage plasticity. Finally, the response of RNP bound RNAs was verified via the genome wide sequencing of RNAs from transgenic mouse macrophages containing or lacking HuR. Through this approach, the researcher identified several pre-RNA/mRNA abberations that relate to states of HuR-deficiency such as inflammatory bowel diseases and intestinal cancers.

RNA-interference experiments for 4 hnRNPs in HuR proficient and deficient settings identified as HuR interactos validated the functionality of HuR:hnRNP connections in HuR-driven macrophage control. Most importantly the ourcome of these knock-downs demonstrated that some of these interactions act in a pro-inflammatory fashion whereas others induce immunoregulatory effects. This pilot studies paved the way for the application of the PAR-Clip for hnRNP components and the generation and analysis of novel transgenic systems assessing their functions.

The tasks undertaken by the researcher yielded multiple and bipartite benefits. Firstly, they introduced the researcher into multidisciplinary fields of immunology, biomedicine and systems biology thus to support modern career choices. Secondly, the lab acquired new technologies strengthening its innovation and exploitation portfolios. Third, these novel technologies are now accessible to Greek research and support of national training programs. In socioeconomic terms multiple benefits emerge from the data produced since they identify novel means for immune regulation during chronic inflammatory dysfunction as well as alternative means for disease monitoring and treatment.

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BIOMEDICAL SCIENCES RESEARCH CENTER ALEXANDER FLEMING
Greece