RNA-binding proteins: novel mediators of inflammation and accelerated aging in Chronic Obstructive Pulmonary Disease

Background. RNA-binding proteins (RBPs) play a key role in post-transcriptional gene regulation (PTGR) of genes involved in numerous biological processes. These proteins act through the binding to specific cis-elements present in their RNA targets and by forming, with other regulatory factors, dynamic ribonucleoprotein complexes (RNPs) that ultimately determine the fate of different type of RNAs. There are several families of RBPs classified according to the type of RNA target that they bind. Multiple studies in the last decade have established that aberrant expression and function of RBPs participate to cancer pathogenesis by altering the stability and translation of genes involved in many mechanism of neoplastic transformation. Many of the cancer-related pathways for which PTGR mediated by RBPs has been established are also critically involved in chronic inflammation; to this end, many important basic and preclinical studies and gene ablation animal models indicate that RBPs are critically involved also in inflammatory responses and immunity. In contrast to the growing number of studies on the role of RBPs in human cancer, translational studies based on chronic inflammatory disease are still scarce. In particular, one of these studies demonstrated the loss of AU-rich element factor 1 (AUF-1) in airway epithelium of patients with stable chronic obstructive pulmonary disease (COPD) and in cytokine-and cigarette smoke-challenged human epithelial cells, along with a global downregulation of RBPs expression. Aim. The thesis aimed at identifying the role of AUF-1 as determinant of increased inflammation and accelerated cellular senescence in COPD pathogenesis, and understanding the mechanisms mediating cytokine-induced AUF-1 loss. On these bases, our long-term aim is to explore RBP therapeutic targeting, for which we plan to develop dedicated experimental models. Results. Using an RNA immunoprecipitation and sequencing (RIP-Seq) approach, we identified AUF-1 targets in the airway epithelial cell line BEAS-2B and identified specific interacting sequences in targeted transcripts. In vitro validation of AUF-1 association to selected transcripts was performed by biotin pulldown, while the levels of AUF-1 targets were evaluated by qRT-PCR, revealing a differential alteration by partial or near-total loss of AUF-1. Cytomix-induced decreased levels of AUF-1 protein were associated with accelerated cell senescence readouts of lysosomal damage, cell cycle arrest and secretion of senescence-associated secretory phenotype (SASP) factors. Interestingly, AUF-1 protein was detected into extracellular vesicles (EVs), suggesting a mechanism of loss of intracellular protein. Finally, AUF-1 targets were also identified in the human SASP proteome atlas and as differentially expressed genes in transcriptomic databases of HSAEC and lung tissue samples from stable COPD patients. Conclusions. The studies performed in this thesis suggested that AUF-1 may play a pathogenic role in COPD by altering post-transcriptional control of epithelial gene expression, thus contributing to increased airway inflammation, also through EVs-related functions. Overall, the identification of these changes can be used to infer putative pathogenetic roles of RBPs and identify novel disease-related regulatory networks, that will be further investigated with dedicated experimental models.