Development of new therapies based on the non-canonical functions of the RNA component of telomerase

Abstract

Telomerase is a ribonucleoprotein complex mainly composed of a catalytic subunit with reverse transcriptase activity (telomerase reverse transcriptase, TERT) and an RNA component (telomerase RNA component, TERC), responsible for telomere maintenance. However, several studies confirm that both components perform others functions beyond telomeric role. Specifically, our laboratory has discovered that TERC regulates myelopoiesis, both in zebrafish and in humans. The specific objectives of the present work are: 1. Molecular characterization of the TERC domains responsible of its non-canonical hematopoietic function. 2. Development of aptamers derived from TERC to treat blood diseases. 3. Identification of TERC interactome. Methodology, results and conclusions: During the development of this Doctoral Thesis, we have exploited the unquestionable advantages of the zebrafish to study the role of terc in myelopoiesis, characterizing the functional involvement of each terc domain in this process. To investigate the function of each domain, different mutations that are frequently found in patients with dyskeratosis congenita (DC) were reproduced and we observed that only the mutations affecting the CR4/CR5 domain impaired myelopoiesis. Taking advantage of the functional knowledge of the roles played by the CR4/CR5 domain of TERC in myelopoiesis, several aptamers (small synthetic oligonucleotides, capable of specifically recognizing target molecules with high affinity) were developed. Two of the aptamers, CR4CR5 and AA, were able to stimulate myelopoiesis by increasing the number of neutrophils and macrophages, without affecting the number of erythrocytes, and independently of terc expression or telomerase activity. In addition, they functioned as terc; that is, they interacted with RNA polymerase II and with the terc binding sequences present in the promoter of master myelopoiesis genes, enhancing their expression in a terc binding site-dependent manner. Importantly, aptamers harbouring CR4/CR5 mutations found in DC patients failed to perform all these functions. In addition, preclinical zebrafish models of DC (terc deficiency) and poikiloderma with neutropenia (usb1 deficiency) diseases demonstrated the therapeutic potential of the developed aptamers to treat neutropenia. Finally, the corresponding human aptamers also increased myeloid gene expression promoting myelopoiesis in human induced pluripotent stem cells. Therefore, two potential therapeutic agents for DC and other neutropenic diseases have been developed in this study. Although we delved into TERC structural and functional characteristics during this thesis, we decided that it was still possible to explore its potential beyond its role in telomeric biology and its non-canonical role in myelopoiesis. For this reason, a proteomic approach was developed to fully exploit the potential of TERC. By this way, it has been discovered that TERC potentially interacts with a multitude of proteins, involved in several cellular processes, such as protein folding, degradation (ubiquitination) and translation, carbon and lipid metabolism, nonsense-mediated mRNA decay, mitochondrial biogenesis and cell cycle. Notably, terc harbouring the mutation of the CR4/CR5 domain found in DC patients showed a similar interactome as wild type terc, despite these mutations impair both telomerase activity and TERC-dependent regulation of myelopoiesis. This study paves the way for future functional studies aimed at the characterization of the non-canonical roles of terc that could help in improving diagnosis and treatment of patients with TERC mutations.