| dc.description.abstracteng | The world’s population is ageing rapidly. Currently, people at the age of 60 and over represent 12.3% of the global population. By 2050, this number will rise to almost 22%. Our knowledge of ageing underlying processes is poor, mostly due to the lack of proper models to study it. The straightforward strategy to identify the key genetic players in ageing is the detection of genes leading to accelerated ageing observed in patients with so called progeroid syndromes (PSs), which are very rare genetic disorders characterised by the fatal and severe course of the disease. Clinical features of PSs resemble the physiological processes of ageing, with early manifestation of ageing-associated conditions, such as osteoporosis, atherosclerosis or cancers. Recently, our group examined a 4½-year-old girl diagnosed with a congenital segmental progeria syndrome. Trio-based whole-exome sequencing (WES) led to the identification of a de novo variant, located in the Nuclear pre-lamin A Recognition Factor (NARF) gene. This mutation, c.1100A>G, changed a highly conserved histidine at position 367 to arginine (p.His367Arg), and is predicted to be damaging by several in silico prediction programs. NARF is an evolutionarily conserved gene that has its homologues in both yeast (Saccharomyces cerevisiae, NAR1) and nematodes (Caenorhabditis elegans, OXY-4 or Y54H5A.4). In mammals, there is an additional homologue of the NARF gene named iron-only hydrogenase-like protein 1 (IOP1), also known as nuclear pre-lamin A recognition factor-like (NARFL). All four proteins are similar to bacterial iron hydrogenases, but they have lost their hydrogenase activity. Functional analysis of the p.His367Arg mutation identified in our patient revealed that it interferes with the nuclear localisation of NARF. I demonstrated that NARF is able to form homodimers, which are probably important for its translocation to the nucleus. I also showed that substitution of the conserved histidine exhibits a dominant negative effect on the wild-type NARF, resulting in complete mislocalisation of both mutated and wild-type NARF to the cytoplasm. This suggests that dimerisation of NARF allows for the generation of unconventional nuclear localisation signal (NLS), and mutation prevents proper conformation and nuclear import. In my studies, I was able to determine the direct interactions of NARF with two proteins: lamin A and CBX5. Both proteins also interact with each other, and have been associated with premature ageing phenotypes. I established that the corresponding mutation causes prolonged DNA repair induced by UV-light lesions in mouse embryonic stem (mES) cells. In addition, I demonstrated impaired proliferation of mES cells carrying the mutation. Both findings could explain the failed attempts at Narf KI mouse line generation in my study. KI mES cell injections into blastocysts gave rise only to low-grade chimeras without germline transmission. I hypothesise that disturbed nuclear transport of the NARF protein and its accumulation in the cytoplasm probably prevent its proper functioning within the nuclear compartment of the cell. Detailed knowledge of the function of the NARF protein in the nucleus is still lacking. The results described within my thesis present another step towards understanding NARF function and the mechanisms underlying ageing and ageing-associated diseases. | de |