Abstract (eng)
The human telomerase is a ribonucleoprotein-complex (RNP) consisting of an RNA part, the human telomerase RNA (hTR), a protein part, the human telomerase reverse transcriptase (hTERT), and accessory proteins like Dyskerin Nop10, Nhp2, Tcab1 and Gar1. The main function of this complex is the maintenance of the telomeres by de novo synthesis of hexameric repeats (5’-TTAGGG-3’) onto the end of chromosomes after each replication cycle to counteract the end replication problem. In somatic cells telomerase is not expressed and cannot elongate telomeres, therefore after a certain number of cell divisions the short telomeres cause senescence and cell death. For highly proliferative cells, like stem cells, the telomerase expression is up-regulated and telomere length is maintained. Also in 90 % of all cancer types the telomerase is highly up-regulated. This has led biomedical research to focus on ways to use this fact in new therapies. A dysfunctional telomerase enzyme can cause diseases, like dyskeratosis congenita, aplastic anemia, idiopathic pulmonary fibrosis, which are all connected to shortened telomeres and the senescence of highly proliferative cell like stem cells. Mutations in hTERT, hTR and accessory proteins can cause such phenotypes. Single mutations in hTR have been identified to cause such phenotypes and reduce or abolish telomerase activity. These changes seem to influence folding and conformation of the human telomerase RNA. Despite the fact that there are great efforts to obtain high resolution data on telomerase RNA, the information on full length hTR is scarce. In this thesis we analyzed the telomerase activity of hTERT bound to mutant hTR in which highly conserved residues or structurally interesting nucleotides were altered. Of the analyzed mutants, C123A affected telomere function most severely. In fact, nucleotide addition processivity was reduced to <10%, while repeat addition processivity was abolished. Exploring the structural consequences of this single point mutation revealed that disrupting the G91-C123 base pair in P2a perturbs formation of the pseudoknot and associated triple helix, thereby inducing misfolding of the core domain. In contrast, this mutation does not appear to affect binding of hTERT to the CR4/CR5 domain and to interfere with telomere biogenesis. As pseudouridines are known to play a role in stabilizing and folding of non-coding RNAs, it was of interest to identify potential pseudouridines within hTR. In fact, a total of 29 pseudouridines were observed clustering in the pseudoknot/template domain. Specifically, all Us within the triple helical scaffold appear to be pseudouridines, potentially involved in stabilizing this structural element essential for telomerase function.