| dc.description.abstracteng | Telomere length homeostasis is a prerequisite for cell viability. It is challenged through successive shortening, which is due to the “end-replication-problem”. This limits the cellular life span in multicellular organisms enabling senescence and antagonizing unlimited growth, important for the prevention of cancer. Telomerases, are specialized and conserved enzymes, that evolved to counteract telomere shortening in stem and germ cells as well as in the single cell eukaryote Saccharomyces cerevisiae, through their reverse transcriptase activity. By using budding yeast as a model organism, we aimed to define the stepwise order in which the scaffolding telomerase RNA TLC1 matures and how the telomerase ribonucleoparticle (RNP) is assembled. As shown earlier, first, an immature ~ 1.3 kb long precursor of TLC1 is synthesized by RNAP II and the transcript is subsequently exported into the cytoplasm. Export occurs via Mex67-Mtr2 and Xpo1. Both transport factors do not contact the RNA directly but rather require adaptor proteins. In this work we have shown that the Mex67-adaptor Npl3 and the Xpo1 interacting m7G-cap-binding factor Cbp20 contact TLC1. Furthermore, we confirm a model in which the Est- and Pop-protein loading as well as the association of the Sm-ring occurs in the cytoplasm. These proteins are important for the functionality of the enzyme as their correct assembly on TLC1 is crucial for the reverse transcriptase activity, and thus for telomere elongation. Re-import of the pre-RNP into the nucleus is facilitated via the import receptor Mtr10. We identified a second TLC1 import factor, Cse1, that supports nuclear re-import of pre-TLC1 in an importin α independent pathway. Both import factors cooperate in the nuclear re-import of pre-TLC1, and we discovered additional functions for them in TLC1 protection. Mutation of CSE1 leads to the destabilization of the Sm-ring on TLC1, while mutation of MTR10 leads to less mature TLC1, although the Sm-ring was bound properly. Both import receptors re-import TLC1 through contact with the Sm-ring. Therefore, we suggest that this resembles a quality control step in the life cycle of TLC1, because only Sm-ring containing pre-TLC1 RNPs can enter the nucleus. The re-imported pre-TLC1 is subsequently trimmed by the nuclear exosome up to the Sm-ring to form the 1157 nt long mature TLC1. Maturation of TLC1 is finalized by the trimethylation of the m7G-cap, which prevents repeated Xpo1 contact and export. Unrevealing this stepwise maturation process helps to explain why and how immature TLC1 does not disturb telomere maintenance before the mature and functional ribonucleoenzyme is assembled. | de |