E008 Study on the mechanism of human telomerase assembly

Telomerase, a ribonucleoprotein enzyme, catalyses the synthesis of repeated telomeric sequences. Telomeres are required for chromosome stability and cell viability and a decrease of their length was found to be associated with several cardiovascular diseases. Telomerase contains the hTR RNA, and several proteins, including the reverse transcriptase hTERT. The hTR 5’ domain contains the template sequence for DNA synthesis and binds hTERT. The H/ACA 3’ domain is required for stability and associates with 4 proteins (Dyskerin, GAR1, NOP10 and NHP2 proteins). Limited information is available on telomerase assembly. A pre-complex containing Dyskerine, NOP10, NHP2 and an assembly factor, called NAF1, is likely formed in the cytoplasm and transported to the nucleus, where it associates with the H/ACA domain of nascent RNA. Exchange of NAF1 for GAR1 allows the production of the functional H/ACA RNP domain. We showed that several factors are implicated in H/ACA RNP assembly : NUFIP, the R2TP complex, and HSP90. The SMN complex, containing the SMN, Gemin2 to Gemin8 and Unrip proteins may also be involved, since protein SMN interacts with GAR1. For further analysis on the role of the SMN complex in telomerase assembly, we tested by yeast two hybrid assays whether other proteins of this complex can interact with H/ACA RNP proteins and their assembly factors. We discovered a possible interaction between NAF1, Gemin 3 and Gemin8. Hence, the SMN complex may be involved in the replacement of NAF1 by GAR1: it may associate with GAR1 through an interaction with SMN, and with the pre-H/ACA RNP complex through interaction of NAF1 with Gemin3 and 8, allowing the replacement of NAF1 by GAR1. We are testing this hypothesis. The SMN complex may also be involved in hTERT binding to the 5’ domain of hTR and we will also explore this possibility. Through these studies we should bring insight into telomerase assembly that may be useful to understand telomere attrition in cardiovascular diseases.