Identification of proteins associated with TERT that are required for telomerase function would provide us with better understanding of telomerase regulation as well as the mechanism of its action. AH3 tagged TERT was therefore expressed in HeLa S3 cells and telomerase RNP complex was isolated and purified by tandem affinity approach. Telomerase complexes were prepared from both S100 extracts (n(velence)2) and nuclear extracts (n(velence)2) to ensure thorough analysis of TERT associated proteins. TERT was reproducibly detected using nanoRPLC-MS/MS in all four sample preparations. Besides known TERT-associated proteins, two novel proteins, Pontin and Reptin, were identified within a single, prominently stained coomassie gel band (Figure 1 (A)). Both proteins were identified in all four sample preparation with greater than 50percent protein coverage. Pontin and Reptin are ATPases associated with various cellular activities. They have been previously associated with chromatin remodeling and been identified within small nucleolar RNA (snoRNA) complexes. Their exact functions in these protein complexes, however, were not clear. We found that pontin and reptin were associated with both endogenous TERT and endogenous dyskerin in human cancer cells. Depletion of pontin and reptin using RNA interference revealed that these ATPases serve an essential role in telomerase biogenesis.
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