The Insertion in Fingers Domain in Human Telomerase Can Mediate Enzyme Processivity and Telomerase Recruitment to Telomeres in a TPP1-Dependent Manner

Abstract

In most human cancer cells, cellular immortalization relies on the activation and recruitment of telomerase to telomeres. The telomere-binding protein TPP1 and the TEN domain of the telomerase catalytic subunit TERT regulate telomerase recruitment. TERT contains a unique domain, called the insertion in fingers domain (IFD), located within the conserved reverse transcriptase domain. We report the role of specific hTERT IFD residues in the regulation of telomerase activity and processivity, recruitment to telomeres, and cell survival. One hTERT IFD variant, hTERT-L805A, with reduced activity and processivity showed impaired telomere association, which could be partially rescued by overexpression of TPP1-POT1. Another previously reported hTERT IFD mutant enzyme with similarly low levels of activity and processivity, hTERT-V791Y, displayed defects in telomere binding and was insensitive to TPP1-POT1 overexpression. Our results provide the first evidence that the IFD can mediate enzyme processivity and telomerase recruitment to telomeres in a TPP1-dependent manner. Moreover, unlike hTERT-V791Y, hTERT-V763S, a variant with reduced activity but increased processivity, and hTERT-L805A, could both immortalize limited-life-span cells, but cells expressing these two mutant enzymes displayed growth defects, increased apoptosis, DNA damage at telomeres, and short telomeres. Our results highlight the importance of the IFD in maintaining short telomeres and in cell survival.

ACKNOWLEDGMENTS

We thank Gerardo Ferbeyre (Université de Montréal, Montréal, Canada) for the plasmid construct pMSCV-puromycin, Thomas R. Cech (University of Colorado Boulder, Boulder, CO) for the pVAN3XFLAG-hTPP1, pVAN3XFLAG-hPOT1 and pVAN3XFLAG-hTERT constructs, and Joachim Lingner (Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland) for the pcDNA6/myc-His C-hTERT and pBluescript II SK(+)-hTR plasmids. We thank Silvia Bacchetti (Istittuto Regina Elena, Rome, Italy) for the HA5 cells. We are grateful to Peter Lansdorp (British Columbia Cancer Center, Vancouver, Canada) for providing us with the TFL-Telo, version 2.0, software. We thank Michael Terns (The University of Georgia, Athens, GA) for providing us with the anticoilin antibody. We thank Stephane Richard (McGill University, Montreal, Canada) for providing the Axio Imager M1 microscope. We are thankful to Paul Maddox (University of North Carolina, Chapel Hill, NC) for providing help with microscopy analysis and Christian Young for assisting us with FACS analysis.

C.A. was supported by a Chercheur National of the Fonds de Recherche du Québec-Santé (FRQS). Support to T.W.C. was provided by a McGill University Faculty of Medicine Internal Studentship, a Canadian Institute for Health Research McGill Integrated Cancer Research Training Program Studentship, and an FRQS studentship, and support to Y.D. was provided by a Canadian Institute for Health Research Canada graduate scholarship.

ADDENDUM

While the manuscript was under review, a cryo-electron microscopy structure of Tetrahymena telomerase subunits was published (46) in which the authors suggest that p50 is a structural and functional paralog of TPP1. Furthermore, the cryo-electron microscopy density attributed to the IFD apparently independently contacts both p50 and the TERT TEN domain. Based on the Tetrahymena telomerase structure, the authors propose that the human TERT IFD may interact with the TPP1 OB fold and hTERT TEN domain, in support of our findings that the human TERT IFD regulates telomerase and telomere function in a TPP1-dependent manner.