TIN2 Functions with TPP1/POT1 To Stimulate Telomerase Processivity

ABSTRACT

TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short-telomere syndromes. While the genetics underscore the importance of TIN2, the mechanism through which TIN2 regulates telomere length remains unclear. Here, we tested the effects of human TIN2 on telomerase activity. We identified a new isoform in human cells, TIN2M, that is expressed at levels similar to those of previously studied TIN2 isoforms. All three TIN2 isoforms localized to and maintained telomere integrity in vivo, and localization was not disrupted by telomere syndrome mutations. Using direct telomerase activity assays, we discovered that TIN2 stimulated telomerase processivity in vitro. All of the TIN2 isoforms stimulated telomerase to similar extents. Mutations in the TPP1 TEL patch abrogated this stimulation, suggesting that TIN2 functions with TPP1/POT1 to stimulate telomerase processivity. We conclude from our data and previously published work that TIN2/TPP1/POT1 is a functional shelterin subcomplex.

KEYWORDS:

• POT1
• TIN2
• TPP1
• alternative splicing
• processivity
• shelterin
• telomerase
• telomere

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SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00593-18.

ACKNOWLEDGMENTS

We thank Jonathan Alder, Deborah Wuttke, Sarah Wheelan, Carla Connelly, and Leslie Glustrom for suggestions and help with experiments; Mary Armanios for human LCL cell lines; and Andrew Holland for FLP-in cell lines. We thank Jonathan Alder, Valerie Gaysinskaya, and Deborah Wuttke for critical reading of the manuscript. We also thank the Johns Hopkins Deep Sequencing & Microarray Core Facility for advice and core services.

This work was supported by NIH grants R37AG009383 and R35CA209974 to C.W.G. and a Turock Scholar award to A.M.P.

A.M.P. and C.W.G. designed the project and wrote the manuscript. A.M.P. performed all cloning, cell line generation, telomerase assays, and data analysis. M.A.S. performed immunofluorescence, lentiviral transductions, qRT-PCR, TIF analysis, and cell line growth. J.P.T.O. and A.M.P. performed 3′RACE and PacBio sequencing.

We declare that we have no competing interests.