Chaperone-mediated nuclear translocation of human telomerase and post-translational regulation of telomere-associated protein, TRF1

Abstract

Telomerase is a unique ribonucleoprotein enzyme that is required for continued cell proliferation. To generate catalytically active telomerase, hTERT should be transported to the nucleus and assembled with the telomerase RNA component. The molecular chaperones Hsp90 and p23 have been shown to maintain hTERT in a conformation enabling nuclear translocation. However, the regulatory role of chaperones in nuclear transport of hTERT remains unclear. In this work, we demonstrate that immunophilin FKBP52 links the hTERT-Hsp90 complex to dynein/dynactin motor, thereby promoting the cytoplasmic transport of hTERT to the nucleus along microtubule tracks. FKBP52 interacts with the hTERT-Hsp90 complex through the TPR domain binding to Hsp90 as well as with the dynamitin component of the dynein-associated dynactin complex through the PPIase domain. Depletion of FKBP52 inhibits nuclear transport of hTERT and results in a cytoplasmic accumulation. The resulting cytoplasmic hTERT is rapidly degraded through ubiquitin-dependent proteolysis, thereby abrogating telomerase activity. In addition, overexpression of dynamitin, which is known to dissociate dynein/dynactin motor from its cargoes, reduced telomerase activity. These results provide a molecular mechanism by which FKBP52 modulates telomerase activity by regulating nuclear transport of hTERT.

The human telomeric protein TRF1 is one of the telomere-associated proteins, which is coated at the chromosome ends to protect telomere. In particular, TRF1 negatively regulates telomere extension by inhibiting telomerase access to the ends of the telomeres, suggesting that the TRF1 at the telomeres is tightly regulated for controlling telomere length. Here, we identified NEDD8 Ultimate Burster1 (NUB1), a negative regulator of the NEDD8 conjugation system, by recruiting free NEDD8 and its conjugates to the proteasome for degradation as a novel TRF1-interacting protein. NUB1 interacts with TRF1 in vitro and in vivo through its N-terminal region in an NEDD8-independent manner. We found that overexpression of NUB1 decreased levels of TRF1 and also reduced the half-life of TRF1, whereas depletion of endogenous NUB1 expression stabilized levels of endogenous TRF1. A previous report found that the ubiquitin-like domain at the N-terminal region of NUB1 interacted with the 26S proteasome subunit (S5a/PSMD4). We also found that the interaction between TRF1 and PSMD4 was enhanced by NUB1 overexpression, but decreased by NUB1 knockdown, suggesting that NUB1 might play an adaptor role between TRF1 and proteasome for degradation. These findings demonstrate that NUB1 plays a critical role in controlling the proteasomal degradation of TRF1 through physical interaction for telomere homeostasis.

A telomere is a specialized heterochromatic structure found at the end of eukaryotic chromosomes. It is composed of telomeric DNA and a six-subunit protein complex, named shelterin, which is required for maintaining and protecting the telomere from nucleolytic attacks. Human TRF1 is one of the shelterin proteins directly involved in telomere length regulation. Post-translational modifications of TRF1 play important roles in modulating telomere length homeostasis by regulating the abundance of TRF1 at telomeres through the interactions with various proteins. Here, we demonstrate the novel post-translational modification of TRF1 called neddylation. We find that TRF1 is subject to NEDD8-conjugation in vitro and in vivo, and we also identify and characterize NEDP1, which is a human NEDD8-specific protease that efficiently abolishes the neddylation of TRF1. NEDD8 fusion to the C-terminus of TRF1 to mimic the effects of mono-neddylation is found to cause the cytoplasmic localization of TRF1 and reduce the stability of TRF1, suggesting that the neddylation of TRF1 is involved in the subcellular localization and cellular abundance of TRF1. Moreover, the level of TRF1 is increased and cytoplasmic localization is inhibited by MLN4924 treatment, which is a novel NEDD8-Activating Enzyme (NAE) inhibitor, implying that neddylation is a novel post-translational modification mechanism for controlling TRF1 at telomeres.