Nucleolin Interacts with Telomerase

Shilagardi Khurts,Kenkichi Masutomi,Luvsanjav Delgermaa,Kuniaki Arai,Naoki Oishi,Hideki Mizuno,Naoyuki Hayashi,William C Hahn,Seishi Murakami

doi:10.1074/jbc.m407643200

Abstract

Telomerase is a specialized reverse transcriptase composed of core RNA and protein subunits which plays essential roles in maintaining telomeres in actively dividing cells. Recent work indicates that telomerase shuttles between subcellular compartments during assembly and in response to specific stimuli. In particular, telomerase colocalizes with nucleoli in normal human fibroblasts. Here, we show that nucleolin, a major nucleolar phosphoprotein, interacts with telomerase and alters its subcellular localization. Nucleolin binds the human telomerase reverse transcriptase subunit (hTERT) through interactions with its RNA binding domain 4 and carboxyl-terminal RGG domain, and this binding also involves the telomerase RNA subunit hTERC. The protein-protein interaction between nucleolin and hTERT is critical for the nucleolar localization of hTERT. These findings indicate that interaction of hTERT and nucleolin participates in the dynamic intracellular localization of telomerase complex.

Highlights

Telomerase is a specialized reverse transcriptase composed of core RNA and protein subunits which plays essential roles in maintaining telomeres in actively dividing cells
IMR90 cells expressing enhanced green fluorescence protein (EGFP)-human telomerase reverse transcriptase subunit (hTERT) showed a pan-nuclear localization of EGFPhTERT with enriched nucleolar localization as assessed by costaining with an anti-nucleolin-specific antibody
The results show that the interaction of nucleolin with hTERT and hTERC regulates the subcellular localization of telomerase

Summary

EXPERIMENTAL PROCEDURES

Plasmids—The bacterial and mammalian expression vectors for full sized nucleolin and nucleolin mutants as well as the mammalian expression vectors pNKZFLAG-hTERT (amino-terminal FLAG-tagged hTERT), pNCZFLAG-hTERT (carboxyl-terminal FLAG-tagged hTERT), and pNKZGST-hTERT (amino-terminal GST-fused hTERT) have been described previously (22–25). PGRN164 vector containing hTERC cDNA was linearized with FspI and used as a template for hTERC RNA preparation as described previously (5, 25). After centrifugation of the sonicated lysates, the supernatants were passed through DEAESepharose, and the GST fusion proteins were recovered using glutathione-Sepharose 4B beads (Amersham Biosciences). The resin was washed, and the GST fusion proteins were eluted with glutathione. Preparation of Cell Extracts, Immunoprecipitation, and Immunoblotting—Cells were harvested, washed with PBS (Ϫ), and sonicated in a lysis buffer (50 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM leupeptin, 10 mM aprotinin, 1 mM dithiothreitol). Lysates derived from 5 ϫ 106 cells were diluted 10-fold in the lysis buffer containing 1% bovine serum albumin and precleared by incubation with GammaBind G-Sepharose (Amersham Biosciences) at 4 °C for 1 h. After an extensive washing (50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1 mM

Nucleolin Interacts with hTERT

RESULTS

DISCUSSION