The Nucleolar Localization Domain of the Catalytic Subunit of Human Telomerase

Abstract

Telomerase is the enzyme essential to complete the replication of the terminal DNA of most eukaryotic chromosomes. In humans, this enzyme is composed of the telomerase reverse transcriptase (hTERT) and telomerase RNA (hTR) subunits. hTR has been found in the nucleolus, a site of assembly of ribosomes as well as other ribonucleoproteins (RNPs). We therefore tested whether the hTERT component is also found in the nucleolus, where it could complex with the hTR RNA to form a functional enzyme. We report here that hTERT does indeed localize to the nucleolus, and we mapped the domain responsible for this localization to the hTRbinding region of the protein by deletion analysis. Substitution mutations in two of the three conserved hTRbinding domains in this nucleolar localization domain (NoLD) abolished nucleolar localization. However, another mutation that impeded hTR binding did not alter this subcellular localization. Additionally, wild type hTERT was detected in the nucleolus of cells that failed to express hTR. Taken together, we propose that the nucleolar localization of hTERT involves more than just the association with the hTR subunit. Furthermore, the coincidental targeting of both the hTR and hTERT subunits to the nucleolus supports the premise that the assembly of telomerase occurs in the nucleolus.Telomerase is a reverse transcriptase ribonucleoprotein (RNP) 1 complex composed of a reverse transcriptase catalytic protein subunit (TERT) that copies a template region of an accompanying RNA subunit (TR) onto telomeres as DNA (1). In humans, this enzyme is of great medical importance because of its pivotal role in unlimited cellular proliferation, a hallmark of cancer cells (2). The union of the RNA and protein subunits to form an RNP is essential for telomerase activity (3).Based on a comparison of the amino acid sequence of TERT from organisms of many different kingdoms and on mutational analysis, it has been possible to identify a number of discrete domains in TERT. The central region of the catalytic subunit contains seven motifs found in reverse transcriptases, which define the catalytic core (4 -17). The C terminus of TERT is highly divergent, both at the sequence and the functional level (18,19). The N-terminal region is more conserved, containing domain I and the DAT (dissociates activities of telomerase) domain (18, 20) followed by domains II and III (18,20,21) and the T motif (1, 5, 6), which are essential for telomere elongation. Substitution mutations in domains II and III or the T motif decrease TERT binding to the telomerase RNA in yeast (18), ciliate (22, 23), or human cells (20,24) and correspondingly result in a dysfunctional enzyme. Deletion analysis has also defined the region extending from amino acids 326 to 613, which harbors all three of the aforementioned domains, as the minimum region required for hTR binding (22), although mutations in domain I can also have some effect on hTR binding (24).The site of assembly of the telomerase RNP has not been determined; however, there is growing e...

Telomerase promotes efficient cell cycle kinetics and confers growth advantage to telomerase-negative transformed human cells

“…The DAT mutants were previously reported to retain the capacity to promote the non-canonical functions of TERT in chromatin remodeling and are therefore competent to promote DNA-damage repair, despite defects in their canonical TERT functions . We also included three telomerase-RNA-binding-defective TERT mutants (FYAA561, RBD386 and RBD512; Etheridge et al, 2002). Although our earlier experiments demonstrated that the expression of both TERT and TER components were necessary to support telomerase growth-promoting functions in ALT cells, we could not rule out the possibility that these two telomerase components were working independently.…”

hTERT associates with human telomeres and enhances genomic stability and DNA repair

“…It is yet to be determined whether hTERT binds to any specific sequence of total genomic DNA. However, Etheridge et al (2002) have demonstrated that hTERT localizes to the nucleolus. In contrast to this, hTERT binding to telomeres is dependent on hTR as well as the RT motif, which suggests that hTERT must be functionally reconstituted in order to interact with telomeres so that it could provide the function of replenishing the telomeric DNA.…”

“…One possibility is that hTERT interactions with telomeres may influence the association between telomeres and nuclear matrix. Recently, we and others have shown that hTERT is present throughout the nucleus but in certain instances hTERT is concentrated in the nucleolar organizing region (Tahara et al, 1999;Vaziri et al, 1999;Kawakaim et al, 2000;Wood et al, 2001;Etheridge et al, 2002). More directly, telomeres have been shown cytologically as well as biochemically to be tethered to the nuclear matrix (Smith and de Lange, 1997;Pandita, 2002).…”

Telomerase stimulates ribosomal DNA transcription under hyperproliferative conditions

“…Notably, a recent report identified a nucleolar localization signal in TERT 9 . Interestingly, the nucleolar localization of TERT appears to be unrelated to its telomerase function 10 .…”