• RESEARCH

RESEARCH

1. Telomere and telomerase biology

Telomeres, the specialized nucleoprotein complexes at the ends of linear eukaryotic chromosomes, are essential for the maintenance of chromosome integrity and have been implicated in aging and cancer. Mammalian telomeres consist of long tracts of duplex TTAGGG repeats with 3’ single stranded G overhangs and are tightly associated with the six-subunit protein complex shelterin (TRF1, TRF2, RAP1, TIN2, TPP1, and POT1) that provides telomere protection by preventing chromosome ends from being recognized as DNA damage. In the absence of a telomere maintenance pathway, most human somatic cells show a progressive loss of telomeric DNA due to incomplete replication of chromosome ends. Although homologous recombination-mediated DNA synthesis has been demonstrated for replenishing telomeric DNA, the maintenance of telomere repeats in most eukaryotic organisms requires the enzyme telomerase which adds telomeric repeats onto the 3’ ends of linear chromosomes by reverse transcription. Human telomerase consists of telomerase reverse transcriptase (hTERT), telomerase RNA component (TERC) and several additional proteins including dyskerin, TCAB1, pontin and reptin. Telomerase expression is very low in most human somatic cells but upregulated in many human cancer cells and stem cells, suggesting that activation of telomerase supports the continued cell proliferation. During the decade, our laboratory has focused to identify the new telomere-binding proteins and the novel components of telomerase holoenzyme and elucidate their physiological functions in aging and cancer.


2. Catalytically active telomerase holoenzyme is assembled in the nucleolus during S phase

Telomerase undergoes a highly elaborate, stepwise process of assembly and trafficking within the nucleus. After transcription, a TERC RNA molecule assembles with a preformed H/ACA protein complex of dyskerin, NOP10, NHP2, and the H/ACA RNP assembly factor NAF1 and is exonucleolytically processed at its 3′ end to the boundary of the H/ACA motif. NAF1 is exchanged for the mature H/ACA RNP protein GAR1. The subsequent assembly of the TERC-dyskerin RNP with hTERT generates the catalytically active telomerase RNP. However, this complex cannot act on telomeres without additional assembly and trafficking processes. The telomerase RNP has been shown to accumulate in Cajal bodies by direct association of the TERC CAB box motif with TCAB1. Telomerase-containing Cajal bodies associate with a subset of telomeres. Mutations in TCAB1 prevent telomerase from elongating telomeres by disrupting telomerase localization to Cajal bodies, thereby causing dyskeratosis congenita, a human disease due to telomerase deficiency. Telomerase recruitment to telomeres requires the interaction between the OB-fold domain of TPP1 and hTERT. This interaction recruits telomerase from a Cajal body reservoir to telomeric chromatin for telomere elongation.

It has been well documented that telomerase acts on telomeres specifically during S phase, when telomere synthesis occurs. Moreover, the assembly and intranuclear trafficking of telomerase holoenzyme are also regulated to restrict telomere synthesis to S phase. However, it is unclear how the assembly and trafficking of telomerase holoenzyme are linked to the cell cycle. We show that the catalytically active holoenzyme is initially assembled in the dense fibrillar component of the nucleolus exclusively during S phase. The telomerase RNP is retained in nucleoli through the interaction between hTERT and nucleolin. When the telomerase RNP associates with TCAB1 in S phase, it separates from nucleoli and is transported to Cajal bodies for subsequent recruitment to telomeric chromatin. Consistently, the amount of telomerase RNP associated with nucleolin is increased following depletion of TCAB1. This suggests that the TCAB1-dependent trafficking of telomerase to Cajal bodies occurs in a step separate from the holoenzyme assembly in nucleoli.

3. Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles

Telomerase is upregulated in human cancer cells but repressed in normal somatic cells, suggesting that the activation of telomerase supports tumor proliferation and survival by maintaining functional telomeres. Telomerase undergoes a highly elaborate, stepwise process for the assembly and trafficking of the telomerase holoenzyme. After the preassembly in nucleoli, the telomerase ribonucleoprotein (RNP) is transported to Cajal bodies by the direct interaction of the telomerase RNA component (TERC) CAB box sequence with TCAB1. Depletion of TCAB1 does not affect telomerase RNP assembly and telomerase enzymatic activity but reduces telomerase localization to Cajal bodies, resulting in a failure to maintain functional telomeres. To elongate telomere repeats, Cajal bodies containing the telomerase RNP transiently associate with telomeric chromatin. It has been recently reported that the OB-fold domain of TPP1 is required for telomerase recruitment to telomeres through the interaction with telomerase reverse transcriptase (TERT), and this interaction is an essential step in telomere length maintenance. Nonetheless, many open questions remain about the precise molecular mechanisms of telomerase recruitment and how telomerase efficiently finds the site of action in the context of chromatin architecture.

In a search for proteins capable of interacting with TERC using a RNA affinity chromatography, we identify SRSF11 (also named SRp54) as a TERC-interacting factor that localizes to nuclear speckles, subnuclear structures that are enriched in pre-messenger RNA splicing factors. SRSF11 has been shown to function as a splicing factor that is a member of the highly conserved family of serine/arginine (SR) proteins. In this work, we show that SRSF11 associates with active telomerase through the interaction with TERC and directs it to nuclear speckles specifically during S phase. We also show that SRSF11 can associate with telomeres through the interaction with TRF2, which are constitutively present at nuclear speckles. Our data suggest that nuclear speckle is the S phase-specific nuclear site where telomerase is loaded on telomeres, and that SRSF11functionsasanuclearspeckle-targetingfactorthat is essential for telomerase recruitment to telomeres.

4. Telomerase activates transcription of cyclin D1 gene through an interaction with NOL1

Although overexpression of telomerase is sufficient to overcome replicative senescence, recent studies have suggested that besides its reverse transcriptase activity, telomerase has the noncanonical functions that contribute to cancer development and progression. Ectopic expression of telomerase in human mammary epithelial cells results in enhanced expression of growth-promoting genes. Transgenic induction of TERT in mouse skin epithelium has been shown to cause proliferation of quiescent stem cells. This function for TERT is independent of reverse transcriptase activity. In addition, TERT has been found to directly interact with BRG1 and activate transcription of Wnt/β-catenin-dependent genes such as cyclin D1 and Myc. However, the proposed noncanonical role of TERT in the Wnt/β-catenin signaling cascade has been controversial. Several studies have reported a lack of physical association of TERT with BRG1 or β-catenin, as well as no apparent effect of TERT deficiency on phenotypes associated with Wnt signaling in TERT-knockout mice. Although TERT appears to regulate the expression of growth-promoting genes, this event might not be solely promoted by Wnt signaling.

Given that the large size of human telomerase suggests the existence of additional components, we performed a large-scale affinity purification to identify proteins that interact with telomerase. Here, we identify proliferation-associated nucleolar antigen p120 (NOL1, also known as NOP2) as a TERC-binding protein. NOL1 was originally identified as a RNA-binding and nucleolar-specific protein that is highly expressed in the majority of human malignant tumor cells but not in normal resting cells. Although NOL1 has been implicated as a tumor cell marker, the molecular mechanism by which NOL1 contributes to tumorigenesis is poorly understood. We show that NOL1 binds to the T-cell factor (TCF)-binding element (TBE) of the cyclin D1 promoter and activates its transcription. Telomerase is also recruited to the cyclin D1 promoter through the interaction with NOL1, further enhancing transcription of cyclin D1 gene. These results suggest a new role for telomerase as a modulator of NOL1-dependent transcriptional activation in human cancer cells.