Human cells lacking coilin and Cajal bodies are proficient in telomerase assembly,trafficking and telomere maintenance

The RNA component of human telomerase (hTR) localizes to Cajal bodies, and it has been proposed that Cajal bodies play a role in the assembly of telomerase holoenzyme and telomerase trafficking. Here, the role of Cajal bodies was examined in Human cells deficient of coilin (i.e. coilin-knockout (KO) cells), in which no Cajal bodies are detected. In coilin-KO cells, a normal level of telomerase activity is detected and interactions between core factors of holoenzyme are preserved, indicating that telomerase assembly occurs in the absence of Cajal bodies. Moreover, dispersed hTR aggregates and forms foci specifically during S and G2 phase in coilin-KO cells. Colocalization of these hTR foci with telomeres implies proper telomerase trafficking, independent of Cajal bodies. Therefore, telomerase adds similar numbers of TTAGGG repeats to telomeres in coilin-KO and controls cells. Overexpression of TPP1-OB-fold blocks cell cycle-dependent formation of hTR foci and inhibits telomere extension. These findings suggest that telomerase assembly, trafficking and extension occur with normal efficiency in Cajal bodies deficient human cells. Thus, Cajal bodies, as such, are not essential in these processes, although it remains possible that non-coilin components of Cajal bodies and/or telomere binding proteins (e.g. TPP1) do play roles in telomerase biogenesis and telomere homeostasis.     

A Cajal body-independent pathway for telomerase trafficking in mice

The intranuclear trafficking of human telomerase involves a dynamic interplay between multiple nuclear sites, most notably Cajal bodies and telomeres. Cajal bodies are proposed to serve as sites of telomerase maturation, storage, and assembly, as well as to function in the cell cycle-regulated delivery of telomerase to telomeres in human cells. Here, we find that telomerase RNA does not localize to Cajal bodies in mouse cells, and instead resides in separate nuclear foci throughout much of the cell cycle. However, as in humans, mouse telomerase RNA (mTR) localizes to subsets of telomeres specifically during S phase. The localization of mTR to telomeres in mouse cells does not require coilin-containing Cajal bodies, as mTR is found at telomeres at similar frequencies in cells from wild-type and coilin knockout mice. At the same time, we find that human TR localizes to Cajal bodies (as well as telomeres) in mouse cells, indicating that the distinct trafficking of mTR is attributable to an intrinsic property of the RNA (rather than a difference in the mouse cell environment such as the properties of mouse Cajal bodies). We also find that during S phase, mTR foci coalesce into short chains, with at least one of the conjoined mTR foci co-localizing with a telomere. These findings point to a novel, Cajal body-independent pathway for telomerase biogenesis and trafficking in mice.     

Telomerase enzymatic component hTERT shortens long telomeres in human cells

Telomere lengths are tightly regulated within a narrow range in normal human cells. Previous studies have extensively focused on how short telomeres are extended and have demonstrated that telomerase plays a central role in elongating short telomeres. However, much about the molecular mechanisms of regulating excessively long telomeres is unknown. In this report, we demonstrated that the telomerase enzymatic component, hTERT, plays a dual role in the regulation of telomere length. It shortens excessively long telomeres and elongates short telomeres simultaneously in one cell, maintaining the optimal telomere length at each chromosomal end for efficient protection. This novel hTERT-mediated telomere-shortening mechanism not only exists in cancer cells, but also in primary human cells. The hTERT-mediated telomere shortening requires hTERT’s enzymatic activity, but the telomerase RNA component, hTR, is not involved in that process. We found that expression of hTERT increases telomeric circular DNA formation, suggesting that telomere homologous recombination is involved in the telomere-shortening process. We further demonstrated that shelterin protein TPP1 interacts with hTERT and recruits hTERT onto the telomeres, suggesting that TPP1 might be involved in regulation of telomere shortening. This study reveals a novel function of hTERT in telomere length regulation and adds a new element to the current molecular model of telomere length maintenance.     

PinX1 is involved in telomerase recruitment and regulates telomerase function by mediating its localization

Telomerase recruitment to telomere is the prerequisite for telomere extension, but the proteins involved in this process are still largely unknown. PinX1 is a telomerase inhibitor and has been implicated in telomere maintenance. Silencing of PinX1 significantly reduced the localization of telomerase to telomere during mid-late S phase, suggesting the involvement of PinX1 in the cell cycle-dependent trafficking of hTERT to telomere. We also revealed that PinX1 mediated the chromosomal localization of hTERT during anaphase. This study revealed the role of PinX1 in telomerase function regulation by mediating its localization inside cells.     

Cell cycle-dependent recruitment of telomerase RNA and Cajal bodies to human telomeres

Telomerase is a ribonucleoprotein enzyme that counteracts replicative telomere erosion by adding telomeric sequence repeats onto chromosome ends. Despite its well-established role in telomere synthesis, telomerase has not yet been detected at telomeres. The RNA component of human telomerase (hTR) resides in the nucleoplasmic Cajal bodies (CBs) of interphase cancer cells. Here, in situ hybridization demonstrates that in human HeLa and Hep2 S phase cells, besides accumulating in CBs, hTR specifically concentrates at a few telomeres that also accumulate the TRF1 and TRF2 telomere marker proteins. Surprisingly, telomeres accumulating hTR exhibit a great accessibility for in situ oligonucleotide hybridization without chromatin denaturation, suggesting that they represent a structurally distinct, minor subset of HeLa telomeres. Moreover, we demonstrate that more than 25% of telomeres accumulating hTR colocalize with CBs. Time-lapse fluorescence microscopy demonstrates that CBs moving in the nucleoplasm of S phase cells transiently associate for 10-40 min with telomeres. Our data raise the intriguing possibility that CBs may deliver hTR to telomeres and/or may function in other aspects of telomere maintenance.     

Mechanism of dominant-negative telomerase function

Human telomerase uses its integral core components, hTR and hTERT, to maintain telomeres in many cell types. Expression of a dominant-negative mutant of the catalytic subunit of telomerase, DN-hTERT, has been shown to cause telomere shortening and ultimately cell death in a number of tumor-derived cell lines. However, the mechanism of dominant-negative hTERT function and its fate inside the cell are still unknown. In order to understand the effect of the dominant-negative on wild-type hTERT, each was fused with GFP and expressed in telomerase-positive cells. GFP-DN-hTERT expression resulted in cytoplasmic exportation and degradation via ubiquitination. Co-expression of wild-type GFP-hTERT with an untagged DN-hTERT resulted in decreased wild-type hTERT levels, export to the cytoplasm, and increased ubiquitination, suggesting that DN-hTERT complexes with wild-type hTERT to induce cytoplasmic localization. Based on the cytoplasmic degradation, we propose two new mechanisms of dominant-negative hTERT, employing the theory of interactive dimerization. First, the heterodimer of DN-hTERT with wild-type hTERT is exported to the cytoplasm for ubiquitin-mediated protein degradation, and second, the heterodimer may be degraded at a faster rate than the wild-type hTERT homodimer. Understanding mechanisms of telomerase degradation will guide future drug design to target sites on telomerase important for catalytic activity and protein stability.     

Human telomerase RNA accumulation in Cajal bodies facilitates telomerase recruitment to telomeres and telomere elongation

Telomerase is required for telomere maintenance and is responsible for the immortal phenotype of cancer cells. How telomerase is assembled and reaches telomeres in the context of nuclear architecture is not understood. Recently, the telomerase RNA subunit (hTR) was shown to accumulate in Cajal bodies (CBs), subnuclear structures implicated in ribonucleoprotein maturation. However, the functional relevance of this localization for telomerase was unknown. hTR localization to CBs requires a short sequence motif called the CAB box. Here, we reconstitute telomerase in human cells and determine the effects of CAB box mutations on telomere biology. We demonstrate that mutant hTR, which fails to accumulate in CBs, is fully capable of forming catalytically active telomerase in vivo but is strongly impaired in telomere extension. The functional deficiency is accompanied by a decreased association of telomerase with telomeres. Collectively, these data identify subnuclear localization as an important regulatory mechanism for telomere length homeostasis in human cells.     

A human-Tetrahymena pseudoknot chimeric telomerase RNA reconstitutes a nonprocessive enzyme in vitro that is defective in telomere elongation

The phylogenetically-derived secondary structures of telomerase RNAs (TR) from ciliates, yeasts and vertebrates are surprisingly conserved and contain a pseudoknot domain at a similar location downstream of the template. As the pseudoknot domains of Tetrahymena TR (tTR) and human TR (hTR) mediate certain similar functions, we hypothesized that they might be functionally interchangeable. We constructed a chimeric TR (htTR) by exchanging the hTR pseudoknot sequences for the tTR pseudoknot region. The chimeric RNA reconstituted human telomerase activity when coexpressed with hTERT in vitro, but exhibited defects in repeat addition processivity and levels of DNA synthesis compared to hTR. Activity was dependent on tTR sequences within the chimeric RNA. htTR interacted with hTERT in vitro and dimerized predominantly via a region of its hTR backbone, the J7b/8a loop. Introduction of htTR in telomerase-negative cells stably expressing hTERT did not reconstitute an active enzyme able to elongate telomeres. Thus, our results indicate that the chimeric RNA reconstituted a weakly active nonprocessive human telomerase enzyme in vitro that was defective in telomere elongation in vivo. This suggests that there may be species-specific requirements for pseudoknot functions.