Multiple Mechanisms for Elongation Processivity within the Reconstituted Tetrahymena Telomerase Holoenzyme

To maintain telomeres, telomerase evolved a unique biochemical activity: the use of a single-stranded RNA template for the synthesis of single-stranded DNA repeats. High repeat addition processivity (RAP) of the Tetrahymena telomerase holoenzyme requires association of the catalytic core with the telomere adaptor subcomplex (TASC) and an RPA1-related subunit (p82 or Teb1). Here, we used DNA binding and holoenzyme reconstitution assays to investigate the mechanism by which Teb1 and TASC confer high RAP. We show that TASC association with the recombinant telomerase catalytic core increases enzyme activity. Subsequent association of the Teb1 C-terminal domain with TASC confers the capacity for high RAP even though the Teb1 C-terminal domain does not provide a high-affinity DNA interaction site. Efficient RAP also requires suppression of nascent product folding mediated by the central Teb1 DNA-binding domains (DBDs). These sequence-specific high-affinity DBDs of Teb1 can be functionally substituted by the analogous DBDs of Tetrahymena Rpa1 to suppress nascent product folding but only if the Rpa1 high-affinity DBDs are physically tethered into holoenzyme context though the Teb1 C-terminal domain. Overall, our findings reveal multiple mechanisms and multiple surfaces of protein-DNA and protein-protein interaction that give rise to elongation processivity in the synthesis of a single-stranded nucleic acid product.     

The 5' guanosine tracts of human telomerase RNA are recognized by the G-quadruplex binding domain of the RNA helicase DHX36 and function to increase RNA accumulation

Telomerase promotes telomere maintenance by copying a template within its integral RNA subunit to elongate chromosome ends with new telomeric repeats. Motifs have been defined within the telomerase RNA that contribute to mature RNA accumulation, holoenzyme catalytic activity, or enzyme recruitment to telomeres. Here, we describe a motif of human telomerase RNA (hTR), not previously characterized in a cellular context, comprised of several guanosine tracts near the RNA 5' end. These guanosine tracts together are recognized by the DEXH box RNA helicase DHX36. The helicase domain of DHX36 does not mediate hTR binding; instead, hTR interacts with the N-terminal accessory domain of DHX36 known to bind specifically to the parallel-strand G-quadruplex substrates resolved by the helicase domain. The steady-state level of DHX36-hTR interaction is low, but hTR guanosine tract substitutions substantially reduce mature hTR accumulation and thereby reduce telomere maintenance. These findings suggest that G-quadruplex formation in the hTR precursor improves the escape of immature RNP from degradation, but subsequently the G-quadruplex may be resolved in favor of a longer terminal stem. We conclude that G-quadruplex formation within hTR can stimulate telomerase-mediated telomere maintenance.     

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.     

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.     

TPP1 as a versatile player at the ends of chromosomes

Telomeres, the ends of linear eukaryotic chromosomes, are tandem DNA repeats and capped by various telomeric proteins. These nucleoprotein complexes protect telomeres from DNA damage response （DDR）, recombination, and end-to-end fusions, ensuring genome stability. The human telosome/shelterin complex is one of the best-studied telomere-associated protein complexes, made up of six core telomeric proteins TRF1, TRF2, TIN2, RAPI, POT1, and TPPI. TPP1, also known as adrenocortical dysplasia protein homolog （ACD）, is a putative mammalian homolog of TEBP-β and belongs to the oligonucleotide binding （OB）-fold-containing protein family. Three functional domains have been identified within TPP1, the N-terminal OB fold, the POT1 binding recruitment domain （RD）, and the carboxyl-terminal TIN2-interacting domain （TID）. TPP1 can interact with both POT1 and TIN2 to maintain telomere structure, and mediate telomerase recruitment for telomere elongation. These features have indicated TPP1 play an essential role in telomere maintenance. Here, we will review important findings that highlight the functional significance of TPP1, with a focus on its interaction with other telosome components and the telomerase. We will also discuss potential implications in disease therapies.     

The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance

The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Delta strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.