The structure and integrity of telomeres are essential for genome stability. Telomere dysregulation can lead to cell death, cell senescence, or abnormal cell proliferation. The maintenance of telomere repeats in most eukaryotic organisms requires telomerase, which consists of a reverse transcriptase (RT) and an RNA template that dictates the synthesis of the G-rich strand of telomere terminal repeats. Structurally, telomerase reverse transcriptase (TERT) contains unique and variable N- and C-terminal extensions that flank a central RT-like domain. The enzymology of telomerase includes features that are both similar to and distinct from those characteristic of other RTs. Two distinguishing features of TERT are its stable association with the telomerase RNA and its ability to repetitively reverse transcribe the template segment of RNA. Here we discuss TERT structure and function; its regulation by RNA-DNA, TERT-DNA, TERT-RNA, TERT-TERT interactions, and TERT-associated proteins; and the relationship between telomerase enzymology and telomere maintenance.

Dyskerin and its homologs are ancient and conserved enzymes that catalyze the most common post-transcriptional modification found in cells, pseudouridylation. The resulting pseudouridines provide stability to RNA molecules and regulate ribosome biogenesis and splicing events. Dyskerin does not act independently—it is the core component of a protein heterotetramer, which associates with RNAs that contain the H/ACA motif. The variety of H/ACA RNAs that guide the function of this ribonucleoprotein (RNP) complex highlights the diversity of cellular processes in which dyskerin participates. When associated with small nucleolar (sno) RNAs, it regulates ribosomal (r) RNAs and ribosome biogenesis. By interacting with small Cajal body (sca) RNAs, it targets small nuclear (sn) RNAs to regulate pre-mRNA splicing. As a component of the telomerase holoenzyme, dyskerin binds to the telomerase RNA to modulate telomere maintenance. In a disease context, dyskerin malfunction can result in multiple detrimental phenotypes. Mutations inDKC1, the gene that encodes dyskerin, cause the premature aging syndrome X-linked dyskeratosis congenita (X-DC), a still incurable disorder that typically leads to bone marrow failure. In this review, we present the classical and most recent findings on this essential protein, discussing the evolutionary, structural, and functional aspects of dyskerin and the H/ACA RNP. The latest research underscores the role that dyskerin plays in the regulation of gene expression, translation efficiency, and telomere maintenance, along with the impacts that defective dyskerin has on aging, cell proliferation, haematopoietic potential, and cancer.

Human telomerase is a multimer containing two human telomerase RNAs (hTRs) and most likely two human telomerase reverse transcriptases (hTERTs). Telomerase synthesizes multiple telomeric repeats using a unique repeat addition form of processivity. We investigated hTR and hTERT sequences that were essential for DNA synthesis and processivity using a direct primer extension telomerase assay. We found that hTERT consists of two physically separable functional domains, a polymerase domain containing RNA interaction domain 2 (RID2), reverse transcriptase (RT), and C-terminal sequences, and a major accessory domain, RNA interaction domain 1 (RID1). RID2 mutants defective in high-affinity hTR interactions and an RT catalytic mutant exhibited comparable DNA synthesis defects. The RID2-interacting hTR P6.1 helix was also essential for DNA synthesis. RID1 interacted with the hTR pseudoknot-template domain and hTERT9s RT motifs and putative thumb and was essential for processivity, but not DNA synthesis. The hTR pseudoknot was essential for processivity, but not DNA synthesis, and processivity was reduced or abolished in dimerization-defective pseudoknot mutants. trans-acting hTERTs and hTRs complemented the processivity defects of RID1 and pseudoknot mutants, respectively. These data provide novel insight into the catalytic organization of the human telomerase complex and suggest that repeat addition processivity is one of the major catalytic properties conferred by telomerase multimerization.

Functional human telomerase complexes are minimally composed of the human telomerase RNA (hTR) and a catalytic subunit (human telomerase reverse transcriptase [hTERT]) containing reverse transcriptase (RT)-like motifs. The N terminus of TERT proteins is unique to the telomerase family and has been implicated in catalysis, telomerase RNA binding, and telomerase multimerization, and conserved motifs have been identified by alignment of TERT sequences from multiple organisms. We studied hTERT proteins containing N-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase catalytic activity, hTR binding, and hTERT multimerization. Using multiple sequence alignment, we identified two vertebrate-conserved TERT N-terminal regions containing vertebrate-specific residues that were required for human telomerase activity. We identified two RNA interaction domains, RID1 and RID2, the latter containing a vertebrate-specific RNA binding motif. Mutations in RID2 reduced the association of hTR with hTERT by 50 to 70%. Inactive mutants defective in RID2-mediated hTR binding failed to complement an inactive hTERT mutant containing an RT motif substitution to reconstitute activity. Our results suggest that functional hTERT complementation requires intact RID2 and RT domains on the same hTERT molecule and is dependent on hTR and the N terminus.

Effective strategies to reverse or prevent chemotherapeutic resistance are required before cancer therapies can be curative. Telomerase is the ribonucleoprotein responsible for de novo synthesis and maintenance of telomeres, and its activity is predominantly observed in cancer cells. The telomerase enzyme has been successfully inhibited or inactivated to sensitize cells to cellular stresses; however, no studies have determined yet the effect of combining a pharmacological inhibitor of telomerase catalysis and traditional chemotherapeutics for the treatment of drug-sensitive or drug-resistant cancers. Here, we describe the effect of 2-[(E)-3-naphtalen-2-yl-but-2-enoylamino]-benzoic acid (BIBR1532), a small-molecule inhibitor of telomerase catalytic activity, on drug-resistant leukemia and breast cancer cells and their parental counterparts when treated in combination with chemotherapeutics. We observed that BIBR1532-treated cells show progressive telomere shortening, decreased proliferative capacity, and sensitization to chemotherapeutic treatment. These effects are telomere length-dependent, because cells insensitive to BIBR1532 or cells released from telomerase inhibition did not demonstrate changes in growth ability or drug sensitivity. Our novel observations suggest that pharmacological telomerase inhibition in combination therapy may be a valid strategy for the treatment of both drug-sensitive and drug-resistant cancers.

The vast body of literature regarding human telomere maintenance is a true testament to the importance of understanding telomere regulation in both normal and diseased states. In this review, our goal was simple: tell the telomerase story from the biogenesis of its parts to its maturity as a complex and function at its site of action, emphasizing new developments and how they contribute to the foundational knowledge of telomerase and telomere biology.