Functional Characterization of Yeast Telomerase RNA Dimerization

Abstract

Telomerase is the cellular RNA-dependent DNA polymerase (i.e. reverse transcriptase) that uses an integral RNA template to synthesize telomeric DNA repeats at the ends of linear chromosomes. Human telomerase RNA (hTERC) is thought to function as a dimeric complex consisting of two RNAs that interact with each other physically as well as genetically. We show here for the first time that the yeast Saccharomyces cerevisiae telomerase RNA TLC1 likewise forms dimers in vitro. TLC1 dimerization depends on a unique 6-base self-complementary sequence, which closely mimics palindromic sequences that mediate functional dimerization of HIV-1 and other retroviral genomes. We found that dissimilar but comparably located TLC1 palindromes from other sensu stricto yeasts can functionally substitute for that of S. cerevisiae. Yeast cells expressing dimerizationdefective TLC1 alleles have shorter telomeres than those with wild-type TLC1. This study, therefore, highlights dimerization as a functionally conserved feature of the RNA templates utilized by reverse transcriptases of both viral and cellular origins.Telomerase is the cellular reverse transcriptase (RT) 2 that is responsible for adding telomeric DNA repeats onto the ends of linear chromosomes (1). Found in all eukaryotes, it is a multimolecular complex of which the minimal components are a catalytic RT protein (TERT) and an integral RNA template. TERT proteins from diverse mammalian, protozoan, and yeast species appear to be related to one another evolutionarily as well as to RTs from retroviruses and cellular retroelements (2-4). Telomerase RNAs, by contrast, differ widely in length and primary sequence, and their phylogenetic relationships to one another and to retroviral genomes, if any, are unclear. Certain properties that are shared among groups of retroviral or telomerase RNAs may indicate either a common evolutionary origin or the convergent evolution of features involved in RT function. Widely divergent telomerase RNAs, for example, share certain core secondary structures that are critical for telomerase activity but have no obvious counterparts in retroviral RNAs (5, 6). The single-stranded RNA genomes of retroviruses, on the other hand, share a tendency to form stable RNA homodimers that are found in all known retroviral particles and appear to be the natural substrates for viral RTs (reviewed in Ref. 7). The assembly of these viral genomic dimers is generally initiated at a specific palindrome (i.e. a self-complementary base sequence), unique to each virus, that dimerizes readily both in vitro and within cells, and mutations in these palindromes that disrupt genomic dimer formation can markedly impair viral replication and infectivity (7). RNAs derived from at least some cellular retroelements undergo similar dimerization (4). Unexpectedly, studies from our laboratory and others have recently shown that the human telomerase RNA also dimerizes readily and that this capacity to dimerize correlates with telomerase catalytic activity in cells or cell-free extract...

Inhibition of DNA double-strand break repair by the Ku heterodimer in mrx mutants of Saccharomyces cerevisiae

“…The locations of known binding sites for Est1, Est2 and the Yku70/Yku80 complex are depicted schematically in Figure 3A [16,18,19]. To identify the region(s) of TLC1 needed for enhancement of repair, unique restriction sites present in the GAL1p∷TLC1 plasmid pLKL74Y (Figure 3A) were used as breakpoints to create the deletion derivatives pLKL75Y-79Y shown in Figure 3B and 3C (see Materials and methods).…”

“…The RNA subunit of telomerase acts as a scaffold for the binding of several proteins, including Est1, Est2 and Yku70/Yku80, and for association with another TLC1 RNA molecule to form dimers in vivo [16,17,18,19]. Altering cellular levels of telomerase affects cell physiology in different ways.…”

Hypermethylation of yeast telomerase RNA by the snRNA and snoRNA methyltransferase Tgs1

“…These in turn might lead to changes of telomerase activity either by directly altering template function or by disturbing the interaction with Est or Sm proteins, thus causing reduced telomerase activity. However, mutations affecting Sm protein binding (Seto et al, 1999) and TLC1 dimerization (Gipson et al, 2007), and changes in cellular concentrations (Mozdy and Cech, 2006) showed telomerase defects that were distinct from those of tgs1Δ. It is also possible that the intrinsic enzymatic activity of telomerase is unchanged, but that the missing m 3 G cap might alter the positive or negative regulation processes of telomerase action at the telomere.…”

Telomerase reverse transcriptase mediated immortalization of human bone marrow stromal cells

“…In several studies, exogenous virus, or oncogene has been introduced to target the cells to construct the immortalized cells (Kelekar and Cole 1987;Arimura et al 2007;Wu et al 2007), in which the integration of target gene was random and expression of target gene might have interfered with the intracellular physiological processes, which could result in unexpected changes such as loss of differentiation characteristic and lack of control of check point. The cells treated with virus, or oncogene belong to transformed cells but not the normal cells, and thus they are different partially, or completely from the normal cells in the transformation features such as changes in cell morphology, karyotype and tumorigenicity, as well as loss of suspended growth and contact inhibition (Gipson et al 2007). The hTERT gene is an immortalization gene.…”