Functional Analysis of Conserved Residues in the Putative “Finger” Domain of Telomerase Reverse Transcriptase

Abstract

Telomerase is a ribonucleoprotein reverse transcriptase (RT) responsible for the maintenance of one strand of telomere terminal repeats. The catalytic protein subunit of telomerase, known generically as telomerase reverse transcriptase (TERT), exhibits significant homology to RTs encoded by retroviruses and retroelements. The polymerization mechanisms of telomerase may therefore be similar to those of the "conventional" RTs. In this study, we explored the extent of mechanistic conservation by analyzing mutations of conserved residues within the putative "finger" domain of TERT. Previous analysis has implicated this domain of retroviral RTs in nucleotide and RNA binding and in processivity control. Our results demonstrate that residues conserved between TERT and human immunodeficiency virus-1 RT are more likely than TERT-specific residues to be required for enzyme activity. In addition, residues presumed to make direct contact with either the RNA or nucleotide substrate appear to be functionally more important. Furthermore, distinct biochemical defects can be observed for alterations in the putative RNA-and nucleotide-binding TERT residues in a manner that can be rationalized by their postulated mechanisms of action. This study thus supports a high degree of mechanistic conservation between telomerase and retroviral RTs and underscores the roles of distinct aspects of telomerase biochemistry in telomere length maintenance.Telomerase is a ribonucleoprotein that is responsible for maintaining the terminal repeats of telomeres in most organisms (1). It acts as an unusual reverse transcriptase, using a small segment of an integral RNA component as template for the synthesis of the dGT-rich strand of telomeres (2).Telomerase activity has been characterized in a wide range of organisms and genes encoding both the RNA and protein components of the enzyme complex identified (for reviews, see Refs. 3 and 4). Telomerase RNAs found in ciliated protozoa, in addition to having a short templating region, share a common secondary structure. Telomerase RNAs from yeast and mammals are considerably larger; and within each group, conserved structural elements can be identified based on phylogenetic and mutational analysis (5, 6). The catalytic reverse transcriptase protein subunit TERT, 1 first purified from Euplotes aediculatus as p123, was found to be homologous to Est2p, a yeast protein required for telomere maintenance (7-9). Both proteins possess reverse transcriptase (RT)-like motifs, alterations in which can result in inactivation of telomerase activity and reduced telomere length. Subsequently, homologs of TERT were identified in Schizosaccharomyces pombe, human, mouse, Tetrahymena, Oxytricha, and Arabidopsis (10 -17). Mutational analysis of the RT motifs in these latter proteins further supports a role for TERT in directly mediating catalysis (18,19). Because coexpression of TERT and telomerase RNA in vitro in the rabbit reticulocyte lysate system suffices to reconstitute enzyme activity (18,20), these two subunits probabl...

Functional characterization of natural telomerase mutations found in patients with hematologic disorders

“…Surprisingly, we found that deletion of nucleotides 28-34 of the hTERC RNA (ie, the ⌬28-34 variant) produced similar telomerase enzymatic activity ( Figure 4A, lanes 4-6) as compared to cell lysate with either the wild-type hTERC (lanes 1-3) or the known G58A sequence polymorphism (lanes 7-9). On the contrary, other variants, which are located within either the template region (⌬52-55 and A48G) or in the known P2a paired region (⌬79C) effectively abolished telomerase enzymatic function (lanes [13][14][15][16][17][18][19][20][21], consistent with prior observations that both the template's primary sequence and the secondary structural formation of the P2a stem loop to be absolutely required for optimal telomerase enzymatic function. 3,4,39 Our results were not attributable to differences in hTERC synthesis, processing, or stability, because Northern blotting verified that each construct produced comparable steady-state levels of hTERC expression in transfected cells ( Figure 4B).…”

“…The other RTs (ie, P721R and T726M) showed as much telomerase activities (lanes 7-12) as did those with either the wild-type hTERT (lanes 1-3) or the known A279T polymorphic sequence 20 (lanes 19-21). Similarly, both of the remaining variants located within the C-terminal domain of hTERT (ie, R979W and F1127L) showed little to no reduction in telomerase enzymatic activities (lanes [13][14][15][16][17][18]. We also tested a novel hTERT K570N found recently in an AA patient in our cohort ( Table 1).…”

“…The fact that mutations of key residues that are known to affect its conventional RT catalytic activity also negatively influence telomerase activity strongly argues that telomerase RT domain is the catalytic domain of the enzyme complex. 13,[15][16][17][18] Inherited mutations in both hTERC RNA and hTERT protein underlie rare bone marrow failure syndromes, autosomal dominant dyskeratosis congenita (DC) and acquired aplastic anemia (AA). [19][20][21] DC is characterized by abnormal skin pigmentation, nail dystrophy, and oral leukoplakia and is often complicated by life-threatening bone marrow failure and immunodeficiency.…”

Identificación de la secuencia del gen de la subunidad catalítica de la telomerasa en Plasmodium falciparum.

“…El motivo T y otros posibles motivos descritos como CP y CP2 parecen estar involucrados en la unión ARN -TERT (43,44). Teniendo en cuenta la conservación, al menos, del motivo T en todas las TERT, su función de unión al ARN podría ser presumiblemente compartida en todas las especies (45).…”

Genetic Variations in Telomere Maintenance, with Implications on Tissue Renewal Capacity and Chronic Disease Pathologies

“…Mutations in RID2 result in a loss of telomerase catalytic activity due to lost interaction with the RNA template TER [111]. The reverse transcriptase (RT) domain is responsible for catalyzing the assembly of the telomeric DNA repeats using the TER template [113,114]. Mutations in any of its conserved aspartic acid residues ablate telomerase catalytic activity [114].…”