Functional Conservation of the Telomerase Protein Est1p in Humans

Abstract

Eukaryotic telomerase contains a telomerase reverse transcriptase (TERT) and an RNA template component that are essential for telomerase catalytic activity and several other telomerase-associated factors of which only a few appear to be integral enzyme components [1-3]. The first essential telomerase protein identified was S. cerevisiae Est1p, whose deletion leads to ever-shorter telomeres despite the persistence of telomerase activity [4-6]. Extensive genetic and biochemical data show that Est1p, via its interaction with the telomerase RNA and telomere end DNA binding complex Cdc13p/Stn1p/Ten1p, promotes the ability of telomerase to elongate telomeres in vivo [7-22]. The characterization of Est1p homologs outside of yeast has not been documented. We report the characterization of two putative human homologs of Est1p, hEST1A and hEST1B. Both proteins specifically associated with telomerase activity in human cell extracts and bound hTERT in rabbit reticulocyte lysates independently of the telomerase RNA. Overproduction of hEST1A cooperated with hTERT to lengthen telomeres, an effect that was specific to cells containing telomerase activity. Like Est1p, hEST1A (but not hEST1B) exhibited a single-stranded telomere DNA binding activity. These results suggest that the telomerase-associated factor Est1p is evolutionarily conserved in humans.

The conserved Est1 protein stimulates telomerase DNA extension activity

“…Est1 is a fundamental telomere DNA maintenance factor that is conserved from yeast to human (10,21). Genetic experiments in yeast have suggested an Est1-role in both recruiting and activating telomerase (3).…”

“…Prior biochemical studies demonstrate that yeast and human Est1 bind single-stranded G-rich DNA and apparently recognize RNA nonspecifically in vitro (9)(10)(11). In contrast to the in vitro work, in vivo studies indicate that yeast Est1 targets a bulged-stem loop in the TLC1 telomerase RNA (12, 13).…”

Dissociation of telomerase activity and telomere length maintenance in primitive human hematopoietic cells

“…In the absence of detectable changes in the level of telomerase activity, the eventual stabilization of telomere length points to alterations in telomere-associated proteins leading to better utilization of the telomerase present in the cells, possibly through improved recruitment of the enzyme to the telomere. Human orthologs to yeast Est1 and Cdc13, which mediate access of telomerase to telomeres (45), have recently been identified (46,47) and may play a similar role in telomere stabilization in human cells. The additional genetic changes in postcrisis T2a cells (34) provide a starting point for investigating the molecular basis of telomere length regulation in human hematopoietic cells.…”

The Double Life of UPF1 in RNA and DNA Stability Pathways

“…22 Similarly to ScEst1p, human EST1A/SMG6 interacts with active telomerase in human cells. 19,20 While EST1 deletion in yeast leads to a progressive shortening of the telomeres and after 60-80 population doublings to cellular senescence, shRNA-mediated depletion of human EST1A/SMG6 causes a rapid cell cycle arrest after a few population doublings (our unpublished data), rendering the detection of a putative telomere shortening phenotype impossible. Nevertheless, overexpression of EST1A/SMG6 in human cell lines leads to the accumulation of chromosomal end-to-end fusions suggesting a role for EST1A/SMG6 in maintaining the normal structure of human chromosome ends.…”

“…We and others have previously demonstrated that human EST1A, which is identical to the NMD factor SMG6, shares sequence similarity with Est1p from Saccharoymces cerevisiae. [19][20][21] ScEst1p is an essential core component of the telomerase enzyme, which adds telomeric repeats to the termini of chromosomes (the telomeres), to counteract telomere loss due to incomplete end replication. 22 Similarly to ScEst1p, human EST1A/SMG6 interacts with active telomerase in human cells.…”