Abstract
BackgroundThe first telomerase-associated protein (Est1) was isolated in yeast due to its essential role in telomere maintenance. The human counterparts EST1A, EST1B, and EST1C perform diverse functions in nonsense-mediated mRNA decay (NMD), telomere length homeostasis, and telomere transcription. Although Est1 and EST1A/B interact with the catalytic subunit of yeast and human telomerase (Est2 and TERT, respectively), the molecular determinants of these interactions have not been elaborated fully.ResultsTo investigate the functional conservation of the EST1 protein family, we performed protein-protein interaction mapping and structure-function analysis. The domain in hEST1A most conserved between species, containing a TPR (tricotetrapeptide repeat), was sufficient for interaction of hEST1A with multiple fragments of hTERT including the N-terminus. Two mutations within the hTERT N-terminus that perturb in vivo function (NAAIRS92, NAAIRS122) did not affect this protein interaction. ScEst1 hybrids containing the TPR of hEST1A, hEST1B, or hEST1C were expressed in yeast strains lacking EST1, yet they failed to complement senescence. Point mutations within and outside the cognate ScEst1 TPR, chosen to disrupt a putative protein interaction surface, resulted in telomere lengthening or shortening without affecting recruitment to telomeres.ConclusionsThese results identify a domain encompassing the TPR of hEST1A as an hTERT interaction module. The TPR of S. cerevisiae Est1 is required for telomerase-mediated telomere length maintenance in a manner that appears separable from telomere recruitment. Discrete residues in or adjacent to the TPR of Est1 also regulate telomere length homeostasis.
Highlights
The first telomerase-associated protein (Est1) was isolated in yeast due to its essential role in telomere maintenance
Mapping the interactions between hEST1A and hTERT it was known that hEST1A interacts with hTERT in vitro independently of hTR [33], and that tetratricopeptide repeat (TPR) typically mediate protein-protein interactions [45], it was unknown whether the TPR was sufficient for the interaction between hEST1A and hTERT
The hTERT-hEST1A interactions were not perturbed by micrococcal nuclease, indicating that they did not require nucleic acids (Figure 1A, B)
Summary
The first telomerase-associated protein (Est1) was isolated in yeast due to its essential role in telomere maintenance. In S. cerevisiae, telomeres are usually maintained by telomerase or RAD52-dependent recombination. Cdc and Est are critical for the recruitment of the telomerase core complex (Est2-Tlc1) to telomeres in S phase [reviewed in [11,12,13,14]]. Cdc binds to singlestranded telomeric DNA [15,16] and associates with telomeres throughout the cell cycle, with a peak in association during S phase [17,18]. Est binds to singlestranded telomeric DNA [19] and associates with telomeres in S phase [17,18,20].
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