The Est1 Subunit of Yeast Telomerase Binds the Tlc1 Telomerase RNA

Abstract

Est1 is a component of yeast telomerase, and est1 mutants have senescence and telomere loss phenotypes. The exact function of Est1 is not known, and it is not homologous to components of other telomerases. We previously showed that Est1 protein coimmunoprecipitates with Tlc1 (the telomerase RNA) as well as with telomerase activity. Est1 has homology to Ebs1, an uncharacterized yeast open reading frame product, including homology to a putative RNA recognition motif (RRM) of Ebs1. Deletion of EBS1 results in short telomeres. We created point mutations in a putative RRM of Est1. One mutant was unable to complement either the senescence or the telomere loss phenotype of est1 mutants. Furthermore, the mutant protein no longer coprecipitated with the Tlc1 telomerase RNA. Mutants defective in the binding of Tlc1 RNA were nevertheless capable of binding single-stranded TG-rich DNA. Our data suggest that an important role of Est1 in the telomerase complex is to bind to the Tlc1 telomerase RNA via an RRM. Since Est1 can also bind telomeric DNA, Est1 may tether telomerase to the telomere.Telomeres are the natural ends of linear chromosomes.Telomeres are maintained at a characteristic length by a balance between two forces, loss of telomeres during DNA replication and synthesis of telomeres by an enzyme called telomerase. Telomerase is a special reverse transcriptase which contains not only a reverse transcriptase catalytic subunit but also an RNA molecule which serves as the template for telomere elongation (11). In yeast, the catalytic subunit is called Est2 (7,20,25,26), and the RNA template is called Tlc1 (39).Telomerases from several organisms have been partially characterized (3,7,12,13,24,26,29,30). In general, these complexes contain components in addition to the catalytic subunit and the RNA template (10,12,24,30,36). For the yeast Saccharomyces cerevisiae, genetic screens have identified five genes (EST1, EST2, EST3, EST4/CDC13, and TLC1) (20,27,39) whose mutations lead to progressive telomere shortening and eventual loss of viability (i.e., senescence). EST2 and TLC1 encode the reverse transcriptase (25, 26) and the RNA template (39), respectively. The Cdc13 or Est4 protein can bind the single-stranded G-rich telomeric sequence both in vitro and in vivo (2,23,32). This protein apparently caps the telomere, protecting it from nucleolytic digestion. The functions of the other two genes, EST1 and EST3, are less clear. Neither of them is required for in vitro telomerase activity (5, 25), even though mutants exhibit the same senescence phenotype as TLC1 or EST2 mutants (20). There is evidence that Est1 is associated with telomerase, since Est1 coprecipitates with Tlc1 and telomerase activity (21,40). In addition, Est1 may be associated with the telomere since, like Cdc13, Est1 can bind single-stranded G-rich telomeric DNA in vitro (43). However, the affinity of Est1 for such DNA is low, much lower than the affinity of Cdc13. Unlike Cdc13, Est1 requires a free end for binding to DNA (43). We noticed a possible RNA-bind...

PP2A and Aurora differentially modify Cdc13 to promote telomerase release from telomeres at G2/M phase

“…EST2 and TLC1 encode the reverse transcriptase catalytic protein subunit and the templating RNA, respectively 9,11,12 . In addition, the protein encoded by EST1 is associated with the RNA component of telomerase, TLC1 (refs [13][14][15][16]. Other accessory factors, such as Cdc13, are required for the in vivo action of telomerase.…”

A bulged stem tethers Est1p to telomerase RNA in budding yeast

“…Est1p has been reported to contain a region with weak sequence homology with the consensus RRM, and mutants in this region do not complement an est1⌬ strain and no longer co-immunoprecipitate TLC1 RNA (Zhou et al 2000). The function of this region is not entirely clear, however, because a subsequent report showed that Est1p containing a deletion in this region was still able to partially co-immunoprecipitate the RNA (Evans and Lundblad 2002).…”

“…This interaction occurs independently of most of the RNA, as a TLC1-535-707 fragment is sufficient for coimmunoprecipitation of Est1p. Co-immunoprecipitation of the telomerase RNA and Est1p has been shown to be independent of Est2p (Zhou et al 2000), so it is unlikely that Est2p mediates this interaction. Furthermore, coimmunoprecipitation of the RNA with Est3p is dependent on the presence of Est2p (Hughes et al 2000); therefore, Est1p interaction with the RNA should be independent of Est3p as well.…”

The genome-wide expression response to telomerase deletion in Saccharomyces cerevisiae

“…The TDR included only a few genes previously implicated in telomere function. Three of these were down-regulated when telomeres were shortest: RAP1, a central negative regulator of telomere length (19); EBS1, a gene that contains an RRM RNA recognition motif and is also homologous to the telomerase component EST1 (20); and TBF1, which encodes a DNA-binding protein (21). Rap1p and its associated proteins regulate access of telomerase to the telomere in a lengthdependent manner (19); thus, RAP1 down-regulation could be an adaptive response to shortened telomeres.…”