Telomerase activation after recruitment in fission yeast

Abstract

Current models depict that telomerase recruitment equates to activation. Telomeric DNA-binding proteins and the telomerase accessory proteins coordinate the recruitment of telomerase to the ends of chromosomes in a telomere length- and cell-cycle-dependent manner [1-4]. Recent studies have demonstrated that the telomeric protein TPP1 and its binding protein TIN2 are key proteins for both telomerase recruitment and processivity in mammalian cells [5-7]. Although the precise molecular mechanism of telomerase recruitment has not yet been established, targeted point mutations within the oligonucleotide/oligosaccharide-binding (OB)-fold domain of TPP1 have been shown to impair telomerase association and processivity [8-10]. In fission yeast, telomerase is recruited through an interaction between the telomerase subunit Est1 and Ccq1, a component of the Pot1-Tpz1 telomere complex (POT1-TPP1 orthologs) [11-15]. Here, we demonstrate that association of telomerase with telomeres does not engage activity. We describe a mutation of Tpz1 that causes critical telomere shortening despite telomeric accumulation of the telomerase catalytic subunit, Trt1. Furthermore, Est1-directed telomerase association with Ccq1 is transient, and the Est1-Ccq1 interaction does not remain the bridge between telomeres and telomerase. Rather, direct interaction of Trt1 with Tpz1 is critical for telomere elongation. Moreover, Ccq1, which has been well characterized as a telomerase recruiter, is also required for the activation of telomere-associated telomerase. Our findings reveal a layer of telomerase regulation that controls activity after recruitment.

Figure 1

The Association of Est1 with Ccq1 during Telomerase Recruitment Is Likely to Be Transient (A) Associations of Trt1 with Ccq1 and with Tpz1 are retained in the absence of TER1. Whole-cell extracts (WCEs) were immunoprecipitated (IP) with anti-PK (aPK) antibody in the presence or absence of RNase to purify Ccq1, Tpz1, or Est1 complexes. The resulting immunoprecipitates were hybridized with either anti-PK or anti-Myc. Cdc2 was used as a control for sample input. Histone H3 was used as a control for the presence of DNA. (B) RNase treatment reduces TER1 levels. RNA extracted from 5% of each IP sample was subjected to RT-PCR. TER1 PCR products were visualized on a 2% agarose gel. As a control, the RT enzyme was substituted with water (−RT). (C) Yeast three-hybrid analysis: coexpression of Tpz1 or TER1 disrupts Ccq1-Est1 binding. Equal amounts of cells were spotted on selection plates (−His −Ade) and a nonselective plate to control for loading (+His +Ade). Coexpression of Tpz1 or TER1 from the MET17 promoter was induced by removal of methionine (Met) from the media. Expression of gene products is shown in Figure S1.

Figure 2

Mutation of the OB-Fold Domain of Tpz1 Results in Impaired Telomerase Activity (A–D) Telomere Southern blots of genomic DNA digested with EcoRI and hybridized with a telomeric probe. A slice of the EtBr-stained gel image at 2.5 kb is shown below the blots as a loading control. (B) Genomic DNA was harvested at multiple intervals (as indicated) over the course of >2 weeks after sporulation of diploid strains.

Figure 3

Tpz1 K75 Is Not Involved in Telomerase Recruitment (A) Association efficiency of Trt1 with Tpz1 increases when K75 is mutated to alanine. WCEs were immunoprecipitated with anti-HA antibody to purify Tpz1. The resulting immunoprecipitates were hybridized with anti-PK. Cdc2 was used as a control for sample input. (B) Telomere ChIP: Trt1 is present at the telomere in strains carrying the tpz1-K75A mutation. Strains were crosslinked, and WCEs were subjected to immunoprecipitation with anti-PK antibody. The trt1-PK tpz1-K75A cells were prepared soon after germination from the heterozygous diploid. DNA fragments in the immunoprecipitate were quantified using quantitative PCR (qPCR). Data were obtained from four independent experiments, and normalized to qPCR values were obtained from a control gene sequence (act1) and expressed as fold enrichment over the values obtained from crosslinked WT (untagged) cells; the average and SD of four independent experiments are shown. ^∗p = 0.0289 for “no-tag versus Trt1-PK.” ^∗p = 0.0209 for “Trt1-PK versus Trt1-PK tpz1-K75A.” (C) Association efficiency of Trt1 with Ccq1 increases when K75 of Tpz1 is mutated to alanine. The interaction of Ccq1 with Tpz1 is not affected by substitution of Tpz1 K75 with alanine. WCEs were immunoprecipitated with anti-Flag antibody to purify Ccq1. The resulting immunoprecipitates were hybridized with either anti-HA or anti-PK. Cdc2 was used as a control for sample input.

Figure 4

Direct Interaction of Tpz1 with Trt1 Rescues Telomerase Activity in tpz1-K75A Mutants (A–C) Telomere Southern blots of genomic DNA digested with EcoRI and hybridized with a telomeric probe. A slice of the EtBr-stained gel image at 2.5 kb is shown below the blots as a loading control. (A) Fused Tpz1 and Trt1 are functional: the fusion can maintain telomeres in the absence of endogenous Tpz1 or Trt1. (B) Fusion of Trt1 with Tpz1 rescues the tpz1-K75A mutant phenotype. (C) Fusion of Trt1 to Tpz1 bypasses the need for Ccq1 in telomerase recruitment, but Ccq1 is still required for telomerase activity.