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. 2012 Sep;18(9):1597-604.
doi: 10.1261/rna.034447.112. Epub 2012 Jul 30.

The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements

Johnathan W Lubin  1 , Timothy M Tucey, Victoria Lundblad
Affiliations

The interaction between the yeast telomerase RNA and the Est1 protein requires three structural elements

Johnathan W Lubin et al. RNA. 2012 Sep.

Abstract

In the budding yeast Saccharomyces cerevisiae, the telomerase enzyme is composed of a 1.3-kb TLC1 RNA that forms a complex with Est2 (the catalytic subunit) and two regulatory proteins, Est1 and Est3. Previous work has identified a conserved 5-nt bulge, present in a long helical arm of TLC1, which mediates binding of Est1 to TLC1. However, increased expression of Est1 can bypass the consequences of removal of this RNA bulge, indicating that there are additional binding site(s) for Est1 on TLC1. We report here that a conserved single-stranded internal loop immediately adjacent to the bulge is also required for the Est1-RNA interaction; furthermore, a TLC1 variant that lacks this internal loop but retains the bulge cannot be suppressed by Est1 overexpression, arguing that the internal loop may be a more critical element for Est1 binding. An additional structural feature consisting of a single-stranded region at the base of the helix containing the bulge and internal loop also contributes to recognition of TLC1 by Est1, potentially by providing flexibility to this helical arm. Association of Est1 with each of these TLC1 motifs was assessed using a highly sensitive biochemical assay that simultaneously monitors the relative levels of the Est1 and Est2 proteins in the telomerase complex. The identification of three elements of TLC1 that are required for Est1 association provides a detailed view of this particular protein-RNA interaction.

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Figures

FIGURE 1.
FIGURE 1.
Deletion of sub-helix IVc cannot be suppressed by overexpression of Est1. (A) Proposed secondary structure of Helix IV of TLC1, with the boundaries indicated for two mutations, tlc1-101 and tlc1-201, which delete sub-helices IVc and IVb, respectively; the previously described Est1-binding bulge (nucleotides 660–664), which is deleted by the tlc1-47 mutation, is indicated by the smaller box. (B) Telomere length of the indicated strains, examined after ∼75 generations of growth; size markers (in kb) are indicated. (C) Growth characteristics of TLC1, tlc1-47, and tlc1-101 strains in the presence (left) or absence (right) of a high copy plasmid expressing the Est1 telomerase subunit from the constitutive ADH promoter; strains were examined at a point when senescence was most pronounced (∼100 generations). (D) Proposed secondary structures for sub-helix IVc from TLC1 RNAs from selected isolates from the Saccharomyces and Kluveromyces clades; the two clusters of sequences indicated in red are invariant in the Saccharomyces clade, with partial conservation extending into the Kluveromyces clade. Determination of structures was based on sequence alignments to identify conserved elements as well as comparison with previously published proposed secondary structures, aided by RNA Mfold.
FIGURE 2.
FIGURE 2.
A conserved internal loop is required for telomere length maintenance. (A) A depiction of mutations introduced into the conserved internal loop of sub-helix IVc, with blue boxes or triangles indicating sequence changes or deleted nucleotides, respectively. (B) Telomere length of strains bearing the indicated mutations (expressed from single-copy plasmids introduced into the tlc1-Δ strain YVL3554) examined after ∼100 generations, at a point when the tlc1-Δ null strain (lane 11) has given rise to recombination-dependent survivors (Lundblad and Blackburn 1993). (C) Growth characteristics of the indicated mutant strains, which have been propagated for ∼100 generations.
FIGURE 3.
FIGURE 3.
Est1 association with telomerase is abolished by mutations in the conserved internal loop. (A) Telomere length of the indicated strains was examined after ∼100 generations. (B) Growth characteristics determined as described in Figure 1C, although strains were examined at ∼75 generations, when senescence was not as pronounced. (C,D). Anti-myc Western analysis of anti-FLAG immunoprecipitates following 6% PAGE of the indicated strains; the tlc1 mutations were integrated into the genome of YVL3493 (with the exception of tlc1-207 and the accompanying TLC1 control, which were present on single-copy plasmids in the YVL3701 tlc1-Δ strain). (E) Northern analysis monitoring levels of the TLC1 RNA in the same immunoprecipitates shown in D.
FIGURE 4.
FIGURE 4.
A single-stranded region at the base of sub-helix IVc contributes to TLC1–Est1 association. (A) Proposed secondary structure of Helix IV, with various mutations indicated as in Figure 1D; the tlc1-209 mutation is a combination of the mutations shown in tlc1-201 and tlc1-208. (B) Telomere length of strains expressing the mutations shown in A, after 75 generations of growth. (C) Telomere length of the tlc1-209 strain in the presence or absence of a high copy ADH–Est1 plasmid, compared with a TLC1 strain with the same plasmid.
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