Sequence analysis of the amino acid changes in the <i>tel2</i> mutant proteins.

<p>Sequence analysis of the amino acid changes in the <i>tel2</i> mutant proteins.</p

<i>MED15</i>, coding for a subunit of Mediator, found in the present study to be an extragenic suppressor of <i>tel2</i>-<i>ts</i> mutations, also partially rescues their telomere length defect.

<p>(<b>A</b>) The temperature-sensitive <i>tel2</i>-<i>15</i> and <i>tel2</i>-<i>19</i> mutants, as well as a wild-type (wt) strain,were re-transformed, here, with the <i>MED15</i> or <i>TEL2</i> plasmids, either low-copy (1 or 2 copies) <i>CEN</i> (YCplac) or multi-copy 2 µ (YEplac), both originally isolated from the genetic screens performed in the present study, or with the corresponding empty plasmids (YCp or YEp), and their growth characteristics evaluated after spotting ten-fold dilutions (from left to right in each condition) on selective, -Ura, minimal medium at the indicated temperatures. YCp-<i>MED15</i> rescued <i>tel2</i>-<i>15</i> at up to 29°C, while YEp-<i>MED15</i> rescued <i>tel2</i>-<i>19</i> at up to 34°C. (<b>B</b>) Overexpression of <i>MED15</i>, from the plasmids described above, in A, partially rescued the short telomere phenotype of the <i>tel2</i>-<i>15</i> and <i>tel2</i>-<i>19</i> mutant strains. Compare the extent of the rescue with that provided by <i>TEL2</i> overexpression. Telomere length was measured as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030451#pone-0030451-g003" target="_blank">Figure 3</a> after the strains had been propagated at 27.5°C (<i>tel2</i>-<i>15</i>) or 29°C (<i>tel2</i>-<i>19</i>) on minimal medium for 30 days. (<b>C</b>) Overexpression of <i>MED15</i> had no elongation effect on telomere length in wild-type cells, grown here at 29°C. In fact, a slight shortening of telomeres was observed following <i>MED15</i> overexpression compared with expression of vector alone.</p

Temperature-sensitive <i>Saccharomyces cerevisiae tel2</i> mutants.

<p>(<b>A</b>) Growth characteristics of the temperature-sensitive <i>tel2</i> mutants. Ten-fold serial dilutions (from left to right in each condition) of cultures of the indicated relevant genotype were grown for 3 days on YEPD agar at the indicated temperature and photographed. (<b>B</b>) Temperature-sensitive <i>tel2</i>-<i>15</i> and <i>tel2</i>-<i>19</i> mutants fail to arrest at a specific cell cycle stage when incubated at the restrictive temperature for growth of 34°C. Cell lysis was evident under the microscope upon extended incubation at restrictive temperatures (not shown). (<b>C</b>) Overexpression of <i>MED15</i> from a multi-copy plasmid (2 µ, YEp) does not result in an increase in <i>TEL2</i> transcription. (<b>D</b>) Western blot showing that overexpression of <i>MED15</i>, either from a low-copy (CEN) or a multi-copy (2 µ) plasmid, does not result in an increase in the amount of endogenous Tel2 (HA₂-Tel2 or Myc₂-Tel2) in the cell (the lanes labeled “HA₂-Tel2/Myc₂-Tel2+plasmid” represent the controls with plasmid alone). Endogenous Tel2, tagged with either Myc₂ or HA₂ in its N-terminus, was immunoprecipitated from crude extracts with the corresponding monoclonal antibody and Western blotting with the same antibody. Therefore, overexpression of <i>MED15</i> does not rescue the <i>tel2</i>-<i>ts</i> mutants by increasing either <i>TEL2</i> transcription or even indirectly by increasing Tel2 protein levels.</p

Tel2 controls transcription of <i>EST2</i>/telomerase (but not that of the telomerase RNA subunit <i>TLC1</i>) and is regulated by the Med15 Mediator subunit.

<p>(<b>A</b>) Northern blot analysis of endogenous <i>TLC1</i> RNA levels indicates an absence of deregulation in the indicated <i>tel2</i> mutants. <i>rvb2</i> mutants that exhibit slight and stable telomere shortening (NG, MC, submitted for publication) were also used in that experiment. A <i>tlc1</i> null strain was used to ascertain that the highlighted band indeed corresponds to TLC1 RNA. <i>ACT1</i> and <i>SCR1</i> RNA levels were measured to serve as loading controls. (<b>B</b>) Immunoprecipitation-Western experiments aiming at assessing Myc₁₈-Est2 levels (construct integrated at <i>EST2</i> genomic locus, under the control of native promoter) indicate that <i>EST2</i>/telomerase levels are depressed in the <i>tel2</i>-<i>15</i> ts mutant strain grown at the semi-permissive temperature for growth of 29°C for 2 hr or overnight (ON). The <i>rvb2</i>-<i>2</i> and <i>rvb2</i>-<i>24</i> mutants (NG, MC, submitted for publication) were used as controls. (<b>C</b>) Levels of <i>EST2</i> mRNA, coding for the protein subunit of budding yeast telomerase, were measured relative to those of <i>ACT1</i>, in wild-type cells (wt, normalized to 1.0) and in the temperature-sensitive <i>tel2</i>-<i>15</i> (<b>left panel</b>) and <i>tel2</i>-<i>19</i> (<b>right panel</b>) mutants. The mean values ± standard error correspond to 4 experiments performed with the <i>tel2</i>-<i>15</i> mutant and 5 experiments with the <i>tel2</i>-<i>19</i> mutant, each sample being performed in triplicate. (<b>D</b>) <i>MAE1</i> and <i>MEP2</i> mRNA levels in the <i>tel2</i>-<i>15</i> ts mutant, at the indicated temperatures. Results are from two experiments, each sample being performed in triplicate. (<b>E</b>) Continuous overexpression of the <i>MED15</i> Mediator subunit, achieved by transforming the strains of the indicated relevant genotype with an episomal (2 µ) vector harboring <i>MED15</i> ORF flanked by upstream and downstream natural genomic sequences, resulted in a dramatic increase in <i>EST2</i> mRNA levels. Data are from two experiments using the wild type and the two mutants, each sample being performed in triplicate.</p

The temperature-sensitive <i>tel2</i>-<i>15</i> and <i>tel2</i>-<i>19</i> mutants exhibit short telomeres.

<p>(<b>A</b>) <i>tel2</i>-<i>15</i> confers a more severe telomere length defect than <i>tel2</i>-<i>19</i>, compared here with the previously described <i>tel2</i>-<i>1</i> mutant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030451#pone.0030451-Lustig1" target="_blank">[2]</a>, all grown at 24°C. (<b>B</b>) The shortened telomeres in the <i>tel2</i>-<i>19</i> mutant were not furthered shortened when combined with a <i>tel1</i> null mutation. These strains were grown at 29°C. (<b>C</b>). In contrast, both the <i>tel2</i>-<i>19 yku70</i> null and <i>tel2</i>-<i>15 yku70</i> null double mutants, grown here at 24°C, exhibited shorter telomeres than either one of the corresponding single mutants. Average telomere length (corresponding to the DNA smear migrating at around 1.3 kb) of <i>tel2</i> mutant strains with the indicated relevant genotype, grown for at least 22 days to allow telomere length to attain a stable value, was detected by Southern blotting with a TG_1–3 telomeric P³²-labeled probe. The dashed horizontal lines, drawn between the mean length values of two or more wild-type (wt) or mutant strains, as indicated in the margin, allow to get a better appreciation of the variations in telomere length in the mutants.</p

Increased dosage of several Mediator genes partially rescue the temperature sensitivity of the <i>tel2</i>-<i>19</i> mutant.

<p>(<b>A</b>) Overexpression of the indicated genes, under the control of the <i>GAL1</i>-<i>10</i> promoter, was continuously induced by growing on solid media containing galactose as the sole carbon source. Cells transformed with the indicated plasmid were first grown in liquid culture in selective minimal medium containing glucose as the carbon source before being re-streaked on agar-based selective minimal medium containing galactose as the carbon source. Growth was assessed after 3 days at the indicated temperature in the serially diluted cells (ten-fold dilutions from left to right in each condition). At 35°C, <i>MED16</i>, <i>CDK8</i> and <i>MED18</i> overexpression rescued <i>tel2</i>-<i>19</i>, although slightly less efficiently than <i>MED15</i>. (<b>B</b>) Tel2 and Med15 physically interact <i>in vivo</i>. Putative physical interactions between Tel2-Myc₃ and Med15-HA₂ were measured upon immunoprecipitation (IP) with anti-HA or anti-Myc monoclonal antibodies, followed by Western blotting (West.), from crude extracts from strains having transiently overexpressed the indicated constructs after incubation in galactose-based liquid selective media, for 2 hr at 29°C (same conditions for panels B–E). All experiments were conducted in parallel in strains expressing both constructs and in strains with the single constructs to take into account possible background signals. Full length Tel2-Myc₃ specifically binds the first half of Med15 (Med15^1–540-HA₂) in both directions, that is to say that a positive signal was detected whether one or the other of the two proteins was immunoprecipitated. (<b>C</b>) In contrast, the second half of Med15 (Med15^541–1081-HA₂) did not associate <i>in vivo</i> with full length Tel2-Myc₃. (<b>D</b>) Finally, the first quarter of Med15 (Med15^1–270-HA₂) efficiently bound the first half of Tel2 (Tel2^1–343-HA₂). (<b>E</b>) At the maximal permissive temperature for growth for <i>tel2</i>-<i>15</i> of 29°C, the Tel2-15-Myc₃ protein still physically interacted with Med15^1–540-HA₂, as was also the case at 34°C (data not shown).</p