Figure S1. TRF5 overexpression is toxic to air1Δ air2Δ but overproduction of trf5DADA mutant is not. air1Δ air2Δ (AC2232) cells were transformed with empty control vector, or with vectors containing TRF4, trf4DADA, TRF5, or trf5DADA under GAL1,10-inducible promoter (Table 2). Transformants were streaked at 30°C on glucose plates lacking uracil or on raffinose-galactose (2%) plates lacking uracil to induce expression of cloned alleles and allowed to grow for 3 days.
Supplementary Data.
Because overexpression of histones does not have an effect on air1/2 mutants, we looked for evidence of other RNA binding subunits that could regulate the interaction of Trf4 or Trf5 with their targets. Overexpression of TRF5 is toxic to wild type cells (data not shown). Taking advantage of this observation, we reasoned that if Air1 and Air2 are the exclusive RNA binding subunits that mediate the interaction of Trf4 and Trf5 with their substrates, TRF5 overproduction should not be toxic to cells carrying a double deletion of AIR1 and AIR2. We observe that air1 air2 cells are extremely sensitive to TRF5 overexpression. In contrast, when specific TRF5 mutated form in which the highly conserved aspartic acid residues in the predicted catalytic domain are mutated to alanines (DADA mutant), is overproduced in air1 air2, no deleterious effect is observed (Fig. S1). Similar amounts of Trf5 protein are detected in extracts of cells expressing wild type or DADA-mutated version of TRF5 (not shown). The conclusions that can be drawn from this observation are: first, that Trf5 does have a catalytic activity in agreement with recent reports (Egecioglu et al. 2006; Haracska et al. 2005; Houseley and Tollervey 2006); second, the toxicity of excess of Trf5 is not attributable to excess of Trf5 protein itself but is a consequence of Trf5 catalytic activity; and third, that there must be other/s RNA binding proteins that mediate Trf5 (and possibly Trf4) target recognition besides Air1 and Air2 .