Fig. 5: KKE/D domains interact in a homo- and heterotypic manner. | Nature Communications

Fig. 5: KKE/D domains interact in a homo- and heterotypic manner.

From: The dual life of disordered lysine-rich domains of snoRNPs in rRNA modification and nucleolar compaction

Fig. 5

A Schematic representation of the domain organization of Rpa34, Pxr1, Nop56 and Nop58 proteins. B Yeast two-hybrid (Y2H) assays using the indicated combinations of the Gal4 activation domain (Gal4AD) alone or fused to the KKE/D domain of Nop56 (Nop56(441-504)), the KKE/D domain of Nop58 (Nop58(451-511)), or the KKE/D repeats of Pxr1 (Pxr1(172-213)) and the Gal4 DNA-binding domain (Gal4BD) fused to the KKE/D domain of Rpa34 (Rpa34(183-233)). Growth on SDC lacking tryptophan and leucine (TL) allowed to select cells containing both constructs; growth on SDC lacking tryptophan, leucine and histidine (TLH) indicated that the constructs interact; growth on SDC lacking tryptophan, leucine and adenine (TLA) indicated that the constructs interact strongly. n = 3 biologically independent experiments. C Y2H assays using the indicated combinations of the Gal4 activation domain (Gal4AD) fused to full-length Pxr1 (Pxr1), Pxr1 lacking its KKE/D domain (Pxr1(1-149)), Pxr1’s isolated KKE/D domain (Pxr1(149-271)) or Pxr1’s KKE/D repeats (Pxr1(172-213)) and the Gal4 DNA-binding domain (Gal4BD) fused to the KKE/D repeats of Pxr1 (Pxr1(172-213)), the KKE/D repeats of Cbf5 (Cbf5(433-483)), the KKE/D domain of Nop56 (Nop56(441-504)) or the KKE/D domain of Nop58 (Nop58(451-511)). Same legend as in (B). n = 3 biologically independent experiments. D Schematic representation of the interactions detected by Y2H assays between the KKE/D domains of Rpa34, Cbf5, Pxr1, Nop56 and Nop58 proteins. Circles correspond to self-interaction in a KKE/D domain-dependent manner. E KKE/D-GFP (KKE-GFP) or GFP occupancy on rDNA genes at 18S, 25S or intergenic (NTS2) regions in wild-type (WT) or rpa34-Δkk, nop56-Δkk, nop58-Δkk, cbf5-Δkk (ΔΔΔΔkk) quadruple mutant cells. GFP and KKE/D-GFP were immunoprecipitated using anti-GFP antibodies. DNA occupancy was defined as the ratio between the immunoprecipitation (IP) and the input signals. Two-tailed t-test analysis was used for statistics. Data are presented as mean values ± SD. Significant differences are indicated by stars and with the p value. n = 6 biologically independent experiments. Source data are provided as a Source data file.

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