Fig. 7: Functional impact of mutations in the conserved salt-bridge network D2915-R2976-D3042 of the Rea1 middle domain.

a A heterozygous REA1/rea1::kanR diploid strain was transformed with a plasmid directing expression of the Rea1_{D2915A-R2976A-D3042A} mutant. After sporulation of the resulting strains, tetrads were dissected and haploid spores were spotted in rows on YPD medium. Spores containing the Rea1_{D2915A-R2976A-D3042A} mutant could support viability (red rectangles), although growth was strongly impaired. b Impact on pre-rRNA processing. Left panel: Northern analyses of pre-rRNA processing in GAL::rea1 cells expressing Rea1_{D2915A-R2976A-D3042A} (“Rea1_sb”) or wild-type Rea1 (Rea1_wt) from plasmids or transformed with an empty vector (empty), shifted 24 hours on glucose-containing medium. Right panel: Quantification of 27SB/35S ratio. The Rea1_{D2915A-R2976A-D3042A} mutants displays reduced ratios compared to the Rea1_wt control indicative of pre60S maturation defects. Experiment was done once. c Upper panel: Detailed northern pre-rRNA processing analysis of rea1::kanR strain expressing Rea1_{D2915A-R2976A-D3042A} (“Rea1sb”) from a plasmid. Four wt strains and four Rea1_{D2915A-R2976A-D3042A} expressing strains were analysed in parallel. Detection of the indicated (pre-)rRNAs by northern hybridization with anti-sense oligonucleotide probes. Lower panel: levels of the indicated (pre-)rRNAs in the Rea1_{D2915A-R2976A-D3042A} mutant or wild-type (n = 4). Error bars show the standard deviation. d Rea1 depletion affects the association of Rsa4 with pre60S particles. REA1 (Rea1 +) or GAL::rea1 (Rea1 -) strains expressing HA-tagged Ytm1 or Rsa4 were grown in glucose-containing medium. Immunoprecipitation experiments were then carried out with anti-HA agarose beads and the indicated pre-rRNAs in the input and immunoprecipitated (IP samples) were detected by northern analyses. The experiment was repeated twice. e Rea1_{D2915A-R2976A-D3042A} (“Rea1_sb”) affects the association of Rsa4 with pre60S particles. Left panel: Analysis as in d, except that a GAL::rea1 strain expressing HA-tagged Rsa4 and Rea1_{D2915A-R2976A-D3042A} or wild-type Rea1 (Rea1_wt) from plasmids was used. -: strain transformed with an empty vector. Detection of 27SB pre-rRNA by northern hybridization. Right panel: ratios of the levels of precipitated 27SB pre-rRNA over input levels. Experiment was done once. f Disrupting the D2915-R2976-D3042 salt-bridge network leads to nuclear pre60S particle export defects. Experiments were repeated three times. Source data are provided as a Source Data file.