Mutation at T109 disrupts She2 interaction with ASH1 mRNA and its localization at the bud tip. (A) RNA immunoprecipitation of ASH1 mRNA by She2 wild-type or mutants. Myc-tagged wild-type She2 (WT), She2 T109A (T109A), or She2 T109D (T109D) were immunoprecipitated using anti-myc antibody, followed by RNA purification and RT-qPCR analysis. (Left panel) Enrichment of ASH1 mRNA following immunoprecipitation is reported as a ratio of immunoprecipitate versus input (IP/INPUT), with wild-type She2 set as 1.0. (Right panel) Western blot of Myc-tagged wild-type She2 (WT), She2 T109A (T109A), or She2 T109D (T109D) from input or immunoprecipitate (IP). (N = 3). (*) P < 0.05. (B) Fluorescence in situ hybridization (FISH) on ASH1 mRNA in yeast cells expressing wild-type She2 (WT), She2 T109A (T109A), or She2 T109D (T109D) proteins. (C) Quantification of ASH1 mRNA localization phenotypes in yeast cells expressing wild-type She2 (WT), She2 T109A, or She2 T109D proteins. N = 100 cells per strain, from two independent experiments.
