Functional Specificity among Ribosomal Proteins Regulates Gene Expression  Suzanne Komili, Natalie G. Farny, Frederick P. Roth, Pamela A. Silver  Cell  Volume 131, Issue 3, Pages 557-571 (November 2007) DOI: 10.1016/j.cell.2007.08.037 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Loc1 Is Required for the Translational Regulation of ASH1 mRNA (A) Reporter constructs used to assay the regulation of ASH1 mRNA. The reporters contain the promoter and ORF of yeast PGK1, an array of U1A hairpins, and either PGK1's own 3′ UTR or one of ASH1's four localization elements. Each reporter was coexpressed along with U1A-GFP, which specifically binds the U1A hairpins and allows visualization of reporter mRNA location in live cells. The fraction of cells with either bud-tip, bud-cytoplasm, or ubiquitous localization was determined (see Experimental Procedures). (B) Defective anchoring of the E3 construct in loc1Δ cells is due to aberrant translation. Histograms of the E3 reporter construct localizations in wild-type, loc1Δ, and wild-type cells following brief treatment with cycloheximide. Error bars represent standard deviations between replicate experiments. (C) Protein level of myc-Ash1 increases in loc1Δ cells relative to actin (negative control). Western blots of myc-Ash1 and actin were performed from extracts of wild-type and loc1Δ cells. Equal amounts of protein were loaded in each lane. (D) mRNA level of ASH1 decreases in loc1Δ cells. Error bars represent standard error of measurements from individual arrays. Cell 2007 131, 557-571DOI: (10.1016/j.cell.2007.08.037) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Genes Required for Bud-Site Selection in Yeast Are Also Required for the Localization of ASH1 mRNA (A) Representative images of cells expressing ASH1 reporter. (B) Strains that have defective bud-site selection also have defects in localization of the E3 reporter construct. Fraction of cells exhibiting bud-tip, bud-cytoplasm, bud-neck, and “other” (not bud-tip, bud-neck, or bud-cytoplasm) localizations of the E3 reporter construct in cultures lacking the genes indicated is shown. Error bars represent standard deviations of replicate experiments. Cell 2007 131, 557-571DOI: (10.1016/j.cell.2007.08.037) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Regulated Translation of the E3 Reporter Construct Requires a Specific Subset of Duplicated Ribosomal Protein Genes (A and B) Error bars represent standard deviations of replicate experiments. (A) Ribosomal proteins that are required for bud-site selection have a larger defect in anchoring of the E3 reporter construct than their nearly-identical paralogs. Fraction of cells exhibiting either bud-tip or bud-cytoplasmic localization of the E3 reporter construct in cells lacking the gene is indicated. Genes that are required for bud-site selection in diploids are indicated by an asterisk. (B) There is a significantly greater difference in the effect on anchoring of the E3 reporter construct between pairs of duplicated ribosomal protein genes in which one copy is required for bud-site selection than for pairs in which neither copy is required for bud-site selection. The fraction of cells exhibiting bud-tip and bud-cytoplasmic localization of the E3 reporter construct was assayed in strains lacking a variety of duplicated ribosomal protein genes. The difference between the fraction of cells exhibiting bud-tip localization is plotted against the difference in the fraction exhibiting bud-cytoplasmic localization for both members of each pair. Cell 2007 131, 557-571DOI: (10.1016/j.cell.2007.08.037) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Phenotypic Data Reveals Complex Functional Relationships between Duplicated Ribosomal Protein Genes (A) Hierarchical clustering analysis of phenotypic data by ribosomal protein (vertical axis) and phenotype (horizontal axis). Although many ribosomal proteins shared some phenotypes, no two proteins are required for the same set of processes, and different groups are required for each process. (B) Paralogous ribosomal proteins are not phenotypically similar. Rpl2a and Rpl2b cluster with completely different groups of genes, as indicated by the shaded boxes that correspond to (A). (C) Paralogous ribosomal proteins share no more phenotypes than nonparalogous genes. The number of shared phenotypes between all combinations of duplicated ribosomal protein genes was calculated and sorted into paralogous or nonparalogous relationships. Normalized values are displayed. (D) Phenotypic effects are not determined by expression level. mRNA expression levels of all duplicated ribosomal protein genes from transcriptional profiling data was used to determine the relative contribution of each paralog. Genes were sorted into “higher” or “lower” based on whether they contributed more or less than half of the mRNA, respectively. Error bars represent standard deviations. Cell 2007 131, 557-571DOI: (10.1016/j.cell.2007.08.037) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Paralogous Ribosomal Proteins Exhibit Different Localizations and Assembly Requirements in Specific Genetic Backgrounds GFP-tagged Rpl7a, Rpl7b, Rps18a, and Rps18b were expressed from the genome under their own promoters in wild-type, loc1Δ, puf6Δ, and loc1Δpuf6Δ cells. Representative fluorescent (top) and nomarski (bottom) images are shown. Cell 2007 131, 557-571DOI: (10.1016/j.cell.2007.08.037) Copyright © 2007 Elsevier Inc. Terms and Conditions