Fig. 1: Homogenization of yeast ribosomal proteins identifies dose independent paralogs subfunctionalization.

a Gene conversion strategy for “homogenization” of ribosomal protein genes. Genes showing paralog-specific response to different conditions were selected for homogenization. Homogenized strains were created by replacing one paralog locus including all regulatory sequences, introns and UTRs, with sequence of the other. Cells carrying one or two copies of a single paralog were examined for growth in liquid media and maximum growth rates were calculated for each strain. Duplicated ribosomal protein gene expression levels were examined by RT-qPCR to identify dose-dependency of growth defects. b RNA abundance in transcripts per million (TPM) of each paralog obtained by RNA sequencing n = 2 biologically independent samples. Universal and conventional names of each protein are shown below. c Abundance of protein isoforms generated by each duplicated ribosomal protein gene was determined by Swath Multiple Reaction Monitoring (MRM) identifying the major and minor isoforms. No suitable peptides to reliably distinguish between L27 paralogs were found, hence their peptide signal intensities were not determined (ND). d Total RNA generated by each gene pair was detected using primers common to both paralogs in RT-qPCR after deletion or homogenization of duplicated ribosomal protein genes and is shown relative to mRNA detected in wild-type strain. B∆ and A∆ indicate RNA detected in strains lacking the B or A paralog, while AA and BB indicate RNA detected in cells containing two copies of the A or B isoforms respectively. e Growth rates of deletion and homogenized strains were determined and shown relative to wild-type. f Growth rates of deletion and homogenized strains were determined in media containing staurosporine (3 µg/ml), hygromycin (100 µg/ml) or NaCl (0.9 M) and the effect on growth are shown relative to wild-type treated similarly. Bars shown in (c–f) represent the means of n = 3 biologically independent samples shown as data points, p values from two-tailed unpaired t test are indicated in (f).