<p>Rpl22 homologs complemented the extra ribosomal function of Rpl22A in <i>S. cerevisiae.</i></p>

The projection delineates parts that are likely involved in the intron-regulatory mechanism of <i>RPL22.</i> (B) The Rpl22 proteins from related yeast species, <i>S. pombe</i> and <i>H. sapiens</i> were tested for their ability to support WT growth of the <i>rpl22a</i>Δ <i>rpl22b</i>Δ strain. We expressed in <i>rpl22a</i>Δ <i>rpl22b</i>Δ and WT <i>S. cerevisiae</i> cells different <i>RPL22</i> genes as intronless versions under the control of the <i>ADH1</i> promoter (plasmid pVTU260) and evaluated whether the production of different Rpl22 proteins would rescue the growth phenotype.

Transformation with the empty pVTU260 served as a control (-). The Rpl22 proteins tested included Rpl22A (A), the binding-dead mutant of Rpl22 (A_mut) and the homologs from <i>Candida albicans</i> (Ca), <i>Candida glabrata</i> (Cg), <i>Debaryomyces hanseni</i> (Dh), <i>Yarrowia lipolytica</i> (Yl), <i>Schizosaccharomyces pombe</i> (Sp), <i>Kluyveromyces lactis</i> (Kl) and <i>Homo sapiens</i> (Hs). The y-axis represents the generation time of individual strains.

The plotted values are the means and standard deviations of three biological replicates. The results of the Student’s t-test with Bonferroni correction for multiple tests are indicated by (*) or (‡) for the differences between a strain and WT or the double deletant <i>rpl22a</i>Δ <i>rpl22b</i>Δ, respectively (both (*) and (‡) indicate <i>P</i> ≤ 0.001). (C) Endogenous <i>RPL22B splicing</i> can be inhibited by Rpl22 homologs in the <i>rpl22a</i>Δ strain. <i>The RPL22</i> homologs were expressed in the <i>rpl22a</i>Δ strain as in (B).

After the preparation of cDNA from exponentially growing cells, the level of endogenous <i>RPL22B</i> mRNA was measured by qPCR using primers specific for the <i>RPL22B</i> locus. The signal was normalized to <i>SPT15</i> and to the <i>rpl22a</i>Δ strain transformed by an empty vector (-). Splicing inhibition was caused by the expression of <i>S. cerevisiae</i> Rpl22A (A) but not its binding-dead mutant (A_mut).

The Rpl22 homologs were labeled as in (B). Statistical significance of the difference between the <i>rpl22a</i>Δ strain harboring an empty vector (-) and a Rpl22 expressor is indicated as (‡); (*) is used for the difference between (A) and other strains. Both symbols indicate <i>P</i> ≤ 0.005 based on a t-test with Bonferroni correction for multiple comparisons.

(D) All tested Rpl22 proteins can inhibit splicing of the <i>RPL22B</i> reporter in vivo. The <i>rpl22a</i>Δ <i>rpl22b</i>Δ cells expressing the indicated homologues of Rpl22 (see (B)) were transformed with a plasmid carrying the reporter <i>RPL22B-CUP1.</i> The reporter splicing efficiency was measured and the figure was formatted as in <a href="plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.g002" target="_blank">Fig 2</a>. <i>TOM22</i> was used as a loading control.

The figure represents the result of one of three independent experiments.</p>