2026-10-07T17:35:09Z https://api.figshare.com/v2/oai

oai:figshare.com:article/34047647 2026-10-01T17:26:17Z category_1 category_4 category_8 category_13 category_14 category_873 category_734 category_61 category_64 category_134 portal_5 item_type_1 month_year_10_2026

Rpl22 homologs complemented the extra ribosomal function of Rpl22A in S. cerevisiae. Kateřina Abrhámová (4739808) Alexandra Gredová (25157630) Karolína Navrátilová (25157633) Mohamed Boumaiza (5636147) Petr Folk (209900) Biophysics Biochemistry Microbiology Genetics Molecular Biology Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified Developmental Biology Cancer Virology intergenic regulation mediated first catalytic step 5 ’ ss div >< p additional stem loops tested splicing efficiency saccharomyces cerevisiae </ rather stalled splicing enhanced splicing inhibition intron binding using predicted stem alternate stem p structure permitted splicing b </ structural ensemble rpl22 </ ribosomal roles ribosomal proteins regulatory mechanism prevent recognition predicted features particular type others described mutagenesis identified mrna behaves later stage hybrid system fulfill non containing reporter branch point binding capacity allosteric switch (A) Projection of invariant amino acids on the surface of Rpl22A of S. cerevisiae. The structure of Rpl22 and 25S rRNA was extracted from the ribosome structure (PDB: 4V88) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.ref064" target="_blank">64</a>] using the ChimeraX program (<a href="https://www.rbvi.ucsf.edu/chimerax" target="_blank">https://www.rbvi.ucsf.edu/chimerax</a>) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.ref065" target="_blank">65</a>]. Helices 57 and 59 of 25S rRNA are highlighted in blue and orange, respectively. The pooled amino acid differences between Rpl22A and the Rpl22 homologs tested in (B) to (D) are projected onto the surface of the molecule. The colors indicate the identity to Rpl22A (red), conservative substitutions (pink; see ‘:’ and ‘. in the alignment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.s008" target="_blank">S8 Fig</a>), and nonconserved positions (gray). The projection delineates parts that are likely involved in the intron-regulatory mechanism of RPL22. (B) The Rpl22 proteins from related yeast species, S. pombe and H. sapiens were tested for their ability to support WT growth of the rpl22aΔ rpl22bΔ strain. We expressed in rpl22aΔ rpl22bΔ and WT S. cerevisiae cells different RPL22 genes as intronless versions under the control of the ADH1 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 (Amut) and the homologs from Candida albicans (Ca), Candida glabrata (Cg), Debaryomyces hanseni (Dh), Yarrowia lipolytica (Yl), Schizosaccharomyces pombe (Sp), Kluyveromyces lactis (Kl) and Homo sapiens (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 rpl22aΔ rpl22bΔ, respectively (both (*) and (‡) indicate P ≤ 0.001). (C) Endogenous RPL22B splicing can be inhibited by Rpl22 homologs in the rpl22aΔ strain. The RPL22 homologs were expressed in the rpl22aΔ strain as in (B). After the preparation of cDNA from exponentially growing cells, the level of endogenous RPL22B mRNA was measured by qPCR using primers specific for the RPL22B locus. The signal was normalized to SPT15 and to the rpl22aΔ strain transformed by an empty vector (-). Splicing inhibition was caused by the expression of S. cerevisiae Rpl22A (A) but not its binding-dead mutant (Amut). The Rpl22 homologs were labeled as in (B). Statistical significance of the difference between the rpl22aΔ 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 P ≤ 0.005 based on a t-test with Bonferroni correction for multiple comparisons. (D) All tested Rpl22 proteins can inhibit splicing of the RPL22B reporter in vivo. The rpl22aΔ rpl22bΔ cells expressing the indicated homologues of Rpl22 (see (B)) were transformed with a plasmid carrying the reporter RPL22B-CUP1. The reporter splicing efficiency was measured and the figure was formatted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.g002" target="_blank">Fig 2</a>. TOM22 was used as a loading control. The figure represents the result of one of three independent experiments. 2026-10-01T17:25:45Z Image Figure 10.1371/journal.pone.0359197.g007 https://figshare.com/articles/figure/_p_Rpl22_homologs_complemented_the_extra_ribosomal_function_of_Rpl22A_in_i_S_cerevisiae_i_p_/34047647 CC BY 4.0