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oai:figshare.com:article/34047632 2026-10-01T17:26:10Z 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

Splicing-inhibitory mutations block RPL22 intron reporter splicing in vivo irrespective of the presence of the Rpl22 protein. 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) Mutants stabilizing the inhibitory conformation of RPL22Bi inhibited splicing irrespective of the presence of the Rpl22 protein in vivo. The splicing efficiency of RPL22B-CUP1 reporters was tested in rpl22aΔ rpl22bΔ strain harboring the plasmid pVTU260/RPL22A for overexpression of Rpl22A and in the same strain transformed with empty vector. Semi-quantitative PCR was run on cDNA prepared from RNA isolated from exponentially growing cultures using random hexamers. The loading control is included in a separate inset with the indicated gene. One of at least three independent experiments is shown. We detected alternative splicing products, which migrated above the size of the major product and which reflect the usage of alternative 5’ss. The PCR products of the corresponding length were sequenced to confirm this assumption. (B) Summary of RPL22Bi reporter experiments in (A), Rpl22 binding data from the yeast three-hybrid assay, and types of stem loop arrangements predicted by RNAshapes. Pre-mRNA accumulation was approximated on a scale of ‘++ + ’ to ‘-.’ The binding of Rpl22 to a fragment of the RPL22B intron (I2 construct; see text) was tested in a yeast three-hybrid assay. Cell growth corresponding to Rpl22 binding was summarized on a scale from ‘++ + ’ to ‘-.’ For the photographs of spot tests, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.s004" target="_blank">S4 Fig</a>; n.d. - “not done”. The secondary structures were predicted using RNAshapes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.ref054" target="_blank">54</a>]. The structures were sorted according to the stem loop arrangement involving the conserved region and its complementary elements (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.g001" target="_blank">Fig 1</a>), and the number of structure types classified as I, P, or O (other) was expressed in %. See text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.s011" target="_blank">S1</a>-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.s003" target="_blank">S3 Figs</a> for additional information. (*) The 3’COMP/CONS base pairing in ΔCons was sensu stricto not of the I type, but the apical part of the main stem loop and the rest of the prediction remained the same as in the I structures (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.s002" target="_blank">S2 Fig</a>). (C) The perfect complementarity between the conserved element and the 5’sequence element of RPL22Ai inhibited the splicing similarly to RPL22Bi. The elements are annotated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.g001" target="_blank">Fig 1A</a>. The experiment was carried out as in (A), except that the RPL22A-CUP1 reporter was used. (D) Keeping partial complementarity was sufficient to inhibit the splicing of RPL22Bi with scrambled conserved element. The mutant MutConsComp1 did not bind Rpl22 in Y3H (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.s007" target="_blank">S7C Fig</a>) and returned WT-like secondary structure prediction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.s002" target="_blank">S2 Fig</a>). The experiment was done as in (A). 2026-10-01T17:25:45Z Image Figure 10.1371/journal.pone.0359197.g002 https://figshare.com/articles/figure/_p_Splicing-inhibitory_mutations_block_i_RPL22_i_intron_reporter_splicing_i_in_vivo_i_irrespective_of_the_presence_of_the_Rpl22_protein_p_/34047632 CC BY 4.0