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oai:figshare.com:article/34047641 2026-10-01T17:26:15Z 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

The splicing of the RPL22B intron was affected by the pre-mRNA context. 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 The experiment was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.g002" target="_blank">Fig 2</a>. (A) Both main and alternative 5’ss contributed to the RPL22Bi splicing inhibitory mechanism. The removal of alternative 5’ss increased the level of unspliced RNA in the absence of Rpl22 compared to WT (ΔALT5’ss). Mutating the 5’ss sequences to the yeast consensus decreased the ability of Rpl22 to inhibit splicing and increased the use of alternative 5’ss. The alternative splicing products migrated above the size of the major band and the PCR products of the corresponding length were sequenced to confirm this assumption. (B) Predicted secondary structure of the main stem loop of RPL22Bi. The structure was modeled by RNAfold [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.ref052" target="_blank">52</a>], visualized using Forna [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.ref058" target="_blank">58</a>] and annotated according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.ref011" target="_blank">11</a>]. ‘Clamp’ and ‘RNA internal loop’ (RIL) are labeled red and green, respectively. In [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.ref011" target="_blank">11</a>], ‘Clamp’ is referred to as ‘Lower distal stem.’ (C) Structure, but not the sequence of the ‘Clamp’ was important for splicing regulation. Deletion of the 3’strand of the ‘Clamp’ or weakening of its complementarity rendered intron splicing permissive regardless of Rpl22. On the contrary, maintaining complementarity while scrambling the sequence or flipping the stem arms retained the WT behavior. (D) Nucleotides within the ‘RNA internal loop’ were dispensable for the regulation of RPL22Bi splicing. In contradiction to previous findings [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.ref011" target="_blank">11</a>], nucleotides within the ‘RNA internal loop’ (RIL) are dispensable for the regulation of RPL22Bi splicing. CCCU to AAAC mutation in the 5’arm of the loop (‘5’RIL AAAC’), the UGAA to CAUU mutation in its 3’arm (‘3’RIL CAUU’), or the deletion of the loop or its arms did not lose regulation after the overexpression of the Rpl22 protein. ΔRIL indicates the deletion of both 5’arm CCCU and 3’arm UGAA nucleotides; Δ5’RIL and Δ3’RIL indicate the per partes deletions of CCCU and UGAA, respectively. 2026-10-01T17:25:45Z Image Figure 10.1371/journal.pone.0359197.g005 https://figshare.com/articles/figure/_p_The_splicing_of_the_i_RPL22B_i_intron_was_affected_by_the_pre-mRNA_context_p_/34047641 CC BY 4.0