<p>Splicing-inhibitory mutations block <i>RPL22</i> intron reporter splicing <i>in vivo</i> irrespective of the presence of the Rpl22 protein.</p>

The PCR products of the corresponding length were sequenced to confirm this assumption. (B) Summary of <i>RPL22B</i>i 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 <i>RPL22B</i> 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="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="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="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="plosone.org/article/info:doi/10.1371/journal.pone.0359197#pone.0359197.s011" target="_blank">S1</a>-<a href="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 <i>sensu stricto</i> 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="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 <i>RPL22A</i>i inhibited the splicing similarly to <i>RPL22B</i><b>i.</b> The elements are annotated in <a href="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 <i>RPL22A-CUP1</i> reporter was used. (D) Keeping partial complementarity was sufficient to inhibit the splicing of <i>RPL22B</i>i with scrambled conserved element. The mutant MutConsComp1 did not bind Rpl22 in Y3H (<a href="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="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).</p>