Regulation of yeast RPL22B splicing depends on intact pre-mRNA context of the intron

Abstract

Yeast RPL22A and RPL22B genes form an intergenic regulatory loop modulating the ratio of paralogous transcripts in response to changing levels of proteins. Gabunilas and Chanfreau (Gabunilas and Chanfreau, PLoS Genet 12, e1005999, 2016) and our group (Abrhamova et al., PLoS ONE 13, e0190685, 2018) described that Rpl22 proteins bound to the divergent introns of RPL22 paralogs and inhibited splicing in dosage dependent manner. Here, we continued to study the splicing regulation in more detail and designed constructs for in vivo analyses of splicing efficiency. We also tested Rpl22 binding to RPL22B intron in three-hybrid system. We were able to confirm the findings reported originally by Gabunilas and Chanfreau on the importance of a stem loop structure within the RPL22B intron. Mutations which lowered the stability of the structure abolished Rpl22-mediated inhibition. In contrast, we were not able to confirm the sequence specificity with respect to either Rpl22 binding or splicing inhibition within this region, which they reported. We contradict their results that the RNA internal loop of RPL22Bi (nt 178CCCU181 and 221UGAA224) is crucial for mediating the Rpl22 effects. We assume that this discrepancy reflects the difference in constructs, as the reporters used by Gabunilas and Chanfreau lacked the alternative 5 splice site as well as surrounding exons. Our own comparison confirms that deleting the sequence spanning alternative 5 splice site lowers splicing efficiency, hinting to possible disturbances of the regulatory mechanism. We argue that the structural context of the regulatory element may reach across the intron or into the surrounding sequences, similarly to what was found previously for other genes, such as RPL30. Apparently, more detailed analyses are needed to discern this intriguing example of splicing regulation.