Introns provide a platform for intergenic regulatory feedback of RPL22 paralogs in yeast

Abstract

Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs. We found that introns inhibited the expression of their genes in the RPL22 pair, with the RPL22B intron conferring a much stronger effect. While the WT RPL22A/RPL22B mRNA ratio was 93/7, the rpl22aΔi/RPL22B and RPL22A/rpl22bΔi ratios were >99/<1 and 60/40, respectively. The intron in RPL2A stimulated the expression of its own gene, but the removal of the other introns had little effect on expression of the corresponding gene pair. Rpl22 protein abundances corresponded to changes in mRNAs.Using splicing reporters containing endogenous intron sequences, we demonstrated that these effects were due to the inhibition of splicing by Rpl22 proteins but not by their RNA-binding mutant versions. Indeed, only WT Rpl22A/Rpl22B proteins (but not the mutants) interacted in a yeast three-hybrid system with an RPL22B intronic region between bp 165 and 236. Transcriptome analysis showed that both the total level of Rpl22 and the A/B ratio were important for maintaining the WT phenotype. The data presented here support the contention that the Rpl22B protein has a paralog-specific role.The RPL22 singleton of Kluyveromyces lactis, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in K. lactis cells. Vice versa, the overproduction of the K. lactis protein reduced the expression of RPL22A/B in S. cerevisiae. The extraribosomal function of of the K. lactis Rpl22 suggests that the loop regulating RPL22 paralogs of S. cerevisiae evolved from autoregulation.

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

“…Rpl22 was also shown to be required for IME1 mRNA translation and meiotic induction in S. cerevisiae, as rpl22Δ cells were unable to translate the IME1 transcript because of its atypical 5'UTR (Kim and Strich, 2016 . The protein products of either paralog bound to RPL22 introns, which caused moderate inhibition of the major paralog (RPL22A) and strong inhibition of the minor one (RPL22B) (Abrhámová et al, 2018). A/B changes were related to oxidative stress (see above), or disbalances in free RP concentration caused by exposures to MMS or Cd (Gabunilas and Chanfreau, 2016), supporting the interpretation that tuning the isoform ratio helps the cells to react to changing environment.…”

“…These gene pairs were implicated in intergenic regulation (Parenteau et al, 2011). However, only a handful of such pairs were proven to regulate each other's expression, such as RPS14, RPS9, and RPL22 (Fewell and Woolford, 1999;Plocik and Guthrie, 2012;Petibon et al, 2016;Gabunilas and Chanfreau, 2016;Abrhámová et al, 2018). The incorporation of two different paralogs into ribosomes may give rise to ribosome heterogeneity, which became focus of recent attention.…”

“…A/B changes were related to oxidative stress (see above), or disbalances in free RP concentration caused by exposures to MMS or Cd (Gabunilas and Chanfreau, 2016), supporting the interpretation that tuning the isoform ratio helps the cells to react to changing environment. What cellular process or machinery distinguishes the 19 amino acids difference between Rpl22A and Rpl22B remains unknown; the effects on splicing are apparently mediated by both A and B proteins (Abrhámová et al, 2018). Information on the interface between the intron and Rpl22, and between this complex and the spliceosome, is likewise limited.…”

Mating-type specific ribosomal proteins control aspects of sexual reproduction inCryptococcus neoformans

“…Mice lacking RPL22 are 497 viable and have specific αβ T-cell developmental defects, likely attributable to compensation by 498Rpl22-l1 in other tissues (ANDERSON et al 2007). Recent studies in both yeast and mammals 499 suggest an extraribosomal role for Rpl22 paralogs in binding target mRNAs and regulating their 500 expression (GABUNILAS AND CHANFREAU 2016; ZHANG et al 2017;ABRHAMOVA et al 2018).…”

“…This study focused on the MAT ribosomal proteins, demonstrating that both RPL39 and 85 RPL22 α and a alleles are essential in C. neoformans. Because Rpl22 in yeast and vertebrates has 86 been found to play specialized functions and extra-ribosomal roles (GABUNILAS AND CHANFREAU 87 2016; KIM AND STRICH 2016; ZHANG et al 2017;ABRHAMOVA et al 2018), we aimed to 88 characterize the functions of the C. neoformans RPL22α and RPL22a paralogs. We found that 89 ectopic integration of an RPL22 paralog failed to complement the essential phenotype due to the 90 mutation of the RPL22 of opposite mating type.…”