Ribozomálny proteín Rpl22 reguluje zostrih svojich vlastných transcriptov

Kvasinka Saccharomyces cerevisiae patrí medzi organizmy s malým počtom intrónov, ktoré sa nachádzajú v približne 5% jej génov. Najdôležitejšou skupinou takýchto génov sú gény kódujúce ribozomálne proteíny. Tie sú okrem iného vysoko exprimované a častokrát kódované dvoma paralógmi. Parenteau s kolegami popísala intrón-dependentné intergénové regulačné obvody, ku ktorým dochádza medzi ribozomálnymi paralógmi a ktoré kontrolujú pomery ich expresie. V tejto práci sa nám nepodarilo potvrdiť prítomnosť takýchto obvodov u 6 zo 7 testovaných párov paralógov. Jedinou funkčnou je regulácia u RPL22 paralógov. Ukázali sme, že Rp22 proteín blokuje pre-mRNA zostrih oboch RPL22 paralógov, avšak prednostne RPL22B. Proteín Rpl22 to sprostredkováva väzbou na sekundárnu štruktúru v RPL22B intróne, pričom mutácia RNA-väzbovej domény proteínu Rpl22 vedie k strate väzby. Takáto sekundárna štruktúra a väzba na ňu je zachovaná aj v kvasinke Kluyveromyces lactis, ktorá obsahuje iba jednu kópiu RPL22 génu. Výsledky tejto práce vedú k lepšiemu porozumeniu intergénovej regulácie, ku ktorej dochádza medzi funkčne odlišnými RPL22 paralógmi. Kľúčové slová intróny, gény ribozomálnych proteínov, Rpl22, RPL22 paralógy, zostrih pre-mRNA, Saccharomyces cerevisiaeSaccharomyces cerevisiae is an intron-poor organism with introns present in only 5% of its genes. The most prominent group of intron-containing genes are ribosomal protein (RP) genes. They are highly expressed and most of them are present as two paralogs. Parenteau et al. described the existence of intron- dependent intergenic regulatory circuits controlling expression ratios of RP paralogs. In this project, we did not confirm the regulation in 6 out of 7 tested regulatory circuits. We validated the regulation between RPL22 paralogs. We further showed that Rpl22 protein blocks the pre-mRNA splicing of both paralogs, with RPL22B paralog being more sensitive. Rpl22 protein binds to the stem-loop of RPL22B intron - disruption of the binding domain of Rpl22 proteins leads to loss of interaction. Moreover, the regulation seems to be working the same way in yeast Kluyveromyces lactis, which has only a single RPL22 copy. Overall, these results lead to better understanding of intergenic regulation, which adjusts the expression ratio between functionally different RPL22 paralogs. Key words introns, ribosomal protein genes, Rpl22, RPL22 paralogs, pre-mRNA splicing, Saccharomyces cerevisiaeKatedra buněčné biologieDepartment of Cell BiologyPřírodovědecká fakultaFaculty of Scienc

Regulace exprese genů pro ribosomální proteiny

Kvasinka Saccharomyces cerevisiae produkuje za normálnych podmienok približne 2000 ribozómov za minútu. Produkcia ribozomálnych proteínov je rozsiahla - odhadom predstavuje až 50% celkovej transkripcie prevádzanej RNA polymerázou II a 90% celkového zostrihu pre-mRNA v bunkách. Expresia génov ribozomálnych proteínov je koordinovaná, čím zabezpečuje ekvimolárne množstvo jednotlivých ribozomálnych proteínov. Regulácia prebieha prevažne na úrovni transkripcie, avšak rôznorodá post- transkripčná regulácia je potrebná pre odstránenie fluktuácie koncentrácií jednotlivých ribozomálnych proteínov, a teda pre ich stoichiometrickú vyváženosť. Prevládajúcou je regulácia závislá na intrónoch, ktorá okrem vyvažovania jednotlivých ribozomálnych proteínov zabezpečuje ich rýchlu reguláciu (represiu) v odpovedi na enviromentálny stres. KĽÚČOVÉ SLOVÁ gény pre ribozomálne proteíny, RPG, ribozomálne proteíny, regulácia génovej expresie, koregulácia, Saccharomyces cerevisiaeSaccharomyces cerevisiae cells produce 2000 ribosomes per minute under normal conditions. The expression of ribosomal proteins is massive - it takes 50% of RNA polymerase II transcription and 90% of pre-mRNA splicing in rapidly growing cells. Since cells need an equimolar amount of individual ribosomal proteins, the tight coregulation of gene expression is required. The transcription is a main target of regulation, however, it is inherently unable to set a stoichiometric balance of ribosomal proteins. Various types of post-transcriptional regulation deal with fluctuations of individual ribosomal proteins and fine-tune their expression. Intron-dependent regulation appears to by predominant among ribosomal protein genes. Besides balancing their expression, presence of introns provides a rapid global regulation (repression) of ribosomal protein genes in response to environmental stress. KEY WORDS ribosomal protein genes, RPG, ribosomal protein, gene expression regulation, coregulation, Saccharomyces cerevisiaeDepartment of Cell BiologyKatedra buněčné biologiePřírodovědecká fakultaFaculty of Scienc

Gene expression regulation of ribosomal protein genes

Saccharomyces cerevisiae cells produce 2000 ribosomes per minute under normal conditions. The expression of ribosomal proteins is massive - it takes 50% of RNA polymerase II transcription and 90% of pre-mRNA splicing in rapidly growing cells. Since cells need an equimolar amount of individual ribosomal proteins, the tight coregulation of gene expression is required. The transcription is a main target of regulation, however, it is inherently unable to set a stoichiometric balance of ribosomal proteins. Various types of post-transcriptional regulation deal with fluctuations of individual ribosomal proteins and fine-tune their expression. Intron-dependent regulation appears to by predominant among ribosomal protein genes. Besides balancing their expression, presence of introns provides a rapid global regulation (repression) of ribosomal protein genes in response to environmental stress. KEY WORDS ribosomal protein genes, RPG, ribosomal protein, gene expression regulation, coregulation, Saccharomyces cerevisia

Ribosomal protein Rpl22 regulates the splicing of its own transcripts

Saccharomyces cerevisiae is an intron-poor organism with introns present in only 5% of its genes. The most prominent group of intron-containing genes are ribosomal protein (RP) genes. They are highly expressed and most of them are present as two paralogs. Parenteau et al. described the existence of intron- dependent intergenic regulatory circuits controlling expression ratios of RP paralogs. In this project, we did not confirm the regulation in 6 out of 7 tested regulatory circuits. We validated the regulation between RPL22 paralogs. We further showed that Rpl22 protein blocks the pre-mRNA splicing of both paralogs, with RPL22B paralog being more sensitive. Rpl22 protein binds to the stem-loop of RPL22B intron - disruption of the binding domain of Rpl22 proteins leads to loss of interaction. Moreover, the regulation seems to be working the same way in yeast Kluyveromyces lactis, which has only a single RPL22 copy. Overall, these results lead to better understanding of intergenic regulation, which adjusts the expression ratio between functionally different RPL22 paralogs. Key words introns, ribosomal protein genes, Rpl22, RPL22 paralogs, pre-mRNA splicing, Saccharomyces cerevisia

Ribosomal protein Rpl22 regulates the splicing of its own transcripts

Saccharomyces cerevisiae is an intron-poor organism with introns present in only 5% of its genes. The most prominent group of intron-containing genes are ribosomal protein (RP) genes. They are highly expressed and most of them are present as two paralogs. Parenteau et al. described the existence of intron- dependent intergenic regulatory circuits controlling expression ratios of RP paralogs. In this project, we did not confirm the regulation in 6 out of 7 tested regulatory circuits. We validated the regulation between RPL22 paralogs. We further showed that Rpl22 protein blocks the pre-mRNA splicing of both paralogs, with RPL22B paralog being more sensitive. Rpl22 protein binds to the stem-loop of RPL22B intron - disruption of the binding domain of Rpl22 proteins leads to loss of interaction. Moreover, the regulation seems to be working the same way in yeast Kluyveromyces lactis, which has only a single RPL22 copy. Overall, these results lead to better understanding of intergenic regulation, which adjusts the expression ratio between functionally different RPL22 paralogs. Key words introns, ribosomal protein genes, Rpl22, RPL22 paralogs, pre-mRNA splicing, Saccharomyces cerevisia

Introns provide a platform for intergenic regulatory feedback of <i>RPL22</i> paralogs in yeast

<div><p>Ribosomal protein genes (RPGs) in <i>Saccharomyces cerevisiae</i> 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 <i>RPL22</i>, <i>RPL2</i>, <i>RPL16</i>, <i>RPL37</i>, <i>RPL17</i>, <i>RPS0</i>, and <i>RPS18</i> paralog pairs. We found that introns inhibited the expression of their genes in the <i>RPL22</i> pair, with the <i>RPL22B</i> intron conferring a much stronger effect. While the WT <i>RPL22A</i>/<i>RPL22B</i> mRNA ratio was 93/7, the <i>rpl22a</i>Δ<i>i</i>/<i>RPL22B</i> and <i>RPL22A</i>/<i>rpl22b</i>Δ<i>i</i> ratios were >99/<1 and 60/40, respectively. The intron in <i>RPL2A</i> 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.</p><p>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 <i>RPL22B</i> 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.</p><p>The <i>RPL22</i> singleton of <i>Kluyveromyces lactis</i>, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in <i>K</i>. <i>lactis</i> cells. Vice versa, the overproduction of the <i>K</i>. <i>lactis</i> protein reduced the expression of <i>RPL22A/B</i> in <i>S</i>. <i>cerevisiae</i>. The extraribosomal function of of the <i>K</i>. <i>lactis</i> Rpl22 suggests that the loop regulating <i>RPL22</i> paralogs of <i>S</i>. <i>cerevisiae</i> evolved from autoregulation.</p></div

Introns determine the level of sensitivity of <i>RPL22</i> expression to downregulation.

<p>Heatmaps show endogenous mRNA abundances of <i>RPL22A</i> (left panel) and <i>RPL22B</i> (right panel) in five genetic backgrounds (arranged in rows) with overexpression constructs (in columns). Configurations of chimeras at the <i>RPL22A</i> and <i>RPL22B</i> loci are described for each row in the center. All values were normalized to the WT strain bearing an empty vector (top left cell in each heatmap). Leftmost columns (“vector”) reflect only the effect of genetic background, while A/B columns show the effect of overexpression. cDNA was prepared using random hexamers and oligo(dT) 18-mers. The primers used to detect <i>RPL22B</i> mRNA were specific for the transcript derived from the endogenous <i>RPL22B</i> locus. The detection of the <i>RPL22B</i> mRNA transcribed from the A locus (underlined) was accomplished using a specific set of primers (see last row of the right panel). For details and statistical analysis see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190685#pone.0190685.s013" target="_blank">S7 Table</a>.</p

Rpl22A and Rpl22B interact with a specific region of the <i>RPL22B</i> intron.

<p>(A) RNAfold structure prediction of the <i>RPL22B</i> intron (“I”). Regions tested using the three-hybrid system are shown in color. The I1 region (green) represents nucleotides 11 to 123 of the <i>RPL22B</i> intron; I2 (red)–nt 165 to 236; I3 (blue)–nt 256 to 321. (B) Intron region 2 (I2) interacts with WT Rpl22A and Rpl22B but not with their RNA-binding mutants using the yeast three-hybrid system. <i>RPL22A</i> (“A”) and <i>RPL22B</i> (“B”) or their mutated versions (“Amut”, “Bmut”) in combination with different parts of the <i>RPL22B</i> intron were assayed for expression of the <i>HIS3</i> reporter gene, which is activated in the presence of protein-RNA interaction. 10-fold serial dilutions of cells were spotted on plates with increasing concentrations of 3-aminotriazole (3-AT). “-U”, “-L”, and “-H” denote the lack of uracil, leucine, and histidine in the medium. IRE and IRP served as positive controls. p3HR2 is the empty plasmid for bait RNA expression and pACT2 is the plasmid for expression of the Gal4 activation domain.</p

Splicing of <i>Kluyveromyces lactis RPL22</i> is subject to Rpl22-mediated inhibition in both <i>K</i>. <i>lactis</i> and <i>S</i>. <i>cerevisiae</i>.

<p>(A) <i>KlRPL22</i> expression in BY4741 cells leads to the downregulation of endogenous <i>RPL22</i> mRNAs to an extent similar to that observed for the overexpression of <i>RPL22A</i> and <i>RPL22B</i> (compare with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190685#pone.0190685.g003" target="_blank">Fig 3</a>). (B) Negative regulation of <i>RPL22B</i> by Rpl22A is maintained when the <i>RPL22A</i> intron and exon 2 are replaced with the corresponding part of <i>KlRPL22</i>. (C) The abundance of <i>KlRPL22</i> mRNA expressed from the <i>RPL22A</i> locus as in (B) is decreased by the overproduction of either Rpl22A or Rpl22B. The intronless version of <i>KIRPL22</i> in the same locus does not respond to Rpl22A/B overexpression. (D) Endogenous <i>KlRPL22</i> mRNA is reduced by the ectopic expression of <i>KlRPL22</i> as well as <i>RPL22A</i> and <i>RPL22B</i> in <i>K</i>. <i>lactis</i>. cDNA was prepared using random hexamers and oligo(dT) 18-mers. The statistical significance of the difference between empty vector strain and an overexpression strain is indicated as (1) for <i>P≤</i>0.05, (2) for <i>P≤</i>0.01, and (3) for <i>P≤</i>0.001 based on the t-test with Holm correction for multiple testing (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190685#sec002" target="_blank">Methods</a>).</p

Introns mediate intergenic regulation of <i>RPL22</i> paralogs.

<p>(A) Impact of intron deletions on the expression of duplicated ribosomal protein genes. Transcript levels of <i>RPL16</i>, <i>RPL17</i>, <i>RPL37</i>, <i>RPS0</i> and <i>RPS18</i> paralogs are refractory to intron deletions. In contrast, <i>RPL22</i> and <i>RPL2A</i> show intergenic and intragenic intron dependency, respectively. Plots show mean fold changes of “A” (a) and “B” (b) paralog mRNA in mutants with intron deletion (Δi) in “A”, “B” or both paralogs as determined by RT-qPCR relative to WT. Data were normalized to <i>SPT1</i>5 expression and to the RNA level in WT cells. Error bars represent s.d. from two (<i>RPL17</i>, <i>RPS0</i>, <i>RPS18</i>) or three (<i>RPL2</i>, <i>RPL16</i>, <i>RPL37</i>) biological replicates. The statistical significance of the difference between WT strain and a strain bearing intron deletion is indicated as (2) for <i>P</i>≤0.01 and (3) for <i>P</i>≤0.001 based on the t-test with Holm correction for multiple testing (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190685#sec002" target="_blank">Methods</a>). Pre-cultures of colonies from freshly dissected spores were diluted to such a low OD so that they could undergo 10 generations before they were harvested at mid-exponential phase. In independent experiments, intron deletion mutants of <i>RPL22</i>, <i>RPL16</i> and <i>RPL37</i> were grown in YPAD or synthetic medium for 2 or 10 generations, respectively. The two cultivations gave essentially the same results as above (data not shown). (B) <i>RPL22</i> mRNAs are negatively regulated by their introns and respond in an intergenic way to the manipulations of their paralogous counterparts. The plot shows mRNA levels of <i>RPL22</i> paralogs normalized to WT. The <i>RPL22A</i> to <i>RPL22B</i> ratio was calculated from the same data as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190685#pone.0190685.s001" target="_blank">S1 Fig</a>. Mean values ± s.d. from at least three biological replicates are shown (for details and statistical analysis, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190685#pone.0190685.s001" target="_blank">S1 Fig</a>). (C) Rpl22 protein levels reflect the changes in corresponding mRNAs. Relative protein levels were assessed based on mass spectra intensities measured from whole cell lysates. Mean values ± s.d. from 3 biological replicates are shown. <i>P</i> values were obtained for comparisons of log-normalized protein intensities (Label Free Quantification Algorithm; MaxQuant, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190685#sec002" target="_blank">Methods</a>) between WT strain and a mutant strain using the t-test with Holm correction for multiple testing. Significant differences are indicated as (a) <i>P</i> = 0.0182 for Rpl22A (Rpl22B intensity was below detection limit), (b) <i>P</i> = 0.0297/ 0.0026 for Rpl22A/ B, and (c) <i>P</i> = 0.0006 for Rpl22B. (D,E) Splicing efficiency analysis of <i>RPL22A</i> and <i>RPL22B</i> introns, respectively, in <i>RPL22</i>-manipulated strains. Gels show radioactively labeled primer extension products from cells expressing <i>RPL22A-CUP1</i> (D) and <i>RPL22B-CUP1</i> (E) reporter substrates. U14 snoRNA was used as a loading control. Each gel is representative of at least three independent experiments.</p