Rpl22 is required for IME1 mRNA translation and meiotic induction in S. cerevisiae
© The Author(s) 2016
Received: 25 May 2016
Accepted: 8 July 2016
Published: 29 July 2016
The transition from mitotic cell division to meiotic development in S. cerevisiae requires induction of a transient transcription program that is initiated by Ime1-dependent destruction of the repressor Ume6. Although IME1 mRNA is observed in vegetative cultures, Ime1 protein is not suggesting the presence of a regulatory system restricting translation to meiotic cells.
This study demonstrates that IME1 mRNA translation requires Rpl22A and Rpl22B, eukaryotic-specific ribosomal protein paralogs of the 60S large subunit. In the absence of Rpl22 function, IME1 mRNA synthesis is normal in cultures induced to enter meiosis. However, Ime1 protein production is reduced and the Ume6 repressor is not destroyed in rpl22 mutant cells preventing early meiotic gene induction resulting in a pre-meiosis I arrest. This role for Rpl22 is not a general consequence of mutating non-essential large ribosomal proteins as strains lacking Rpl29 or Rpl39 execute meiosis with nearly wild-type efficiencies. Several results indicate that Rpl22 functions by enhancing IME1 mRNA translation. First, the Ime1 protein synthesized in rpl22 mutant cells demonstrates the same turnover rate as in wild-type cultures. In addition, IME1 transcript is found in polysome fractions isolated from rpl22 mutant cells indicating that mRNA nuclear export and ribosome association occurs. Finally, deleting the unusually long 5′UTR restores Ime1 levels and early meiotic gene transcription in rpl22 mutants suggesting that Rpl22 enhances translation through this element. Polysome profiles revealed that under conditions of high translational output, Rpl22 maintains high free 60S subunit levels thus preventing halfmer formation, a translation species indicative of mRNAs bound by an unpaired 40S subunit. In addition to meiosis, Rpl22 is also required for invasive and pseudohyphal growth.
These findings indicate that Rpl22A and Rpl22B are required to selectively translate IME1 mRNA that is required for meiotic induction and subsequent gametogenesis. In addition, our results imply a more general role for Rpl22 in cell fate switches responding to environmental nitrogen signals.
KeywordsTranslation Differentiation Meiosis Ribosome
The budding yeast S. cerevisiae chooses alternative cell fates based on cell type and environmental cues. For example, in response to poor nitrogen sources, haploid and diploid yeast will undergo a dimorphic switch leading to invasive or pseudohyphal growth, respectively. The switch to pseudohyphal growth requires Ras signaling through Protein Kinase A and is inhibited in response to available nitrogen by Tor1 kinase activation [1–4]. Similarly, meiotic induction occurs only in diploid cells deprived of nitrogen and a fermentable carbon source . The switch from mitotic to meiotic cell divisions requires expression of IME1, which induces the meiotic transcription program by binding and triggering the destruction of the Ume6 repressor . Interestingly, Ime1 is also required for pseudohyphal growth  suggesting that the regulatory pathways controlling these two processes exhibit some degree of overlap. IME1 transcription is controlled by a complex and extensive set of cis-acting promoter elements that respond to cell type, carbon and nitrogen signals [8, 9].
In addition to transcriptional control, IME1 mRNA translation is restricted to meiosis although specific mechanisms were not identified [10, 11]. Many translational control mechanisms in eukaryotic cells operate during translation initiation focusing on the formation of a stable pre-initiation complex. Once a stable complex is formed between the mRNA, the 40S subunit and the initiator tRNA, the catalytic 60S large subunit associates with the small subunit to form the functional 80S complex capable of translation [12, 13]. Following formation of a stable pre-initiation complex, translation can still be inhibited through other mechanisms. For example, the presence of short, upstream open reading frames (uORFs) before the protein encoding initiating AUG causes ribosome stalling and disassociation [14, 15].
The roles that ribosomal proteins (RPs) themselves play in regulating translation initiation are less well understood. The ribosome is composed of an rRNA core bound by many RPs that play essential structural roles for ribosome assembly and function [16–18]. Of the 78 ribosomal protein families in eukaryotes, 34 are also found in prokaryotic ribosomes, 67 in archaea  leaving only 11 families that are specific to eukaryotic cells . Despite the critical role of translation for cellular function, 14 RPs in yeast are not essential for viability indicating that not all ribosomal proteins serve a basic translation function .
One of the non-essential RPs only found in eukarya is the large subunit protein family L22e. RPL22 exist as a paralog pair in yeast (RPL22A, RPL22B) and mammals (Rpl22, Rpl22-like) [20, 21]. L22e binds a stem-loop on the rRNA [16, 22]. However, it is neither directly at the interface of the ribosomal subunits, nor does it play a structural role in organizing the protein exit channel [16, 17]. The murine Rpl22 is not essential for viability but is required for the differentiation of αβ T-cells in mice and hematopoietic stem cell emergence in zebrafish indicating it plays a more specialized role in cell fate decisions [23, 24]. Another group has shown differential expression of Rpl22 and Rpl22-like, the latter of which is alternatively spliced in Drosophila spermatocytes . Interestingly, Rpl22 inhibits the expression of Rpl22-like1 in mice suggesting antagonistic functions for these proteins . The current study describes a role of Rpl22 in mediating cell fate decisions in budding yeast. Although a modest defect is observed in mitotic cell division, loss of Rpl22 function results in significant defects in both pseudohyphal growth and execution of meiotic divisions. The latter phenotype is due to the requirement of Rpl22 in translating the mRNA of the IME1 meiotic inducer. These results identify a specific translation role for Rpl22 during yeast cell fate decisions.
Strains and plasmids
Strains used in this study
MAT a cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3
MAT a cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl22B::KANMX
MAT a cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl22A::KANMX
MAT a cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl22A::KANMX rpl22B::KANMX
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl22A::KANMX
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl22B::KANMX
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl22A::KANMX rpl22B::KANMX
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 IME1::3HA
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl22A::KANMX rpl22B::KANMX IME1::3HA
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 5′UTRΔ-IME1:3HA
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl22A::KANMX rpl22B::KANMX 5′UTRΔ-IME1::3HA
MAT a lys2 lys2 trp1::hisG ura3 LYS2::ho
MAT a/MATα lys2 lys2 trp1::hisG ura3 LYS2::ho
MAT a lys2 trp1::hisG ura3 LYS2::ho rpl22A::KANMX rpl22B::KANMX
MAT a lys2 trp1::hisG ura3 LYS2::ho rpl22A::KANMX rpl22B::KANMX
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl39::KANMX
MAT a/MATα cyh2 R-z ho::LYS2 leu2::hisG lys2 trp1::hisG ura3 rpl29::KANMX
Media and phenotypic assays
Cultures were grown in rich YPDA (2 % dextrose, 2 % peptone, 1 % yeast extract supplemented with 10 mg/l adenine). Plasmid selection was maintained in strains using synthetic dextrose (SD) medium containing 0.17 % yeast nitrogen base without amino acids, 0.5 % ammonium sulfate, 2 % dextrose. Pre-sporulation growth was conducted in either YPA (K acetate (1 %) substituted for dextrose in YPDA) or synthetic acetate (SA, 0.17 % yeast nitrogen base without amino acids, 0.5 % ammonium sulfate, 2 % K acetate). Liquid sporulation medium (SPM, 2 % K acetate supplemented with uracil) was utilized for meiotic timecourse experiments. Invasive growth assays were performed by streaking cells on to YPDA agar plates, incubated for 3 days at 30 °C then washed with a gentle stream of water, while clearing cells on the surface with a gloved hand . Pseudohyphal growth was assayed by streaking wild type or rpl22∆ diploid SK1 cells for single colonies onto synthetic, low-ammonia, dextrose (SLAD) agar plates followed by incubation for 5 days at 30 °C [4, 7]. Meiotic timecourse experiments were conducted with cells grown to mid-log phase in YPA or synthetic acetate (SA), washed in water, and resuspended in sporulation medium (SPM) as previously described . Nuclear divisions were monitored by fixing cells with 70 % ethanol at 4°, washed twice with water, then stained for 15 min with 1 μg/ml 4′,6′-diamidino-2′-phenylindole (DAPI). The cells were washed twice with water and visualized by fluorescence microscopy.
Protein extraction, western blotting, cycloheximide chase assay
Approximately 5 × 107 cells were treated with 0.2 M sodium hydroxide, with subsequent extraction in Laemilli buffer accompanied by glass bead lysis . 1 × 107 cell equivalents were loaded for each sample. Proteins were separated by 10 % SDS-PAGE, transferred to PVDF membranes and blots were probed with anti-HA (12CA5, Roche), anti-Tub1p (Developmental Studies Hybridoma Bank, University of Iowa), poly-clonal anti-Ume6 or anti-Pgk1p (Invitrogen) monoclonal antibodies and visualized using AP conjugated anti-mouse secondary antibody and the CDP-Star system. Cycloheximide (CHX) chase assays were performed essentially as described .
In vivo translation analysis
Exponentially growing cells in rich media were depleted of their methionine and cysteine stores through growth in defined medium lacking these amino acids. After an hour of incubation, 125 μCi of 35S labeled methionine and cysteine were introduced to the medium and incubated at 30 °C. Samples were taken, washed, and frozen in liquid nitrogen every 5 min for 20 min. The proteins were extracted in Laemmli buffer and 1 × 107 cell equivalents were either precipitated using methanol and chloroform to remove unincorporated label to measure total isotope incorporation, or run on a polyacrylamide gel for radiography.
Polysome profiles were performed for each of the given nutritional conditions, as described . Cultures were treated with 100 μg/ml (final concentration) cycloheximide. Harvested cells were washed in lysis buffer in the presence of cycloheximide and heparin, and lysed using glass beads at 4º C. Lysates were clarified with sequential centrifugation (5K×g, 5 min; 13K×g, 10 min) and approximately 200 μg of total RNA was loaded on 15–50 % sucrose gradients. Gradients were centrifuged for 4.25 h at 160K×g. Gradients were analyzed using a continuous flow cuvette. For mRNA analysis of polysomes, wild-type and rpl22Δ cells were grown in 50 ml of YPA and shifted to 10 ml of sporulation medium for 9 h. A small sample was taken for total RNA with the remaining cells treated with cycloheximide (100 μg/ml for 5 min), crosslinked (1 % formaldehyde for 5 min), then the crosslinking quenched with glycine (250 mM). The cells were harvested by centrifugation and snap frozen in liquid nitrogen. Lysates were prepared, centrifuged through a sucrose density gradient, and fractionated as previously described . Fractions were treated with 1 % SDS, 16.6 mM EDTA, and 0.1 mg/ml proteinase K, and incubated at 42 °C for 1 h, then 65 °C for 1 h to reverse crosslinks. Fractions were then extracted with an equal volume of phenol–chloroform–isoamyl alcohol (25:24:1), and precipitated with an equal volume of isopropyl alcohol. The RNA pellets recovered were washed twice in 70 % ethanol, and equal proportions of each fraction were analyzed by Northern blot. Five microgram each of total RNA and crude lysate were loaded alongside RNA recovered from the sucrose density gradient. Membranes were probed for IME1 and ENO1 as described above, and imaged on a Typhoon Phosphor Imager (GE Healthcare).
Northern blotting and qRT-PCR
qRT-PCR primers used in this study
GCC GCT GCT GAA AAG AAT GT
TGG AGA GGT CTT GGA CTT AGA CAA
AAT GTT TTG GGT GAT GCC TCT T
TTC TTG GAG TAA AAT CTG GCA TTG
TTC GCG AAG GAG CAT AAT GC
ACA CTT CCA ATT CAT TCA GAA TCG
CCA AGA CCT TTA CCG TCG ATG T
AGG AAG CTG GGT CGA AGA C
GAG TCT TCG ATC CGG CTT CA
TTC CTA CGG CAC CAT CTA CTT TAA T
25S:18S rRNA ratios were determined for logarithmic cultures or 9 h following the shift to SPM. Total RNA dilutions (2, 1, or 0.5 μg) from each condition were analyzed by Northern blot probing with the Y503 and Y500 rRNA probes as described . The signals were quantitated by phosphorimager and the 25S:18S ratios calculated for each condition. Ratios were averaged for each condition and normalized to the wild-type rich growth (YPD) value. The relative error was propagated from the standard deviation obtained from each averaged ratio.
Rpl22 is a non-essential ribosomal protein
To examine this phenotype further, wild type, rpl22A∆, rpl22B∆ and rpl22∆ double mutants were streaked on rich growth medium and incubated at the intermediate temperature of 19 °C for 4 days. The wild type and rpl22B∆ strains grew similarly while the rpl22A∆ and double mutant failed to form colonies (Fig. 1b, left panel). To determine whether these strains were growth arrested or lost viability, the same plates were then placed at 25 °C for 2 days and images obtained. In this experiment, growth was observed for the rpl22A∆ and double mutant (right panel) indicating that a mild cold shock is sufficient to arrest cell division but not kill the cell while a more severe reduction in temperature results in cell death.
The phenotypic differences observed between rpl22A∆ and rpl22B∆ mutants may be explained by the finding that RPL22A is transcribed at much higher levels than RPL22B . Another contributing factor could also be the regulation of one paralog by the other . Therefore, we examined mRNA levels of each gene in logarithmic cultures by qRT-PCR. RPL22A and RPL22B mRNA levels were first standardized to control transcripts (NUP85 and ENO1). Our control genes were expressed at relatively comparable levels, regardless of the gene deletion (Fig. 1c). In the absence of RPL22A, RPL22B expression increased fourfold while deletion of RPL22B did not affect RPL22A mRNA levels. These results indicate that loss of one RPL22 allele does not adversely impact the activity of the other. These findings are consistent with the model that elevated Rpl22A expression levels represent a major cause for our observed phenotypic differences.
Rpl22 is required for invasive and pseudohyphal growth
As described above, Rpl22 regulates metazoan cell differentiation pathways. Similarly, yeast exhibit several alternative cell fates controlled by cell type and environmental cues. A nitrogen-diminished environment triggers changes in cell cycle and cell shape in haploid or diploid cells termed invasive or pseudohyphal growth, respectively . Therefore, we investigated a role for RPL22 in these processes. First, we determined whether Rpl22A and/or Rpl22B were required for invasive growth. Haploid wild-type, rpl22AΔ, rpl22BΔ, and rpl22Δ double mutant cells were grown on rich solid medium then the plate was washed with water. Cells embedded in the agar due to invasive growth will be resistant to washing. Cells lacking RPL22A or both paralogs were unable to significantly penetrate the agar, while wild type and rpl22BΔ cells were embedded (Fig. 1d). These results indicate that Rpl22A is required for invasive growth. Next, we tested the requirement of Rpl22 for pseudohyphal growth by generating diploid strains lacking both RPL22 paralogs in the SK1 strain background. This background was chosen as it exhibits a robust pseudohyphal growth phenotype. Wild type and rpl22Δ double mutant cells were streaked on SLAD plates which contain limiting nitrogen, incubated at 30 °C for 4 days. Microscopic examination revealed a radial growth from the center of in wild-type colonies. Conversely, no rpl22Δ cells exhibited this phenotype (500 cells examined, Fig. 1e). These results indicate that Rpl22 plays a second role in the switch from budding to hyphal forms of cell division. Taken together, these results indicate that Rpl22 plays an important role in the morphogenic switches that respond to a reduced nitrogen environment.
Rpl22 is required for the execution of meiotic development
To determine if this meiotic role for Rpl22 was a general property of non-essential ribosomal proteins, two additional large subunit proteins, Rpl39 and Rpl29, were analyzed. Rpl29 localizes to the 40S interaction face and is required for efficient subunit association . Conversely, Rpl39 resides near the peptide exit channel . Finally, these nonessential RP genes were chosen as their deletion reduced growth rates to a level similar to rpl22A∆ mutants . Unlike RPL22, RPL39 and RPL29 are not duplicated so only single rpl39∆ or rpl29Δ diploids were constructed. Their ability to undergo meiosis was assessed as just described. These experiments revealed a modest but significant (p = 0.03, n = 3–5) difference between wild type and the rpl39∆ diploid while no difference was observed in rpl29∆ cells (Fig. 2b). However, the eightfold difference between rpl39∆ and rpl22∆ mutant culture meiotic efficiencies indicates that a severe meiotic defect is not a general feature of deleting non-essential ribosomal genes.
Rpl22 is required for IME1 mRNA translation
Early meiotic gene induction requires the destruction of the transcriptional repressor Ume6 [6, 36]. The lack of IME2 transcript accumulation suggested that the Ume6 repressor is not destroyed in the rpl22∆ mutant. To test this model, wild type and rpl22∆ double mutant diploids were subjected to a meiotic timecourse experiment and endogenous Ume6 levels were monitored by Western blot analysis of total protein extracts. As observed previously , Ume6 levels are reduced below the limits of detection in the wild-type culture shortly after transfer to SPM (Fig. 3b). However, Ume6 levels remained constant in the rpl22∆ mutant strain until late in the timecourse. These results indicate that Rpl22 is required for Ume6 destruction and subsequent meiotic progression.
We previously reported that Ime1 association is required for the APC/CCdc20 ubiquitin ligase-directed proteolysis of Ume6 [6, 36]. Therefore, we next examined Ime1 levels during meiosis in a wild type and rpl22∆ double mutant strains. The IME1 allele was chromosomally tagged with three copies of the hemagglutinin (3HA) epitope to allow Ime1 detection by Western blot analysis. Sporulation kinetics and efficiency were indistinguishable between the wild-type strain expressing Ime1 or Ime1-3HA indicating that the tagged allele is functional (data not shown). In the wild-type strain, the Ime1-3HA signal was detected by 3 h following transfer to SPM with peak expression occurring at 9 h (Fig. 3c). In the rpl22∆ cells, Ime1-3HA was detected at 3 h but its levels remained flat throughout the timecourse and did not exhibit a spike in expression. These results suggest that Rpl22 is required for normal Ime1 accumulation, which in turn leads to Ume6 destruction and meiotic progression.
Rpl22 is required for efficient IME1 mRNA translation
Our results indicate that Rpl22 does not control IME1 transcription or Ime1 protein stability. These findings point to translation as a potential explanation for reduced Ime1 accumulation in the rpl22∆ strains. Translation defects can be due to failure of the mRNA to successfully exit the nucleus and associate with the ribosome or defects in the translation process itself. To test these possibilities, we probed for the presence of IME1 mRNA in ribosome fractions. Cultures taken 9 h following transfer to SPM and treated with a combination of CHX and formaldehyde to stall and crosslink ribosomes to mRNA. This arrest protocol was employed as we discovered that conventional CHX translation arrest resulted in severe IME1 mRNA degradation (data not shown). Total RNA isolated from these polysomes was subjected to Northern blot analysis probing for IME1 mRNA. These studies revealed that IME1 mRNA was still degraded in both wild type and rpl22∆ diploids when compared to other control transcripts (ENO1 or rRNA). However, IME1 mRNA was still detected in both wild type and rpl22∆ mutant polysome fractions although the levels appeared reduced in the mutant fractions (Fig. 4c). Quantitating the mRNA samples did reveal that IME1 mRNA levels were reduced approximately 40 % in the rpl22∆ sample in this experiment (Fig. 4d). Taking this result into consideration, this experiment indicates that IME1 mRNA is associated with polysomes in meiotic rpl22∆ cells. These findings suggest that Rpl22 functions following ribosome binding but prior to translation initiation (see “Discussion” section). In addition, these results suggest that IME1 mRNA maybe specifically targeted for degradation on stalled ribosomes.
Rpl22 operates through the IME1 5′UTR to promote translation
Next, we determined whether relieving the 5′UTR block to translation was sufficient to restore Ime1 function and complete meiosis in the rpl22∆ mutant. First, as an indicator of Ume6 destruction, IME2 mRNA expression was compared in an rpl22∆ double mutant diploid harboring either wild type IME1 or the 5′UTR∆-IME1 deletion allele. Total RNA was prepared from a meiotic timecourse experiment and IME2 mRNA concentrations were determined by qRT-PCR. As previously described, IME2 mRNA was not induced in rpl22Δ cells containing the intact IME1 5′UTR (hatched box, Fig. 5c). Although delayed by a timepoint, IME2 mRNA was induced to levels higher in the rpl22∆ 5′UTR∆-IME1 mutant (black box) compared to rpl22∆ IME1 cells. These results indicate that the increase in Ime1 observed in the 5′UTR∆-IME1 strain was sufficient to induce Ume6 destruction and subsequent IME2 transcription but not quite to wild-type levels. Interestingly, although Ime1 levels were induced early and stayed elevated in the rpl22∆ 5′UTR∆-IME1 cells, the kinetics of IME2 mRNA accumulation were slower than wild type. These results suggest that Rpl22 has a role in IME2 mRNA induction in addition to IME1 mRNA translation.
We next tested whether deletion of the 5′UTR could rescue the rpl22Δ sporulation phenotype. In the RPL22 strain, the presence of the 5′UTR∆ allele did not alter sporulation efficiency compared to the intact IME1 allele (Fig. 5d) indicating that the differences in Ime1 expression kinetics do not affect the efficiency of meiotic divisions. In the rpl22∆ double mutant, the presence of 5′UTR∆-IME1 allowed a significant increase in sporulation efficiency compared to rpl22∆ cells harboring wild type IME1. These results indicate that deleting the 5′UTR can bypass the meiotic defect in a rpl22∆ double mutant. However, the rescue was not to the levels observed in wild type cells. This observation, combined with the IME2 mRNA analysis in the rpl22∆ 5′UTR∆-IME1 strain, suggests that Rpl22 has additional execution points later in meiosis (see “Discussion” section).
Rpl22 is required for normal polysome assembly
We hypothesized that defects in polysome formation may explain the observed lack of Ime1 accumulation. Therefore, we performed polysome analysis on wild-type and rpl22Δ cells 12 h after shifting to sporulation medium (Fig. 6e). Interestingly, meiotic rpl22Δ cells did not exhibit the halfmer phenotype as the free 40S and 60S subunit peaks in both sporulating wild type or rpl22Δ cells were largely absent. One possibility is that idle ribosomal subunits are catabolized upon entry into meiosis, relieving “halfmer” formation in polysome peaks. Since normal profiles were obtained from meiotic rpl22∆ cells, these results suggest that the halfmer formation and the failure to translate IME1 mRNA represent separate phenotypes associated with loss of Rpl22 function.
Changes in cell fate require remodeling the gene expression program at the level of both transcription and translation [12, 40]. Although transcriptional control has been the focus of extensive study, it is becoming increasing clear that regulated translation also mediates these decisions. In this report, we demonstrate that the non-essential large subunit ribosomal protein Rpl22 is required for the developmental switch from normal mitotic cell division to either invasive/pseudohyphal growth or meiotic entry. As meiosis and hyphal growth are induced under conditions of limiting or depleted environmental nitrogen, Rpl22 may represent a mediator of low-nitrogen dependent translation. To promote meiotic induction, Rpl22 is necessary for efficient translation of the meiotic inducer IME1 mRNA. Importantly, the requirement of Rpl22 for IME1 mRNA translation can be suppressed by deleting the unusually long IME1 5′UTR. Formally, these results indicate that Rpl22 operates through this region. However, only partial restoration of sporulation efficiency was observed in the rpl22∆ mutant expressing IME1 lacking the 5′UTR suggesting that additional execution points for Rpl22 exist during meiosis. Taken together, these results suggest that Rpl22 is the target of a late nitrogen checkpoint. Once this checkpoint is satisfied, Rpl22 is activated allowing efficient IME1 mRNA translation by overriding 5′UTR-mediated inhibition.
In metazoans, L22 is involved in B- and T-cell differentiation and suppressing T-cell transformation [41–43], reviewed in . Based on these reports and results described here, the regulation and function of yeast and vertebrate Rpl22 share both similarities and differences. Neither yeast nor vertebrate Rpl22 are required for bulk translation while both control cell differentiation events. However, unlike vertebrate Rpl22 and Rpl22-like1 that exhibit both overlapping and antagonistic activities, the yeast Rpl22 paralogs have similar functions with Rpl22A being more active. These results are most likely explained by the higher RPL22A expression levels compared to RPL22B . In mice and zebrafish, Rpl22 antagonizes the expression of Rpl22-like1  while deleting RPL22A results in increased RPL22B transcription. Finally, vertebrate Rpl22 controls developmental process through translation independent mechanisms. In yeast, although a non-translational role for Rpl22A has been reported that helps target specific mRNAs to the bud , we find that Rpl22 mediates meiotic entry by translational control of IME1.
Gene transcription is regulated by signaling networks responding to both intrinsic and extrinsic stimuli . Similarly, IME1 transcription is controlled by the PKA and TOR signaling pathways that monitor the nutritional status of the cell [45–48]. IME1 transcription displays three regulatory states namely off, low and high-level expression . These states indicate cellular conditions of mitotic cell division, conditions permissive to enter meiosis or meiotic induction itself, respectively. We find a similar tiered structure for IME1 mRNA translation as well. Cells growing in the absence of glucose but still sensing nitrogen fail to translate IME1 mRNA although the transcript is present (compare 0 h timepoints, Figs. 3, 5). Transfer to sporulation medium induces a low level of IME1 mRNA translation (3–6 h, Fig. 5). Ime1 levels then elevate (9–12 h) to a threshold sufficient to induce Ume6 destruction and subsequent induction of early meiotic genes such as IME2. However, this induction step requires Rpl22 placing specialized translation into the meiotic induction pathway. These results suggest a model that the nitrogen signal inhibits Rpl22 function thus preventing Ime1 accumulation to a level sufficient to induce meiosis (Fig. 7b). Only when the nitrogen signal is completely removed does Rpl22 become fully functional. Consistent with this model, four phosphorylation sites on Rpl22 have been mapped including potential MAPK/Cdk and caseine kinase recognition sites. This step may provide the cell another safeguard to insure that conditions are correct to enter meiosis. It has been previously described that the small subunit and translation initiation factors receive signals that control the initiation process [50–52]. Our results suggest that the large ribosomal subunit is also a recipient of such signals, allowing increased translational efficiency of developmental mRNAs.
Our results indicate that Rpl22 mediates IME1 mRNA translation through its large 5′UTR. This result is consistent with previous studies that identified this region as important for meiotic translation [10, 11]. The 5′UTR is an important regulatory element in the translation of developmentally regulated loci such as the HOX genes in vertebrates [53, 54]. In the case of HOXa5 and HOXa9, the 5′UTR utilizes an internal ribosome entry site (IRES) through an Rpl38-dependent mechanism. In addition to meiosis, we demonstrate that Rpl22 is required for both invasive and pseudohyphal growth. Two genes required for these processes (FLO8, FLO11) also possess long 5′UTRs that utilize IRES elements for translation . These observations reveal a possible role for Rpl22 in IRES utilization. This possibility is supported by the finding that Rpl22 enhances IRES mediated translation in the hepatitis C virus 3′UTR .
Another mechanism by which the 5′UTR restricts translation is through the presence of short upstream open reading frames (uORFs) . Scanning 40S subunits recognize these uORFs and initiate translation only to terminate the process after a short peptide is generated [15, 57]. The IME1 5′UTR does not contain any uORFs with the canonical AUG start codon. However, a previous study identified ribosomal pausing sites within meiotic 5′UTRs that contain proposed non-canonical sites (e.g., CUG, AUU, GUG) . Examination of the IME1 5′ UTR revealed several of these sequences suggesting the possibility that IME1 mRNA translation may be regulated by ribosome pausing. This possibility is supported by the finding that ribosomal pausing is observed at the 5′UTR in IME1 mRNA early in development but is lost as cells progress through meiosis , see Fig. 7b). As would be predicted, ribosome release from the 5′ UTR occurs coincident with Ime1 protein appearance and the Rpl22 execution point. This model is consistent with our results revealing an initial low-level accumulation of Ime1 followed by a rapid elevation in protein concentration that is dependent on Rpl22.
Rpl22 is a conserved component of the eukaryotic ribosome that carries out specialized functions in many organisms. We find that Rpl22 is required for adopting hyphal growth characteristics and meiotic entry in S. cerevisiae. The latter role is due to Rpl22-dependent translation of the meiotic inducer IME1 mRNA.
upstream open reading frame
internal ribosome entry sites
quantitative reverse transcriptase-polymerase chain reaction
yeast extract peptone acetate
yeast extract peptone dextrose
avian myeloblastosis virus-reverse transcriptase
synthetic, low-ammonia, dextrose
SJK carried out the meiotic mRNA expression experiments, ribosome polysome profiling, analysis of hyphal growth studies, analyzed the data and drafted the manuscript. RS conceived the study, performed phenotypic studies of rpl22∆ null alleles, analyzed the data and edited the manuscript. Both authors read and approved the final manuscript.
We thank Dr. Dmitri Pestov and Dr. Katrina F. Cooper for helpful discussions with this work. We also thank Dr. Dmitri Pestov and Dr. Natalia Shcherbik for expert help with the polysome profiling analysis.
The authors declare that they have no competing interests.
Availability of data and materials
All novel reagents described in this manuscript will be freely available to academic scientists.
This work was supported by a grant from the National Institutes of Health (GM086788) to R.S.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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