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Eukaryotic Elongation Factor 2 Kinase Activity Is Required for the Phenotypes of the Rpl24Bst Mouse

Open AccessPublished:July 15, 2022DOI:https://doi.org/10.1016/j.jid.2022.06.019

      Abbreviation:

      eEF2K (eukaryotic elongation factor 2 kinase)
      To The Editor
      The variant murine Rpl24Bst allele reduces gene expression by 40% and results in a white belly spot and kinked tail of variable severity and penetrance (
      • Oliver E.R.
      • Saunders T.L.
      • Tarlé S.A.
      • Glaser T.
      Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse minute.
      ). White belly spots lack melanocytes owing to defects in their motility during development, whereas kinked tails result from fused or wedge-shaped vertebrae (
      • Oliver E.R.
      • Saunders T.L.
      • Tarlé S.A.
      • Glaser T.
      Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse minute.
      ). Rpl24 encodes the protein RPL24, which is incorporated into the ribosome in the cytoplasm. Notably, altered expression of five other ribosomal protein genes—Rps7, Rps21, Rps19, Rps20, and RACK1—result in belly spots (
      • Dinh T.T.H.
      • Iseki H.
      • Mizuno S.
      • Iijima-Mizuno S.
      • Tanimoto Y.
      • Daitoku Y.
      • et al.
      Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway.
      ;
      • McGowan K.A.
      • Li J.Z.
      • Park C.Y.
      • Beaudry V.
      • Tabor H.K.
      • Sabnis A.J.
      • et al.
      Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects.
      ;
      • Volta V.
      • Beugnet A.
      • Gallo S.
      • Magri L.
      • Brina D.
      • Pesce E.
      • et al.
      RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency.
      ;
      • Watkins-Chow D.E.
      • Cooke J.
      • Pidsley R.
      • Edwards A.
      • Slotkin R.
      • Leeds K.E.
      • et al.
      Mutation of the diamond-Blackfan anemia gene Rps7 in mouse results in morphological and neuroanatomical phenotypes [published correction for PLoS Genet 2015;11:e1005682].
      ). Despite this genetic evidence of a link between ribosomal proteins and belly spots, the mechanism(s) behind the phenotype are unknown. The Rpl24Bst mutation reduces protein synthesis in embryonic neural tube cells (
      • Kondrashov N.
      • Pusic A.
      • Stumpf C.R.
      • Shimizu K.
      • Hsieh A.C.
      • Ishijima J.
      • et al.
      Ribosome-mediated specificity in hox mRNA translation and vertebrate tissue patterning.
      ) and models of cancer (e.g., as reported in
      • Knight J.R.P.
      • Vlahov N.
      • Gay D.M.
      • Ridgway R.A.
      • Faller W.J.
      • Proud C.
      • et al.
      Rpl24Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K.
      ), suggesting that reduced protein synthesis underpins the developmental defects. In agreement, reduced RACK1 limited protein synthesis in murine embryonic fibroblasts (
      • Volta V.
      • Beugnet A.
      • Gallo S.
      • Magri L.
      • Brina D.
      • Pesce E.
      • et al.
      RACK1 depletion in a mouse model causes lethality, pigmentation deficits and reduction in protein synthesis efficiency.
      ). However, the other belly-spot variants do not affect protein synthesis (
      • Kondrashov N.
      • Pusic A.
      • Stumpf C.R.
      • Shimizu K.
      • Hsieh A.C.
      • Ishijima J.
      • et al.
      Ribosome-mediated specificity in hox mRNA translation and vertebrate tissue patterning.
      ;
      • Watkins-Chow D.E.
      • Cooke J.
      • Pidsley R.
      • Edwards A.
      • Slotkin R.
      • Leeds K.E.
      • et al.
      Mutation of the diamond-Blackfan anemia gene Rps7 in mouse results in morphological and neuroanatomical phenotypes [published correction for PLoS Genet 2015;11:e1005682].
      ). Thus, to understand the mechanistic link between protein synthesis and belly spots, experiments are required to directly alter protein synthesis rates in variant mice.
      We previously showed that Rpl24Bst mutation suppresses protein synthesis through the activation of eukaryotic elongation factor 2 kinase (eEF2K) (
      • Knight J.R.P.
      • Vlahov N.
      • Gay D.M.
      • Ridgway R.A.
      • Faller W.J.
      • Proud C.
      • et al.
      Rpl24Bst mutation suppresses colorectal cancer by promoting eEF2 phosphorylation via eEF2K.
      ). Suppression of protein synthesis in Rpl24Bst variants is completely reversed by eEF2K inactivation, providing a tool to reverse the protein synthesis defects in Rpl24Bst variants. The Eef2kD273A allele is a germline knockin that dramatically reduces eEF2K catalytic activity throughout the whole mouse from conception (
      • Gildish I.
      • Manor D.
      • David O.
      • Sharma V.
      • Williams D.
      • Agarwala U.
      • et al.
      Impaired associative taste learning and abnormal brain activation in kinase-defective eEF2K mice.
      ). We therefore used the Eef2kD273A allele to assess the influence of eEF2K inactivation on Rpl24Bst phenotypes. To achieve this, belly spot and tail severity were scored using a scale from 0 (normal) to 4 (severe) for mice generated in our previous study (Supplementary Figure S1a). The combined belly-spot and tail score of Rpl24Bst/+ mice has a median of 3 of 8 (Figure 1a). In comparison, the belly-spot and tail score of Rpl24Bst/+ Eef2kD273A/D273A mice was significantly lower at only 1.5. Furthermore, there is a lower incidence of severe phenotypes, with only 7% of Rpl24Bst/+ Eef2kD273A/D273A mice scoring ≥5 compared with 34% of Rpl24Bst/+ mice. Therefore, inactivation of eEF2K suppresses the observable skin and tail phenotypes of the Rpl24Bst mutation. The main contributing factor to this difference is the tail score, with 35% of tails scoring >2 in Rpl24Bst/+ mice compared with 3.6% in Rpl24Bst/+ Eef2kD273A/D273A mice (Supplementary Figure S1b).
      Figure thumbnail gr1
      Figure 1Inactivation of eEF2K reverses the phenotypes of the Rpl24Bst mutation. (a) Quantification of Bst score from mice with Rpl24Bst mutation alone (n = 115 mice) or in combination with Eef2kD273A/D273A (n = 28 mice). Significance was determined by Kolmogorov‒Smirnov test. Right: representative (median Bst score) image of Rpl24Bst/+ and Rpl24Bst/+ Eef2kD273A/D273A mice. (b) Images of transgenic Dct-lacZ mice at E13.5 mice stained for β-galactosidase (blue) within melanocytes. The upper image shows whole embryos, and the lower image shows an expanded view of one forelimb. Bars = 500 μm. Right: quantification of melanocyte migration scored as the distance from torso to furthest melanocyte/length of forelimb. Significance was determined by one-way ANOVA (Tukey’s multiple comparison). Left to right n = 14, 15, 16, and 13. For both graphs, boxes mark the 25th and 75th percentiles, and the central lines mark the median. Each point represents an individual mouse/embryo. Bst, belly-spot and tail; E13.5, embryonic day 13.5; eEF2K, eEF2K, eukaryotic elongation factor 2 kinase.
      Next, we took a complementary approach measuring the effect of the Rpl24Bst and Eef2kD273A mutations on melanocytes using the Dct-lacZ system (
      • Mackenzie M.A.F.
      • Jordan S.A.
      • Budd P.S.
      • Jackson I.J.
      Activation of the receptor tyrosine kinase kit is required for the proliferation of melanoblasts in the mouse embryo.
      ). Genetically engineered Dct-lacZ mice express β-galactosidase from the melanocyte-specific dopachrome tautomerase (Dct) promoter, allowing whole-mount visualization and quantification of melanocyte location. On embryonic day 13.5, melanocytes are transiting the forelimbs of embryos, with the fraction of melanocyte-positive forelimb a metric for changes in melanocyte migration. We observe a significant reduction in melanocyte migration in Rpl24Bst/+ embryos, consistent with their belly spots in adulthood, whereas the inactivation of eEF2K has no effect (Figure 1b). Compared with that in Rpl24Bst/+ embryos, melanocyte migration is significantly reverted in Rpl24Bst/+ Eef2kD273A/D273A embryos (Figure 1b). Thus, eEF2K activity is required for the perturbed melanocyte migration phenotype of Rpl24Bst embryos. Why the reverted migration in Rpl24Bst/+ Eef2kD273A/D273A embryos (Figure 1b) does not correlate with a reversal of adult belly spot phenotype (Supplementary Figure S1b) is unclear. It is possible that migration is reversed at embryonic day 13.5 but subsequently slows to produce a belly spot. Unfortunately, staining of Dct-lacZ is not possible at later embryonic stages owing to reduced skin permeabilization (
      • Mackenzie M.A.F.
      • Jordan S.A.
      • Budd P.S.
      • Jackson I.J.
      Activation of the receptor tyrosine kinase kit is required for the proliferation of melanoblasts in the mouse embryo.
      ).
      We noted that Rpl24Bst/+ and Rpl24Bst/+ Eef2kD273A/D273A mice were weaned at lower than Mendelian frequencies: Rpl24Bst/+ mice at 1 in 3 (32.1%) when expected at 1 in 2 and Rpl24Bst/+ Eef2kD273A/D273A mice even less frequently at 1 in 5 (20.7%) (Figure 2a). Unexpectedly, when we compared the frequency of Rpl24Bst/+ Eef2kD273A/D273A mice with the experimentally determined frequency of Rpl24Bst/+ mice (32.1%), we found this to be significantly different (Figure 2a). Thus, the viability of Rpl24Bst/+ mice is at least in part dependent on eEF2K activity. To analyze this effect further, we calculated the frequencies of the same genotypes in embryonic day 13.5 embryos, finding that the incidence of the Rpl24Bst mutation was close to Mendelian with active or inactive eEF2K (Figure 2b). From this, we conclude that the reduced viability of Rpl24Bst/+ Eef2kD273A/D273A mice occurs between embryonic day 13.5 and weaning. Therefore, despite being responsible for the skin and tail phenotypes of the Rpl24Bst mutation, eEF2K promotes the preweaning survival of Rpl24Bst-variant mice. Embryo viability could not be determined during our analysis. Thus, Rpl24Bst/+ Eef2kD273A/D273A embryos with the greatest reversion in belly-spot phenotype may not survive, potentially explaining the observed discrepancy in melanocyte migration and adult belly spots in this genotype. These data provide substantive evidence of a mechanistic link between RPL24 and eEF2K at an organism level.
      Figure thumbnail gr2
      Figure 2eEF2K is required for preweaning survival of Rpl24Bst/+mice. (a) The percentage of Rpl24Bst/+ mice weaned per litter is plotted in black, with each point representing an individual litter. The box marks the 25th and 75th percentiles, and the central line marks the median incidence of Rpl24Bst mutation. Significance was assessed by chi-square test using the average incidence of Rpl24Bst/+ mice (n = 38 litters) as the expected frequency compared to the actual frequency of Rpl24Bst/+ Eef2kD273A/D273A mice (n = 57 litters). (b) Analysis as in (a) but for embryos at E13.5. For Rpl24Bst/+ embryos n = 5 litters and for Rpl24Bst/+ Eef2kD273A/D273A embryos n = 6. E13.5, embryonic day 13.5; eEF2K, eukaryotic elongation factor 2 kinase.
      Similar to eEF2K inactivation, deletion of p53 in Rpl24Bst variants reversed belly-spot and tail defects while also reducing the ratio of Rpl24Bst/+ mice (
      • Barkić M.
      • Crnomarković S.
      • Grabušić K.
      • Bogetić I.
      • Panić L.
      • Tamarut S.
      • et al.
      The p53 tumor suppressor causes congenital malformations in Rpl24-deficient mice and promotes their survival.
      ). Rpl24Bst/+ p53‒/‒ mice showed reduced embryonic apoptosis and dependence on p21 for viability. Suggesting a shared mechanism, eEF2K activity is required for apoptosis at various stages of development and promotes p21 expression (
      • Chu H.P.
      • Liao Y.
      • Novak J.S.
      • Hu Z.
      • Merkin J.J.
      • Shymkiv Y.
      • et al.
      Germline quality control: eEF2K stands guard to eliminate defective oocytes.
      ;
      • Liao Y.
      • Chu H.P.
      • Hu Z.
      • Merkin J.J.
      • Chen J.
      • Liu Z.
      • et al.
      Paradoxical roles of elongation factor-2 kinase in stem cell survival.
      ). In addition, ribosome biogenesis stress activates both p53 and eEF2K (
      • Gismondi A.
      • Caldarola S.
      • Lisi G.
      • Juli G.
      • Chellini L.
      • Iadevaia V.
      • et al.
      Ribosomal stress activates eEF2K-eEF2 pathway causing translation elongation inhibition and recruitment of terminal oligopyrimidine (TOP) mRNAs on polysomes.
      ;
      • Knight J.R.P.
      • Sbarrato T.
      • Stoneley M.
      • Willis A.E.
      Ribosomes and stress – linked from birth to death.
      ). Interestingly, the belly-spot phenotypes of Rps7, RACK1, Rps19, and Rps20 mutations were also rescued by deletion of p53 (
      • Dinh T.T.H.
      • Iseki H.
      • Mizuno S.
      • Iijima-Mizuno S.
      • Tanimoto Y.
      • Daitoku Y.
      • et al.
      Disruption of entire Cables2 locus leads to embryonic lethality by diminished Rps21 gene expression and enhanced p53 pathway.
      ;
      • McGowan K.A.
      • Li J.Z.
      • Park C.Y.
      • Beaudry V.
      • Tabor H.K.
      • Sabnis A.J.
      • et al.
      Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects.
      ;
      • Watkins-Chow D.E.
      • Cooke J.
      • Pidsley R.
      • Edwards A.
      • Slotkin R.
      • Leeds K.E.
      • et al.
      Mutation of the diamond-Blackfan anemia gene Rps7 in mouse results in morphological and neuroanatomical phenotypes [published correction for PLoS Genet 2015;11:e1005682].
      ). Whether inactivation of eEF2K would also rescue these belly-spot phenotypes should now be determined
      This work extends the relationship between RPL24 and eEF2K to include melanocyte migration, tail deformation, and organism survival. Rpl24Bst/+ mice have been used in cancer studies, to model retinal degeneration, and to study brain development (
      • Herrlinger S.
      • Shao Q.
      • Yang M.
      • Chang Q.
      • Liu Y.
      • Pan X.
      • et al.
      Lin28-mediated temporal promotion of protein synthesis is crucial for neural progenitor cell maintenance and brain development in mice.
      ;
      • Riazifar H.
      • Sun G.
      • Wang X.
      • Rupp A.
      • Vemaraju S.
      • Ross-Cisneros F.N.
      • et al.
      Phenotypic and functional characterization of Bst+/− mouse retina.
      ). This work shows that eEF2K may play a role across these diverse biological settings, with further work merited to investigate its importance.

      Declaration for animal use

      Studies were carried out under license from the UK Home Office (60/4183 and 70/8646). Mice were maintained in open-top cages with a 12-hour light/dark cycle and free access to water and diet. Experiments were initiated on inbred C57BL/6J male and female mice aged between 6 and 12 weeks, without blinding or randomization.

      Data availability statement

      No datasets were generated or analyzed during this study.

      Conflict of Interest

      OJS reports funding unrelated to work in this project from Astra Zeneca, Novartis, RedX, and Cancer Research Horizons. The remaining authors state no conflict of interest.

      Disclaimer

      The funders had no role in study design, data collection, and interpretation or in the decision to submit the work for publication.

      Author Contributions

      Conceptualization: JRPK, OJS; Formal Analysis: Funding Acquisition: GM, TvdH, CMS, AEW, OJS; JRPK; Investigation: JRPK; Methodology: JRPK; Project Administration: JRPK, OJS; Resources: CGP; Supervision: OJS; Writing - Original Draft Preparation: JRPK; Writing - Review and Editing: JRPK, CGP, GM, TvdH, CMS, AEW, OJS

      Acknowledgments

      Funding was provided by Cancer Research UK ( A17196 , A24388 , A21139 , A31287 ) and H2020 European Research Council (311301) to OJS; by the Wellcome Trust (201487) to GRM, TvdH, CMS, OJS, and AEW; and by the National Health and Medical Research Council to CGP.

      Supplementary Materials and Methods

      Materials availability

      All materials are freely available, where legally permitted, on acceptance of a material transfer agreement.

      Belly-spot and tail scoring

      Genotyping was carried out by Transnetyx (Memphis, TN). Scoring was carried out at the endpoint of published experiments that determined sample size, with no power analysis performed for this study. Scoring was performed regardless of the presence of other alleles such as VillinCreER, Apcfl, ApcMin, or KrasG12D because these alleles do not manifest a belly-spot or tail phenotype.

      Dct-lacZ

      Overnight vaginal plugs were designated embryonic day 0.5, and embryos were collected on embryonic day 13.5. Embryo studies were performed blind. Tails were used for genotyping, and the remainder were fixed in ice-cold 0.25% glutaraldehyde (G6257, Sigma-Aldrich, St. Louis, MO) in PBS, on a rotational mixer for 30 minutes at 4 °C. Embryos were washed in cold PBS and permeabilized in two rounds of 2 mM magnesium chloride, 0.1% sodium deoxycholate (D6750, Sigma-Aldrich), and 0.02% NP40 (74385, Sigma-Aldrich) in PBS for a total of 40 minutes at room temperature. Embryos were stained in 2 mM magnesium chloride, 0.1% sodium deoxycholate, 0.02% NP40, 4.7 mM potassium hexacyanoferrate (II) trihydrate (P9387, Sigma-Aldrich), 4.8 mM potassium hexacyanoferrate (III) (P8131, Sigma-Aldrich), and 0.5 mg/ml X-Gal (V394A, Promega, Madison, WI) in PBS at 4 °C on a rotational mixer protected from light for 36 hours. Embryos were washed in PBS, visualized by light microscopy, and stored in formalin. The sample size required for significance was estimated on the basis of pilot studies using G∗Power (
      • Faul F.
      • Erdfelder E.
      • Buchner A.
      • Lang A.-G.
      Statistical power analyses using G∗Power 3.1: tests for correlation and regression analyses.
      ).
      Figure thumbnail fx1
      Supplementary Figure S1The spectrum of belly-spot and tail scores. (a) Examples of mice exhibiting each score on the belly-spot and tail scale. These mice show equivalent scores for each parameter for ease of representation, but equivalent scores between both metrics were not always the case. (b) Graphs showing the belly-spot and the tail scores that comprise the overall belly-spot and tail score shown in a. Rpl24Bst/+ is shown in green; n = 115 mice. Rpl24Bst/+ Eef2kD273A/D273A is shown in blue; n = 28 mice. The box marks the 25th and 75th percentiles, and the central line marks the median. Each point represents an individual mouse. Significance was tested by Kolmogorov‒Smirnov test.

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