Disrupted retinal development in the embryonic belly spot and tail mutant mouse

The Belly spot and tail (Bst) semidominant mutation, mapped to mouse Chromosome 16, leads to developmental defects of the eye, skeleton, and coat pigmentation. In the eye, the mutant phenotype is characterized by the presence of retinal colobomas, a paucity of retinal ganglion cells, and axon misrouting. The severity of defects in the Bst/+ retina is variable among individuals and is often asymmetric. In order to determine the role of the Bst locus during retinal morphogenesis, we searched for the earliest observable defects in the developing eye. We examined the retinas of Bst/+ and +/+ littermates from embryonic day 9.5 (E9.5) through E13.5 and measured retinal size, cell density, cell death, mitotic index, and cell birth index. We have found that development of the Bst/+ retina is notably dilatory by as early as E10.5. The affected retinas are smaller than their wildtype counterparts, and optic fissure fusion is delayed. In the mutant, there is a marked lag in the exit of retinal cells from the mitotic cycle, even though there are no observable differences in the rate of cellular proliferation or cell death between the two groups. We hypothesize that Bst regulates retinal cell differentiation and that variability of structural defects in the mutant, such as those affecting optic fissure fusion, is a reflection of the extent of developmental delay brought about by the Bst mutation.     

The p53 Tumor Suppressor Causes Congenital Malformations in Rpl24-Deficient Mice and Promotes Their Survival

Hypomorphic mutation in one allele of ribosomal protein l24 gene (Rpl24) is responsible for the Belly Spot and Tail (Bst) mouse, which suffers from defects of the eye, skeleton, and coat pigmentation. It has been hypothesized that these pathological manifestations result exclusively from faulty protein synthesis. We demonstrate here that upregulation of the p53 tumor suppressor during the restricted period of embryonic development significantly contributes to the Bst phenotype. However, in the absence of p53 a large majority of Rpl24^Bst/+ embryos die. We showed that p53 promotes survival of these mice via p21-dependent mechanism. Our results imply that activation of a p53-dependent checkpoint mechanism in response to various ribosomal protein deficiencies might also play a role in the pathogenesis of congenital malformations in humans.Nascent ribosome biogenesis is required during cell growth, proliferation and differentiation (42, 47). It is temporally and spatially organized within the nucleolus, where rRNAs are transcribed, processed, modified, and assembled with ribosomal proteins (RPS) to generate the mature 40S and 60S ribosomal subunits (13). RPS participate in additional steps in ribosome biogenesis in the nucleoplasm and the cytoplasm, such as the transport of ribosomal precursors, stabilization of ribosome structure, and regulation of different steps in protein synthesis (15).The critical role of at least some RPS in mammals is underscored by the pathological or lethal consequences of the deficiency of just one allele. Only a few heterozygous mutations of RP genes have been shown to be viable in mammals, and each of them has shown a relatively specific phenotype. Germ line heterozygous mutation for RPS19, RPS24, RPS17, RPL35a, RPL5, and RPL11 genes have been found in patients with Diamond-Blackfan anemia, which is characterized by absent or decreased erythropoiesis, and less frequently by small stature and various somatic malformations that mostly occur in the cephalic region, as well as an increased incidence of leukemia, osteogenic sarcoma, and myelodisplastic syndrome (7, 9, 12, 17, 18). Recently, a link between heterozygous mutations in Rps19 and Rps20 and dark skin phenotype in mice has been demonstrated (29).Another heterozygous RP mutant is the Belly Spot and Tail (Bst) mouse (34). This is a semidominant, hypomorphic mutation caused by an intronic deletion in the Rpl24 gene, affecting Rpl24 mRNA splicing. Rpl24^Bst/+ mice are characterized by reduced body size, a white ventral middle spot, white hind feet, retinal abnormalities, a kinked tail, and other skeletal abnormalities. Since Rpl24^Bst/+ mouse embryonic fibroblasts from these mice showed a significant reduction in the rate of overall protein synthesis, it has been suggested that their phenotype result exclusively from faulty translation of mRNAs in tissues that depend on rapid and flawless protein synthesis (34).It has been argued that the differential phenotypes of heterozygous mutants of RP genes in mammals might be attributable to the expression levels of the respective RP and the consequent decrease in the amount of ribosomes, impairment of specific steps in protein synthesis, and potential extraribosomal function. However, it should be pointed out that the relative contribution of the impaired protein synthesis or extraribosomal function to these phenotypes remains to be determined (9, 17, 34, 36, 37, 43, 57).Recent evidence indicates that deficiencies in individual RPS could lead to pathological consequences via activation of a p53-dependent checkpoint regulatory mechanism. We demonstrated that inducible deletion of the Rps6 gene in the liver of adult mice inhibits the synthesis of the 40S ribosomal subunit, as well as proliferation of liver cells, after partial hepatectomy, despite seemingly unaffected protein synthesis (54). These observations suggested the existence of a novel checkpoint, downstream of the deficiency in ribosome biogenesis. Likewise, the perigastrulation lethality of Rps6 heterozygote embryos appears to reflect the triggering of a p53-dependent checkpoint response rather than a deficit in protein synthesis (37). We have assumed that activation of a p53-dependent checkpoint is triggered by impaired rRNA processing in the nucleolus in Rps6-deficient cells, since the nucleolar structure and function are compromised by almost all known p53-inducing stresses (37, 41, 50). Based on all of these observations, it could be speculated that the rare occurrence of RP heterozygosity in mammals reflects the fatal consequences of p53-dependent checkpoint activation (36, 37). In addition to function in development, this checkpoint may also play a role in other processes. Since various RP deficiencies in Drosophila melanogaster, zebrafish, and humans pose a great risk for development of malignant tumors, it is possible that induction of a p53-dependent checkpoint response prevents expansion of such potentially hazardous cells (1, 9, 11, 56).Recently, we initiated an RNA interference screen for RP deficiencies that upregulate the p53 tumor suppressor in A549 cells. It has been previously suggested that a defect in ribosome biogenesis in the nucleolus caused by a RP deficiency triggers the p53 response (36, 37, 50). A number of studies in yeast showed that Rpl24 does not participate in ribosome biogenesis in the nucleolus, but it assembles late with the nascent 60S ribosomes in the cytoplasm and regulates the 60S subunit joining step during translation initiation and other steps in protein synthesis (10, 25, 45). Thus, it was surprising to observe that RPL24 deficiency triggered the p53 response in our screen. This observation led us to consider the possibility that p53 is upregulated in Rpl24^Bst/+ mice. In contrast to previous opinion that the phenotype of these mice results exclusively from impaired protein synthesis (34), we demonstrate here that it is largely caused by the aberrant upregulation of p53 protein expression during embryonic development.     

The Ribosomal Protein Rpl22 Controls Ribosome Composition by Directly Repressing Expression of Its Own Paralog,Rpl22l1

Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22^−/− mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22^−/− mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog.     

Developmental arrest of T cells in Rpl22-deficient mice is dependent upon multiple p53 effectors

αβ and γδ lineage T cells are thought to arise from a common CD4(-)CD8(-) progenitor in the thymus. However, the molecular pathways controlling fate selection and maturation of these two lineages remain poorly understood. We demonstrated recently that a ubiquitously expressed ribosomal protein, Rpl22, is selectively required for the development of αβ lineage T cells. Germline ablation of Rpl22 impairs development of αβ lineage, but not γδ lineage, T cells through activation of a p53-dependent checkpoint. In this study, we investigate the downstream effectors used by p53 to impair T cell development. We found that many p53 targets were induced in Rpl22(-/-) thymocytes, including miR-34a, PUMA, p21(waf), Bax, and Noxa. Notably, the proapoptotic factor Bim, while not a direct p53 target, was also strongly induced in Rpl22(-/-) T cells. Gain-of-function analysis indicated that overexpression of miR-34a caused a developmental arrest reminiscent of that induced by p53 in Rpl22-deficient T cells; however, only a few p53 targets alleviated developmental arrest when individually ablated by gene targeting or knockdown. Co-elimination of PUMA and Bim resulted in a nearly complete restoration of development of Rpl22(-/-) thymocytes, indicating that p53-mediated arrest is enforced principally through effects on cell survival. Surprisingly, co-elimination of the primary p53 regulators of cell cycle arrest (p21(waf)) and apoptosis (PUMA) actually abrogated the partial rescue caused by loss of PUMA alone, suggesting that the G1 checkpoint protein p21(waf) facilitates thymocyte development in some contexts.     

Defining the order in which Nmd3p and Rpl10p load onto nascent 60S ribosomal subunits

The large ribosomal subunit protein Rpl10p is required for subunit joining and 60S export in yeast. We have recently shown that Rpl10p as well as the cytoplasmic GTPase Lsg1p are required for releasing the 60S nuclear export adapter Nmd3p from subunits in the cytoplasm. Here, we more directly address the order of Nmd3p and Rpl10p recruitment to the subunit. We show that Nmd3p can bind subunits in the absence of Rpl10p. In addition, we examined the basis of the previously reported dominant negative growth phenotype caused by overexpression of C-terminally truncated Rpl10p and found that these Rpl10p fragments are not incorporated into subunits in the nucleus but instead sequester the WD-repeat protein Sqt1p. Sqt1p is an Rpl10p binding protein that is proposed to facilitate loading of Rpl10p into the 60S subunit. Although Sqt1p normally only transiently binds 60S subunits, the levels of Sqt1p that can be coimmunoprecipitated by the 60S-associated GTPase Lsg1p are significantly increased by a dominant mutation in the Walker A motif of Lsg1p. This mutant Lsg1 protein also leads to increased levels of Sqt1p in complexes that are coimmunoprecipitated with Nmd3p. Furthermore, the dominant LSG1 mutant also traps a mutant Rpl10 protein that does not normally bind stably to the subunit. These results support the idea that Sqt1p loads Rpl10p onto the Nmd3p-bound subunit after export to the cytoplasm and that Rpl10p loading involves the GTPase Lsg1p.     

Eukaryotic cells producing ribosomes deficient in Rpl1 are hypersensitive to defects in the ubiquitin-proteasome system

It has recently become clear that the misassembly of ribosomes in eukaryotic cells can have deleterious effects that go far beyond a simple shortage of ribosomes. In this work we find that cells deficient in ribosomal protein L1 (Rpl1; Rpl10a in mammals) produce ribosomes lacking Rpl1 that are exported to the cytoplasm and that can be incorporated into polyribosomes. The presence of such defective ribosomes leads to slow growth and appears to render the cells hypersensitive to lesions in the ubiquitin-proteasome system. Several genes that were reasonable candidates for degradation of 60S subunits lacking Rpl1 fail to do so, suggesting that key players in the surveillance of ribosomal subunits remain to be found. Interestingly, in spite of rendering the cells hypersensitive to the proteasome inhibitor MG132, shortage of Rpl1 partially suppresses the stress-invoked temporary repression of ribosome synthesis caused by MG132.     

In situ expression of ribosomal protein L21 in developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles at embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to investigate the key regulator genes in odontogenesis. Ribosomal protein L21 (Rpl21) is one of differentially expressed genes in the E12.0 mandible. This study examined the precise expression pattern of Rpl21 mRNA in the mouse mandibular first molar by in situ hybridization. Rpl21 mRNA was expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0. Strong in situ signals were observed in the epithelial bud at E14.0, and in the enamel organ at E15.0. However, either no (E14.0) or only a weak (E15.0) in situ signal was found in the primary enamel knot at these gestational days. Rpl21 was strongly expressed in the inner enamel epithelium, cervical loop and dental lamina from E16.0 to E18.0. In addition, Rpl21 mRNA was also demonstrated in various developing cranio-facial organs. These results suggest that Rpl21 participates in the synthesis of various polypeptides which might be related to the initiation and the development of such tooth germ, and also in the synthesis of enamel components in the presecretory stage of the ameloblast. Rpl21 for protein synthesis might also be related to the morphogenesis of the developing cranio-facial organs.     

Gene structure and chromosomal localization of mouse <Emphasis Type="Italic">Opa1</Emphasis> : its exclusion from the Bst locus

Background  

Autosomal dominant optic atrophy type 1 (DOA) is the most common form of hereditary optic atrophy in human. We have previously identified the OPA1 gene and shown that it was mutated in patients with DOA. OPA1 is a novel member of the dynamin GTPase family that play a role in the distribution of the mitochondrial network. The Bst (belly spot and tail) mutant mice show atrophy of the optic nerves and previous mapping data raise the possibility that Bst and OPA1 are orthologs. In order to analyse the Bst mouse as a model for DOA, we therefore characterized mouse Opa1 and evaluated it as a candidate for the Bst mutant mouse.     

Pbp1 Is Involved in Ccr4- and Khd1-Mediated Regulation of Cell Growth through Association with Ribosomal Proteins Rpl12a and Rpl12b

The Saccharomyces cerevisiae Pbp1 [poly(A)-binding protein (Pab1)-binding protein] is believed to be involved in RNA metabolism and regulation of translation, since Pbp1 regulates a length of poly(A) tail and is involved in stress granule (SG) formation. However, a physiological function of Pbp1 remains unclear, since the pbp1Δ mutation has no obvious effect on cell growth. In this study, we showed that PBP1 genetically interacts with CCR4 and KHD1, which encode a cytoplasmic deadenylase and an RNA-binding protein, respectively. Ccr4 and Khd1 modulate a signal from Rho1 in the cell wall integrity pathway by regulating the expression of RhoGEF and RhoGAP, and the double deletion of CCR4 and KHD1 confers a severe growth defect displaying cell lysis. We found that the pbp1Δ mutation suppressed the growth defect caused by the ccr4Δ khd1Δ mutation. The pbp1Δ mutation also suppressed the growth defect caused by double deletion of POP2, encoding another cytoplasmic deadenylase, and KHD1. Deletion of the gene encoding previously known Pbp1-interacting factor Lsm12, Pbp4, or Mkt1 did not suppress the growth defect of the ccr4Δ khd1Δ mutant, suggesting that Pbp1 acts independently of these factors in this process. We then screened novel Pbp1-interacting factors and found that Pbp1 interacts with ribosomal proteins Rpl12a and Rpl12b. Similarly to the pbp1Δ mutation, the rpl12aΔ and rpl12bΔ mutations also suppressed the growth defect caused by the ccr4Δ khd1Δ mutation. Our results suggest that Pbp1 is involved in the Ccr4- and Khd1-mediated regulation of cell growth through the association with Rpl12a and Rpl12b.     

Toxin-dependent utilization of engineered ribosomal protein L3 limits trichothecene resistance in transgenic plants

The contamination of agricultural products with Fusarium mycotoxins is a problem of world-wide importance. Fusarium graminearum and related species, which are important pathogens of small grain cereals and maize, produce an economically important and structurally diverse class of toxins designated trichothecenes. Trichothecenes inhibit eukaryotic protein synthesis. Therefore, a proposed role for these fungal toxins in plant disease development is to block or delay the expression of defence-related proteins induced by the plant. Using yeast as a model system, we have identified several mutations in the gene encoding ribosomal protein L3 (Rpl3), which confer semi-dominant resistance to trichothecenes. Expression of an engineered tomato RPL3 (LeRPL3) cDNA, into which one of the amino acid changes identified in yeast was introduced, improved the ability of transgenic tobacco plants to adapt to the trichothecene deoxynivalenol (DON), but did not result in constitutive resistance. We show here that, in the presence of wild-type Rpl3 protein, the engineered Rpl3 protein is not utilized, unless yeast transformants or the transgenic plants are challenged with sublethal amounts of toxin. Our data from yeast two-hybrid experiments suggest that affinity for the ribosome assembly factor Rrb1p could be altered by the toxin resistance-conferring mutation. This toxin-dependent utilization of the resistance-conferring Rpl3 protein could seriously limit efforts to utilize the identified target alterations in transgenic crops to increase trichothecene tolerance and Fusarium resistance.     

Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11

PICT1 (also known as GLTSCR2) is considered a tumor suppressor because it stabilizes phosphatase and tensin homolog (PTEN), but individuals with oligodendrogliomas lacking chromosome 19q13, where PICT1 is located, have better prognoses than other oligodendroglioma patients. To clarify the function of PICT1, we generated Pict1-deficient mice and embryonic stem (ES) cells. Pict1 is a nucleolar protein essential for embryogenesis and ES cell survival. Even without DNA damage, Pict1 loss led to p53-dependent arrest of cell cycle phase G(1) and apoptosis. Pict1-deficient cells accumulated p53, owing to impaired Mdm2 function. Pict1 binds Rpl11, and Rpl11 is released from nucleoli in the absence of Pict1. In Pict1-deficient cells, increased binding of Rpl11 to Mdm2 blocks Mdm2-mediated ubiquitination of p53. In human cancer, individuals whose tumors express less PICT1 have better prognoses. When PICT1 is depleted in tumor cells with intact P53 signaling, the cells grow more slowly and accumulate P53. Thus, PICT1 is a potent regulator of the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus.     

Methylation of Ribosomal Protein L42 Regulates Ribosomal Function and Stress-adapted Cell Growth

Lysine methylation is one of the most common protein modifications. Although lysine methylation of histones has been extensively studied and linked to gene regulation, that of non-histone proteins remains incompletely understood. Here, we show a novel regulatory role of ribosomal protein methylation. Using an in vitro methyltransferase assay, we found that Schizosaccharomyces pombe Set13, a SET domain protein encoded by SPAC688.14, specifically methylates lysine 55 of ribosomal protein L42 (Rpl42). Mass spectrometric analysis revealed that endogenous Rpl42 is monomethylated at lysine 55 in wild-type S. pombe cells and that the methylation is lost in Δset13 mutant cells. Δset13 and Rpl42 methylation-deficient mutant S. pombe cells showed higher cycloheximide sensitivity and defects in stress-responsive growth control compared with wild type. Genetic analyses suggested that the abnormal growth phenotype was distinct from the conserved stress-responsive pathway that modulates translation initiation. Furthermore, the Rpl42 methylation-deficient mutant cells showed a reduced ability to survive after entering stationary phase. These results suggest that Rpl42 methylation plays direct roles in ribosomal function and cell proliferation control independently of the general stress-response pathway.     

Ectopic mineralization in the middle ear and chronic otitis media with effusion caused by RPL38 deficiency in the Tail-short (Ts) mouse

Inflammation of the middle ear cavity (otitis media) and the abnormal deposition of bone at the otic capsule are common causes of conductive hearing impairment in children and adults. Although a host of environmental factors can contribute to these conditions, a genetic predisposition has an important role as well. Here, we analyze the Tail-short (Ts) mouse, which harbors a spontaneous semi-dominant mutation that causes skeletal defects and hearing loss. By genetic means, we show that the Ts phenotypes arise from an 18-kb deletion/insertion of the Rpl38 gene, encoding a ribosomal protein of the large subunit. We show that Ts mutants exhibit significantly elevated auditory-brain stem response thresholds and reduced distortion-product otoacoustic emissions, in the presence of normal endocochlear potentials and typical inner ear histology suggestive of a conductive hearing impairment. We locate the cause of the hearing impairment to the middle ear, demonstrating over-ossification at the round window ridge, ectopic deposition of cholesterol crystals in the middle ear cavity, enlarged Eustachian tube, and chronic otitis media with effusion all beginning at around 3 weeks after birth. Using specific antisera, we demonstrate that Rpl38 is an ～8-kDa protein that is predominantly expressed in mature erythrocytes. Finally, using an Rpl38 cDNA transgene, we rescue the Ts phenotypes. Together, these data present a previously uncharacterized combination of interrelated middle ear pathologies and suggest Rpl38 deficiency as a model to dissect the causative relationships between neo-ossification, cholesterol crystal deposition, and Eustachian tubes in the etiology of otitis media.     

Comparative mapping of RPL3, a gene overexpressed in multiple obesity models.

The ribosomal protein 3 gene is differentially expressed in hypothalamus and brown adipose tissue between mouse lines divergently selected for heat loss, and in skeletal muscle of the ob/ob mouse model. Unfortunately, multiple Rpl3-processed pseudogenes have hampered mapping of the functional gene copy in mammalian species. Using PCR amplification with intronic primer binding, we have mapped Rpl3 to MMU15, and have also localized RPL3 to BTA5 in cattle. Comparative mapping implicates a previously mapped copy of RPL3 on HSA22 as the functional copy of human RPL3, while predictive mapping places the porcine homologue on SSC5.     

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

We have shown that Rpl3, a protein of the large ribosomal subunit from baker''s yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-³H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.     

Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.