Evolution of RPS4Y

Sequence variation within RPS4Y, a ribosomal protein gene located in the nonpseudoautosomal region of the Y chromosome, was used to elucidate the origin of this gene in primates. Complete coding and additional flanking sequences (949 bp) of the RPS4Y locus were determined in four nonhuman primate species. Phylogenetic reconstruction of RPS4 sequence evolution supports the monophyly of mammalian RPS4 and RPS4Y. Molecular evolutionary rate estimation reveals significantly elevated rates of DNA and protein evolution in RPS4Y compared with its X-chromosome homologs. These rates enable us to estimate the timing of the transposition of RPS4X to the Y chromosome (95% confidence interval, 32 MYA-74 MYA), and this estimate was verified by Southern hybridization analysis of prosimian and simian genomic DNA. These data support a transposition event of ancestral primate RPS4X to the Y chromosome prior to the divergence of Prosimii.     

The yeast ribosomal protein S7 and its genes.

Ribosomal protein S7 of Saccharomyces cerevisiae is encoded by two genes RPS7A and RPS7B. The sequence of each copy was determined; their coding regions differ in only 14 nucleotides, none of which leads to changes in the amino acid sequence. The predicted protein consists of 261 amino acids, making it the largest protein of the 40 S ribosomal subunit. It is highly basic near the NH2 terminus, as are most ribosomal proteins. Protein S7 is homologous to both human and rat ribosomal protein S4. RPS7A and RPS7B contain introns of 257 and 269 nucleotides, respectively, located 11 nucleotides beyond the initiator AUG. The splicing of the introns is efficient. Either RPS7A or RPS7B will support growth. However, deletion of both genes is lethal. RPS7A maps distal to CDC11 on chromosome X, and RPS7B maps distal to CUP1 on chromosome VIII.     

Yar1 protects the ribosomal protein Rps3 from aggregation

2000 ribosomes have to be synthesized in yeast every minute. Therefore the fast production of ribosomal proteins, their efficient delivery to the nucleus and correct incorporation into ribosomal subunits are prerequisites for optimal growth rates. Here, we report that the ankyrin repeat protein Yar1 directly interacts with the small ribosomal subunit protein Rps3 and accompanies newly synthesized Rps3 from the cytoplasm into the nucleus where Rps3 is assembled into pre-ribosomal subunits. A yar1 deletion strain displays a similar phenotype as an rps3 mutant strain, showing an accumulation of 20S pre-rRNA and a 40S export defect. The combination of an rps3 mutation with a yar1 deletion leads to an enhancement of these phenotypes, while increased expression of RPS3 suppresses the defects of a yar1 deletion strain. We further show that Yar1 protects Rps3 from aggregation in vitro and increases its solubility in vivo. Our data suggest that Yar1 is a specific chaperone for Rps3, which serves to keep Rps3 soluble until its incorporation into the pre-ribosome.     

Ribosomal proteins Rps0 and Rps21 of Saccharomyces cerevisiae have overlapping functions in the maturation of the 3' end of 18S rRNA

The Rps0 proteins of Saccharomyces cerevisiae are components of the 40S ribosomal subunit required for maturation of the 3′ end of 18S rRNA. Drosophila and human homologs of the Rps0 proteins physically interact with Rps21 proteins, and decreased expression of both proteins in Drosophila impairs control of cellular proliferation in hematopoietic organs during larval development. Here, we characterize the yeast RPS21A/B genes and show that strains where both genes are disrupted are not viable. Relative to the wild type, cells with disrupted RPS21A or RPS21B genes exhibit a reduction in growth rate, a decrease in free 40S subunits, an increase in the amount of free 60S subunits, and a decrease in polysome size. Ribosomal RNA processing studies reveal RPS21 and RPS0 mutants have virtually identical processing defects. The pattern of processing defects observed in RPS0 and RPS21 mutants is not a general characteristic of strains with suboptimal levels of small subunit ribosomal proteins, since disruption of the RPS18A or RPS18B genes results in related but distinct processing defects. Together, these data link the Rps0 and Rps21 proteins together functionally in promoting maturation of the 3′ end of 18S rRNA and formation of active 40S ribosomal subunits.     

Ullrich-Turner syndrome is not caused by haploinsufficiency of RPS4X

Ullrich-Turner syndrome (UTS) is frequently associated with monosomy X but may also occur with structural aberrations of the X and the Y chromosomes. It has been hypothesized that the ribosomal protein genes RPS4X and RPS4Y play a critical role in the prevention of UTS. Individual patients with a 46,X,i(Xq) karyotype cannot be differentiated phenotypically from 45,X UTS patients and carry three gene copies of RPS4X. Since haploinsufficiency of one or several gene(s) is thought to cause the UTS phenotype, direct assessment of RPS4X expression levels in these patients should establish whether RPS4X is involved in UTS. We have investigated fibroblasts of four 46,X,i(Xq) UTS patients with typical symptoms and a non-mosaic chromosome complement, and have found significantly increased RPS4X mRNA levels in all patients. Based on our results, we conclude that haploinsufficiency of RPS4X is not the cause of UTS.     

Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7^Mtu and Rps7^Zma) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.     

Analysis of RPS15aE,an Isoform of a Plant-Specific Evolutionarily Distinct Ribosomal Protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>, Reveals its Potential Role as a Growth Regulator

There is increasing evidence for ribosome heterogeneity in biological systems. In Arabidopsis thaliana, the ribosomal protein S15a is encoded by six separate genes, which fall into two evolutionarily distinct categories (Type I and Type II). Type I S15a is a universally conserved component of cytosolic ribosomes, whereas there is ambiguity as to the specific subcellular location of Type II S15a (cytosolic and/or mitochondrial ribosomes). In this study, we investigated the functional significance of the distinct form of ribosomal protein S15a (Type II) in Arabidopsis by examining: the evolutionary relationship of eukaryotic S15a proteins with respect to organellar homologs, the expression of individual Type II S15a genes during various developmental stages by RT-PCR, and the phenotypes of an insertional mutation into the RPS15aE gene. The Type II S15a proteins are plant specific, and the duplication event that gave rise to the Type II S15a genes appears to have occurred during the evolution of land plants. The genes encoding Type II S15a in Arabidopsis are differentially expressed, and mutant plants in which the gene encoding S15aE is knocked down produce larger leaves, longer roots, and possess larger cells than wild-type plants suggesting that the RPS15aE isoform of Type II S15a may act as a regulator of translational activity. Our results add significantly to the understanding of the protein constitution of plant ribosomes and the functional significance of ribosome heterogeneity.     

Alterations in Ribosomal Protein Rps28 Can Diversely Affect Translational Accuracy in Saccharomyces Cerevisiae

Three small-subunit ribosomal proteins shown to influence translational accuracy in Saccharomyces cerevisiae are conserved in structure and function with their procaryotic counterparts. One of these, encoded by RPS28A and RPS28B (RPS28), is comparable to bacterial S12. The others, encoded by sup44 (RPS4) or, sup46 and YS11A (RPS13), are homologues of procaryotic S5 and S4, respectively. In Escherichia coli, certain alterations in S12 cause hyperaccurate translation or antibiotic resistance that can be counteracted by other changes in S5 or S4 that reduce translational accuracy. Using site-directed and random mutagenesis, we show that different changes in RPS28 can have diametrical influences on translational accuracy or antibiotic sensitivity in yeast. Certain substitutions in the amino-terminal portion of the protein, which is diverged from the procaryotic homologues, cause varying levels of nonsense suppression or antibiotic sensitivity. Other alterations, found in the more conserved carboxyl-terminal portion, counteract SUP44- or SUP46-associated antibiotic sensitivity, mimicking E. coli results. Although mutations in these different parts of RPS28 have opposite affects on translational accuracy or antibiotic sensitivity, additive phenotypes can be observed when opposing mutations are combined in the same protein.     

Increased expression of ribosomal protein S2 in liver tumors,posthepactomized livers,and proliferating hepatocytes in vitro

The ribosomal protein S2 (RPS2) is encoded by a gene from the highly conserved mammalian repetitive gene family LLRep3. It participates in aminoacyl-transfer RNA binding to ribosome, potentially affecting the fidelity of mRNA translation. These studies were designed to measure the expression of RPS2 during increased cell proliferation. Using Western and Northern blot analyses, we found that the levels of RPS2 protein and its corresponding mRNA were higher in mouse hepatocellular carcinoma, in mouse livers after one-third partial hepatectomy, and in serum-starved cultured hepatocytes following serum treatment. Our study shows that the increased expression of RPS2 correlates with increased cell proliferation. However, whether the altered expression of this protein reflects its involvement in cellular proliferation or represents an associated phenomena is still a key question that needs to be explored.     

Targeted disruption of the ribosomal protein S19 gene is lethal prior to implantation

The ribosomal protein S19 (RPS19) is located in the small (40S) subunit and is one of 79 ribosomal proteins. The gene encoding RPS19 is mutated in approximately 25% of patients with Diamond-Blackfan anemia, which is a rare congenital erythroblastopenia. Affected individuals present with decreased numbers or the absence of erythroid precursors in the bone marrow, and associated malformations of various organs are common. We produced C57BL/6J mice with a targeted disruption of murine Rps19 to study its role in erythropoiesis and development. Mice homozygous for the disrupted Rps19 were not identified as early as the blastocyst stage, indicating a lethal effect. In contrast, mice heterozygous for the disrupted Rps19 allele have normal growth and organ development, including that of the hematopoietic system. Our findings indicate that zygotes which are Rps19(-/-) do not form blastocysts, whereas one normal Rps19 allele in C57BL/6J mice is sufficient to maintain normal ribosomal and possibly extraribosomal functions.     

Specific Role for Yeast Homologs of the Diamond Blackfan Anemia-associated Rps19 Protein in Ribosome Synthesis

Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3'-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells under-expressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemia-associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis.     

The histone-like C-terminal extension in ribosomal protein S6 in Aedes and Anopheles mosquitoes is encoded within the distal portion of exon 3

In eukaryotic cells, ribosomal protein S6 (RPS6) is the major phosphorylated protein on the small ribosomal subunit. In the mosquitoes Aedes aegypti and Aedes albopictus, the cDNA encoding RPS6 contains 300 additional nucleotides, relative to the Drosophila homolog. The additional sequence encodes a 100-amino acid, lysine-rich C-terminal extension of the RPS6 protein with 42-49% identity to histone H1 proteins from the chicken and other multicellular organisms. Using mass spectrometry we now show that the C-terminal extension predicted by the cDNA is present on RPS6 protein isolated from ribosomal subunits purified from Ae. albopictus cells. To expand our analysis beyond the genus Aedes, we cloned the rpS6 cDNA from an Anopheles stephensi mosquito cell line. The cDNA also encoded a lysine-rich C-terminal extension. However, in An. stephensi rpS6 the extension was approximately 70 amino acids longer than that in Ae. albopictus, and at the nucleotide level, it most closely resembled histone H1 proteins from the unicellular eukaryotes Leishmania and Chlamydomonas, and the bacterium Bordetella pertussis. To examine how the histone-like C-terminal extension is encoded in the genome, we used PCR-based approaches to obtain the genomic DNA sequence encoding Ae. aegypti and Ae. albopictus rpS6. The sequence encoding the histone-like C-terminal extension was contiguous with upstream coding sequence within a single open reading frame in Exon 3, indicating that the lysine-rich extension in mosquito RPS6 is not the result of an aberrant splicing event. An in silico investigation of the Anopheles gambiae genome based on the cDNA sequence from An. stephensi allowed us to map the An. gambiae gene to chromosome 2R, to deduce its exon-intron organization, and to confirm that Exon 3 encodes a C-terminal histone-like extension. Because the C-terminal extension is absent from Drosophila melanogaster, we examined a partial cDNA clone from a Psychodid fly, which shares a relatively recent common ancestor with the mosquitoes. The absence of the C-terminal extension in the Psychodid rpS6 cDNA suggests that the unusual RPS6 structure is restricted to a relatively small group of flies in the Nematocera.     

Amino acid residues in the PSI domain and cysteine-rich repeats of the integrin beta2 subunit that restrain activation of the integrin alpha(X)beta(2)

The leukocyte integrin alpha(X)beta(2) (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. alpha(X)beta(2) is more resistant to activation than other beta(2) integrins and is inactive in transfected cells. However, when human alpha(X) is paired with chicken or mouse beta(2), alpha(X)beta(2) is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human alpha(X)/human beta(2) integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding.