Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro

Diamond-Blackfan anemia (DBA) is caused by aberrant ribosomal biogenesis due to ribosomal protein (RP) gene mutations. To develop mechanistic understanding of DBA pathogenesis, we studied CD34⁺ cells from peripheral blood of DBA patients carrying RPL11 and RPS19 ribosomal gene mutations and determined their ability to undergo erythroid differentiation in vitro. RPS19 mutations induced a decrease in proliferation of progenitor cells, but the terminal erythroid differentiation was normal with little or no apoptosis. This phenotype was related to a G₀/G₁ cell cycle arrest associated with activation of the p53 pathway. In marked contrast, RPL11 mutations led to a dramatic decrease in progenitor cell proliferation and a delayed erythroid differentiation with a marked increase in apoptosis and G₀/G₁ cell cycle arrest with activation of p53. Infection of cord blood CD34⁺ cells with specific short hairpin (sh) RNAs against RPS19 or RPL11 recapitulated the two distinct phenotypes in concordance with findings from primary cells. In both cases, the phenotype has been reverted by shRNA p53 knockdown. These results show that p53 pathway activation has an important role in pathogenesis of DBA and can be independent of the RPL11 pathway. These findings shed new insights into the pathogenesis of DBA.     

Ribosomal Protein L5 and L11 Mutations Are Associated with Cleft Palate and Abnormal Thumbs in Diamond-Blackfan Anemia Patients

Diamond-Blackfan anemia (DBA), a congenital bone-marrow-failure syndrome, is characterized by red blood cell aplasia, macrocytic anemia, clinical heterogeneity, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital anomalies that are present in ~30%–50% of patients. The disease has been associated with mutations in four ribosomal protein (RP) genes, RPS19, RPS24, RPS17, and RPL35A, in about 30% of patients. However, the genetic basis of the remaining 70% of cases is still unknown. Here, we report the second known mutation in RPS17 and probable pathogenic mutations in three more RP genes, RPL5, RPL11, and RPS7. In addition, we identified rare variants of unknown significance in three other genes, RPL36, RPS15, and RPS27A. Remarkably, careful review of the clinical data showed that mutations in RPL5 are associated with multiple physical abnormalities, including craniofacial, thumb, and heart anomalies, whereas isolated thumb malformations are predominantly present in patients carrying mutations in RPL11. We also demonstrate that mutations of RPL5, RPL11, or RPS7 in DBA cells is associated with diverse defects in the maturation of ribosomal RNAs in the large or the small ribosomal subunit production pathway, expanding the repertoire of ribosomal RNA processing defects associated with DBA.     

Ribosomal Protein Genes RPS10 and RPS26 Are Commonly Mutated in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart, and urinary system defects that are present in ∼30%–50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.     

SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles

The SRY (sex-determining region on the Y chromosome) is essential for male development; however, the molecular mechanism by which the SRY induces testis development is still unclear. To elucidate the mechanism of testis development, we identified SRY-interacting proteins using a yeast two-hybrid system. We found two ribosomal proteins, RPS7 (ribosomal protein S7) and RPL13a (ribosomal protein L13a) that interact with the HMG (high-mobility group) box domain of SRY. Furthermore, we confirmed the intracellular distributions of RPS7, RPL13a and SRY and found that the three proteins were co-expressed in COS1 cells. SRY, RPS7 and RPL13a were co-localized in nuclear speckles. These findings suggest that SRY plays an important role in activities associated with nuclear speckles via an unknown mechanism.     

Novel deletion of RPL15 identified by array-comparative genomic hybridization in Diamond–Blackfan anemia

Diamond–Blackfan anemia (DBA) is an inherited red blood cell aplasia that usually presents during the first year of life. The main features of the disease are normochromic and macrocytic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. The patients also present with growth retardation and craniofacial, upper limb, heart and urinary system congenital malformations in ~30–50 % of cases. The disease has been associated with point mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, and RPL26 and GATA1 in about 60–65 % of patients. Here, we report a novel large deletion in RPL15, a gene not previously implicated to be causative in DBA. Like RPL26, RPL15 presents the distinctive feature of being required both for 60S subunit formation and for efficient cleavage of the internal transcribed spacer 1. In addition, we detected five deletions in RP genes in which mutations have been previously shown to cause DBA: one each in RPS19, RPS24, and RPS26, and two in RPS17. Pre-ribosomal RNA processing was affected in cells established from the patients bearing these deletions, suggesting a possible molecular basis for their pathological effect. These data identify RPL15 as a new gene involved in DBA and further support the presence of large deletions in RP genes in DBA patients.     

Ribosomal proteins S13 and L23 promote multidrug resistance in gastric cancer cells by suppressing drug-induced apoptosis

Ribosomal proteins (RP) S13 and RPL23 were previously identified as two upregulated genes in a multidrug-resistant gastric cancer cell line SGC7901/VCR compared to its parental cell SGC7901 by differential display PCR. The aim of this study was to explore the roles of RPS13 and RPL23 in multidrug resistance (MDR) in gastric cancer cells. RPS13 and RPL23 were genetically overexpressed in SGC7901 cells, respectively. Either RPS13 or RPL23 enhanced resistance of SGC7901 cells to vincristine, adriamycin, and 5-fludrouracil. RPL23 also enhanced resistance of SGC7901 cells to cisplatin. Overexpression of either RPS13 or RPL23 did not alter the population doubling time, [³H]leucine incorporation, and intracellular adriamycin accumulation of SGC7901 cells. However, either RPS13 or RPL23 could protect SGC7901 cells from undergoing vincristine-induced apoptosis. Western blot analysis revealed that both RPS13 and RPL23 significantly increased the expression level of Bcl-2 and Bcl-2/Bax ratio in SGC7901 cells. In addition, overexpression of RPL23 enhanced glutathione S-transferase (GST) activity and intracellular glutathione content in SGC7901 cells. Together, this work demonstrates that either RPS13 or RPL23 can promote MDR in gastric cancer cells by suppressing drug-induced apoptosis, and that RPL23 may also promote MDR in gastric cancer cells through regulation of glutathione S-transferase-mediated drug-detoxifying system.     

Silencing of Ribosomal Protein S9 Elicits a Multitude of Cellular Responses Inhibiting the Growth of Cancer Cells Subsequent to p53 Activation

Background

Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9).

Methodology/Principal Findings

Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis.

Conclusions

p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.     

The role of human ribosomal proteins in the maturation of rRNA and ribosome production

Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA.     

The two Arabidopsis RPS6 genes, encoding for cytoplasmic ribosomal proteins S6, are functionally equivalent

Many eukaryotic genomes have experienced ancient whole-genome duplication (WGD) followed by massive gene loss. These eliminations were not random since some gene families were preferentially retained as duplicates. The gene balance hypothesis suggests that those genes with dosage reduction can imbalance their interacting partners or complex, resulting in decreased fitness. In Arabidopsis, the cytoplasmic ribosomal proteins (RP) are encoded by gene families with at least two members. We have focused our study on the two RPS6 genes in an attempt to understand why they have been retained as duplicates. We demonstrate that RPS6 function is vital for the plant. We also show that reducing the level of RPS6 accumulation (in the knock-out rps6a or rps6b single mutants, or in the double heterozygous RPS6A/rps6a,RPS6B/rps6b), confers a slow growth phenotype (haplodeficiency). Importantly, we demonstrate that the functions of two RPS6 genes are redundant and interchangeable. Finally, like in most other described Arabidopsis rp mutants, we observed that a reduced RPS6 level slightly alters the dorsoventral leaf patterning. Our results support the idea that the Arabidopsis RPS6 gene duplicates were evolutionarily retained in order to maintain an expression level necessary to sustain the translational demand of the cell, in agreement with the gene balance hypothesis.     

Molecular adaptation and expression evolution following duplication of genes for organellar ribosomal protein S13 in rosids

Background  

Gene duplication has been a fundamental process in the evolution of eukaryotic genomes. After duplication one copy (or both) can undergo divergence in sequence, expression pattern, and function. Two divergent copies of the ribosomal protein S13 gene (rps13) of chloroplast origin are found in the nucleus of the rosids Arabidopsis, Gossypium, and Glycine. One encodes chloroplast-imported RPS13 (nucp rps13), while the other encodes mitochondria-imported RPS13 (numit rps13). The rps13 gene has been lost from mitochondrial DNA (mt rps13) of many rosids.     

Bt毒素诱导下小菜蛾实时定量PCR 内参基因的筛选

【目的】筛选出Bt毒素诱导后的小菜蛾Plutella xylostella (L.)的实时定量PCR最适内参基因。【方法】选取核糖体18S rRNA (18S rRNA)、 肌动蛋白（ACTB）、 延伸因子（EF1）、3-磷酸甘油醛脱氢酶（GAPDH）、 核糖体蛋白L32 (RPL32)、 核糖体蛋白S13 （RPS13）、 核糖体蛋白S20 （RPS20）和β-微管蛋白(TUB)基因作为候选内参基因, 以geNorm、 Normfinder和BestKeeper软件分析这8个基因在Bt毒素诱导后的小菜蛾不同品系中肠组织中的表达稳定性。并应用筛选出来的内参基因分析小菜蛾氨肽酶2（aminopeptidase N2, APN2）基因的表达水平。【结果】geNorm软件以RPS13和EF1为最稳定内参基因, NormFinder和BestKeeper软件均以RPS13和RPL32为最稳定基因。使用3种不同内参基因分析Bt毒素诱导后的小菜蛾Bt抗性和敏感品系中ANP2表达水平时, 新的内参基因EF1和传统内参基因RPL32表现了良好的稳定性, 二者作为标准化因子, ANP2表达量结果基本一致, 而使用18S rRNA作为内参基因, 却导致部分表达量分析结果有所误差。【结论】筛选出PRS13,RPL32和EF1可以作为小菜蛾某些试验条件下的内参基因, 对小菜蛾基因表达研究奠定了一定基础, 也对其他昆虫内参基因的筛选具有参考价值。     

Ribosomal Proteins RPL37, RPS15 and RPS20 Regulate the Mdm2-p53-MdmX Network

Changes to the nucleolus, the site of ribosome production, have long been linked to cancer, and mutations in several ribosomal proteins (RPs) have been associated with an increased risk for cancer in human diseases. Relevantly, a number of RPs have been shown to bind to MDM2 and inhibit MDM2 E3 ligase activity, leading to p53 stabilization and cell cycle arrest, thus revealing a RP-Mdm2-p53 signaling pathway that is critical for ribosome biogenesis surveillance. Here, we have identified RPL37, RPS15, and RPS20 as RPs that can also bind Mdm2 and activate p53. We found that each of the aforementioned RPs, when ectopically expressed, can stabilize both co-expressed Flag-tagged Mdm2 and HA-tagged p53 in p53-null cells as well as endogenous p53 in a p53-containing cell line. For each RP, the mechanism of Mdm2 and p53 stabilization appears to be through inhibiting the E3 ubiquitin ligase activity of Mdm2. Interestingly, although they are each capable of inducing cell death and cell cycle arrest, these RPs differ in the p53 target genes that are regulated upon their respective introduction into cells. Furthermore, each RP can downregulate MdmX levels but in distinct ways. Thus, RPL37, RPS15 and RPS20 regulate the Mdm2-p53-MdmX network but employ different mechanisms to do so.     

Self-mating in the definitive host potentiates clonal outbreaks of the apicomplexan parasites Sarcocystis neurona and Toxoplasma gondii

Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause devastating disease outbreaks.     

Plant mitochondrial rps2 genes code for proteins with a C-terminal extension that is processed

A gene (rps2) coding for ribosomal protein S2 (RPS2) is present in the mitochondrial (mt) genome of several monocot plants, but absent from the mtDNA of dicots. Confirming that in dicot plants the corresponding gene has been transferred to the nucleus, a corresponding Arabidopsis thaliana nuclear gene was identified that codes for mitochondrial RPS2. As several yeast and mammalian genes coding for mt ribosomal proteins, the Arabidopsis RPS2 apparently has no N-terminal targeting sequence. In the maize mt genome, two rps2 genes were identified and both are transcribed, although at different levels. As in wheat and rice, the maize genes code for proteins with long C-terminal extensions, as compared to their bacterial counterparts. These extensions are not conserved in sequence. Using specific antibodies against one of the maize proteins we found that a large protein precursor is indeed synthesized, but it is apparently processed to give the mature RPS2 protein which is associated with the mitochondrial ribosome.