Ribosomal Protein S19 Gene Research Articles

Gene expression of ribosomal protein mRNA in Chironomus riparius: Effects of endocrine disruptor chemicals and antibiotics

Ribosomal protein genes are essential for cellular development. To examine the effects of ribosomal protein genes under various cellular stress conditions in chironomids, ribosomal protein S3 (RpS3) and S6 (RpS6) cDNA from Chironomus riparius were characterized and their expression was analyzed during development. A comparative and phylogenetic study among different orders of insects was carried out by analysis of sequence databases. C. riparius RpS3 was highly conserved at the protein level and shared over 85% amino acid identity with homologous sequences from other insects. RpS6 also showed approximately 80% amino acid identity. The RpS3 and S6 transcripts were present during different developmental stages but were most abundant during the embryonic stage. Furthermore, expression of the previously reported ribosomal proteins RpL11, L13, and L15, as well as RpS3 and S6 was analyzed following exposure to various concentrations of three endocrine disruptor chemicals (EDCs), di(2-ethylhexyl) phthalate, bisphenol A, and 4-nonylphenol (4NP), and the veterinary antibiotics (VAs) fenbendazole, sulfathiazole, and lincomycin. Only RpS3 gene expression was up-regulated significantly in response to EDCs and fenbendazole. However, the C. riparius ribosomal proteins showed a limited response to cellular stress, following exposure to EDCs and VAs.

Comparative Evolution of S7 Intron 1 and Ribosomal Internal Transcribed Spacer in Coilia nasus (Clupeiformes: Engraulidae)

Coilia nasus is widely distributed in the Yangtze River, the coastal waters of China, Korea and the Ariake Sound of Japan. Several ecotypes exist and this provides a useful model for the study of comparative diversity between molecular markers. Here we analyze and compare the nucleotide sequences between single-copy ribosomal protein S7 gene intron 1 (rpS7) and multiple-copy ribosomal internal transcribed spacer 1 (ITS1) in this species to compare the phylogenetic signal of the two nuclear genes. Nucleotide substitutions among the two gene sequences and partial sequence of mitochondrial cytochrome c oxidase subunit I (COI) gene were also analyzed. A total of 115 clones for rpS7 and 122 clones for ITS1 were obtained from 37 specimens. The nucleotide sequence length is 741 to 743 bp for rpS7 and 334 to 348 bp for ITS1. Intra- and inter-specimen variation in rpS7 results from nucleotide substitution, while such variation in ITS1 is mainly due to different numbers of short base repeats. The content of G + C is lower in rpS7 (43.5%) than in ITS1 (68.2%). Our results indicate that the proportion of the sequence variable sites is higher in rpS7 (61) than in ITS1 (23); the informative parsimony of rpS7 is evidently higher than that of ITS1 (26 vs. 2); the overall ratio between transitions and transversions in ITS1 is slightly lower than in rpS7, but remarkably lower than in COI. These results suggest that rpS7 is more suitable than ITS1 as a marker for genetic divergence of this group. Furthermore, gene flow is observed between the different geographic populations of C. nasus from the phylogeny of this species based on rpS7, showing that rpS7 has more evolutionary characteristics for understanding the processes of genomic evolution at the intraspecific level.

Establishing Rps6 hemizygous mice as a model for studying how ribosomal protein haploinsufficiency impairs erythropoiesis

Diamond-Blackfan anemia is a congenital hypoproliferative macrocytic anemia and 5q- syndrome myelodysplastic syndrome is an acquired hypoproliferative macrocytic anemia. Their common erythroid phenotype reflects a shared pathophysiology-haploinsufficiency of one of many ribosomal proteins and somatic deletion of one allele of the ribosomal protein S14 gene, respectively. Although these abnormalities lead to defective ribosome biogenesis, why ribosomal protein hemizygosity results in anemia is not certain. Here, we characterize the hematopoietic phenotype of mice lacking one allele of the ribosomal protein S6 gene. The mice have an erythroid phenotype similar to both Diamond-Blackfan anemia and the 5q- syndrome and lenalidomide therapy improves their anemia.

Northern refugia and recent expansion in the North Sea: the case of the wrasse Symphodus melops (Linnaeus, 1758)

Pleistocene climate changes have imposed extreme conditions to intertidal rocky marine communities, forcing many species to significant range shifts in their geographical distributions. Phylogeographic analyses based on both mitochondrial and nuclear genetic markers provide a useful approach to unravel phylogeographic patterns and processes of species after this time period, to gain general knowledge of how climatic changes affect shifts in species distributions. We analyzed these patterns on the corkwing wrasse (Symphodus melops, Labridae), a rocky shore species inhabiting North Sea waters and temperate northeastern Atlantic Ocean from Norway to Morocco including the Azores, using a fragment of the mitochondrial control region and the first intron of the nuclear S7 ribosomal protein gene. We found that S. melops shows a clear differentiation between the Atlantic and the Scandinavian populations and a sharp contrast in the genetic diversity, high in the south and low in the north. Within each of these main geographic areas there is little or no genetic differentiation. The species may have persisted throughout the last glacial maximum in the southern areas as paleotemperatures were not lower than they are today in North Scandinavia. The North Sea recolonization most likely took place during the current interglacial and is dominated by a haplotype absent from the south of the study area, but present in Plymouth and Belfast. The possibility of a glacial refugium in or near the English Channel is discussed.

Mice with ribosomal protein S19 deficiency develop bone marrow failure and symptoms like patients with Diamond-Blackfan anemia

AbstractDiamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

Ribosomal protein S3 gene expression of Chironomus riparius under cadmium, copper and lead stress

Heavy metals are of interest because they are often present at significant levels in the environment and can have severe effects on the aquatic environment. To examine the effects of oxidative stress induced by heavy metals on chironomids, the full-length cDNA of ribosomal protein S3 (RpS3) from Chironomus riparius was determined using molecular cloning. The basal expression of the RpS3 gene was not affected by changes in environmental rearing conditions such as temperature, media, and sediment type. However, RpS3 gene expression in C. riparius increased significantly in response to exposure to cadmium, copper, and lead, regardless of the exposure level. These results suggest that expression of the RpS3 gene, which is associated with the DNA repair process, may be used as an indicator of oxidative stress induced by heavy metals during environmental risk assessment. Key words: Chironomus riparius, ribosomal protein S3, cadmium, copper; lead, environmental risk assessment.

Ribosomal protein S3 gene expression of Chironomus riparius under cadmium, copper and lead stress

Ribosomal protein S3 gene expression of Chironomus riparius under cadmium, copper and lead stress

Reduced ribosomal protein gene dosage and p53 activation in low-risk myelodysplastic syndrome

AbstractReduced gene dosage of ribosomal protein subunits has been implicated in 5q− myelodysplastic syndrome and Diamond Blackfan anemia, but the cellular and pathophysiologic defects associated with these conditions are enigmatic. Using conditional inactivation of the ribosomal protein S6 gene in laboratory mice, we found that reduced ribosomal protein gene dosage recapitulates cardinal features of the 5q− syndrome, including macrocytic anemia, erythroid hypoplasia, and megakaryocytic dysplasia with thrombocytosis, and that p53 plays a critical role in manifestation of these phenotypes. The blood cell abnormalities are accompanied by a reduction in the number of HSCs, a specific defect in late erythrocyte development, and suggest a disease-specific ontogenetic pathway for megakaryocyte development. Further studies of highly purified HSCs from healthy patients and from those with myelodysplastic syndrome link reduced expression of ribosomal protein genes to decreased RBC maturation and suggest an underlying and common pathophysiologic pathway for additional subtypes of myelodysplastic syndrome.

Phylogeographic patterns and cryptic speciation across oceanographic barriers in South African intertidal fishes

Biogeographic boundaries are the meeting zone of broadly distributed faunas, or the actual cause of a faunal break. In the latter case, closely related sister species should be found across such a boundary. To achieve such a situation, preliminary stages are expected, where phylogeographic breaks followed by genetic cryptic speciation would be observed. Biogeographic boundaries, in the Cape Point/Cape Agulhas region of southern Africa, offer an ideal system to test such predictions. Here, we studied two intertidal clinid fish species that are endemic to southern Africa, Clinus superciliosus (n = 127) and Muraenoclinus dorsalis (n = 114). Using mitochondrial control region, 16S rRNA, 12S rRNA and NADH2 genes and the nuclear rhodopsin and the first intron of the S7 ribosomal protein gene, we show both phylogeographic breaks and likely cryptic speciation in each species. Pairwise Φ(st) results suggest population genetic structuring for both species, with higher levels for M. dorsalis (Φ(st) = 0.34-0.93) than for C. superciliosus (Φ(st) = 0.1-0.74). Further, we recover two and three distinct lineages within M. dorsalis and C. superciliosus, respectively. Phylogenetic topologies, concordance between nuclear and mitochondrial markers and levels of sequence divergence, which are consistent with closely related sister species pairs, suggest the presence of cryptic species. Our results therefore meet the expectation for reduced gene flow at a biogeographic barrier, which translates into significant genetic breaks and cryptic sister species.

Phylogeography of the shanny Lipophrys pholis (Pisces: Blenniidae) in the NE Atlantic records signs of major expansion event older than the last glaciation

The study of the phylogeography of inshore fish from West Europe is revealing diverse geographical and demographical patterns. Some species conform to the phylogeographic patterns typical of terrestrial organisms, with marked signatures of the last glaciation and a decline of genetic diversity to the north of the species range. Other species, however, reveal no decline in diversity with latitude and signatures of expansions older than the last glaciation. The shanny Lipophrys pholis is a common intertidal resident fish in west European rocky shores. It is unable to leave the rocky stretch where it settled as a juvenile, so that dispersal depends entirely on the planktonic larval stage. These life-history and behavioural traits make the shanny an interesting species for phylogeographical analysis, as long-range movements by adults, which could blur historical signals, are absent. In this paper the phylogeography of L. pholis was studied using a fragment of the mitochondrial control region and one from the first intron of the S7 ribosomal protein gene. The European samples (ranging from SW Spain to the Netherlands) did not display population differentiation, isolation-by-distance or latitudinal declines in genetic diversity. Iberia was proposed as having operated as the main glacial refugium for the shanny. The genealogy of the European population showed that the largest expansion detected was older than the last glaciation, with lineages persisting from the early Pleistocene, which does not conform to colonisation by a few founders in the current interglacial. It is argued that if fishes have very large population sizes and high dispersal rates, populations can efficiently track climatic shifts so that little or no genetic structure remains after each range expansion and latitudinal gradients of genetic diversity tend to be weak or non-existent.

Erythropoiesis failure due to RPS19 deficiency is independent of an activated Tp53 response in a zebrafish model of Diamond–Blackfan anaemia

Diamond-Blackfan anaemia (DBA) is a cancer-prone genetic disorder characterized by pure red-cell aplasia and associated physical deformities. The ribosomal protein S19 gene (RPS19) is the most frequently mutated gene in DBA (~25%). TP53-mediated cell cycle arrest and/or apoptosis in erythroid cells have been suggested to be major factors for DBA development, but it is not clear why mutations in the ubiquitously expressed RPS19 gene specifically affect erythropoiesis. Previously, we showed that RPS19 deficiency in zebrafish recapitulates the erythropoietic and developmental phenotypes of DBA, including defective erythropoiesis with severe anaemia. In this study, we analysed the simultaneous loss-of-function of RPS19 and Tp53 in zebrafish to investigate the role of Tp53 in the erythroid and morphological defects associated with RPS19 deficiency. Co-inhibition of Tp53 activity rescued the morphological abnormalities, but did not alleviate erythroid aplasia in RPS19-deficient zebrafish. In addition, knockdown of two other RP genes, rps3a and rpl36a, which result in severe morphological abnormalities but only mild erythroid defects, also elicited an activated Tp53 response. These results suggest that a Tp53-independent but RPS19-dependent pathway could be responsible for defective erythropoiesis in RPS19-deficient zebrafish.

Treatment of Zebrafish Models of Ribosomopathies (Diamond Blackfan Anemia (DBA) and 5q- Syndrome) with L-Leucine Results In An Improvement of Anemia and Developmental Defects: Evidence for a Common Pathway?

AbstractAbstract 195A growing number of disorders associated with impaired ribosome biogenesis and function have been recently recognized and termed ribosomopathies. The first of these to be identified was Diamond Blackfan Anemia (DBA), a congenital autosomal dominant bone marrow failure syndrome of childhood manifested as profound macrocytic anemia. DBA is characterized by enhanced sensitivity of hematopoietic progenitors to apoptosis with evidence of stress erythropoiesis. In addition to bone marrow defects, DBA patients often have craniofacial, genitourinary, cardiac and limb abnormalities and are at an increased risk of developing hematopoietic malignancies and osteosarcoma. Twenty-five percent of patients with DBA have heterozygous mutations in the ribosomal protein S19 (RPS19) gene, which encodes a component of the 40S ribosomal subunit. Additionally, a growing percentage of DBA patients lacking a mutation in the RPS19 gene have been shown to have mutations in other ribosomal protein genes. A second ribosomopathy, 5q- syndrome is a subtype of myelodysplasia, with an erythroid phenotype strikingly similar to DBA. The erythroid phenotype of 5q- syndrome has been recently demonstrated to be attributable to acquired haploinsufficiency of the ribosomal protein RPS14 gene associated with clonal loss of the long arm of chromosome 5. In both DBA and 5q- syndrome, it is unclear why defects in ribosomal proteins, which would be expected to have widespread consequences, have such a specific effect on erythroid maturation. It has been demonstrated that the efficiency of mRNA translation is significantly depressed in cells derived from DBA patients, consistent with a pathogenic ribosomal defect. L-leucine is an essential branched chain amino acid that is known to modulate protein synthesis by enhancing translation. L-leucine has been used to treat DBA patients, and has been shown to lead to improvement in hemoglobin levels and to transfusion independence in some patients. Based on this observation we hypothesized that a common pathway associated with defective mRNA translation underlies both DBA and 5q- MDS, and that activation of translation by L-Leucine would alleviate the anemia associated with both ribosomopathies. In order to address this hypothesis, we have modeled DBA and 5q- MDS in zebrafish embryos using an antisense morpholino appraoach and have demonstrated that similar to the human diseases, Rps19 and Rps14 deficiency lead to defective erythropoiesis and to developmental abnormalities (specific to DBA). Treatment of Rps19 and Rps14 deficient embryos with L-Leucine resulted in partial reversal of both the anemia and the developmental defects. L-Leucine is thought to regulate protein synthesis by acting as a nutrient signal involving the mTOR (mammalian target of rapamycin) signaling pathway. It has been suggested that L-Leucine enhances translation by activating translation initiation factors and by specific up-regulation of the ribosomal protein S6 kinase. Activation of the mTOR pathway is currently being analyzed in the L-Leucine treated Rps19 and Rps14 deficient zebrafish embryos. Additionally, our observations are also being validated in in vitro human models for DBA and 5q-MDS using human hematopoietic progenitor CD34+ cells and shRNAs specific for Rps19 and Rps14 genes followed by L-Leucine treatment. Our studies should provide evidence for activation of pathways involved in mRNA translation and validate the use of the orally administered amino acid L-Leucine as a therapeutic agent in the treatment of DBA as well as 5q- syndrome without the side-effects associated with current therapies for both diseases. Disclosures:No relevant conflicts of interest to declare.

A transgenic mouse model demonstrates a dominant negative effect of a point mutation in the RPS19 gene associated with Diamond-Blackfan anemia

AbstractDiamond Blackfan anemia (DBA) is an inherited erythroblastopenia associated with mutations in at least 8 different ribosomal protein genes. Mutations in the gene encoding ribosomal protein S19 (RPS19) have been identified in approximately 25% of DBA families. Most of these mutations disrupt either the translation or stability of the RPS19 protein and are predicted to cause DBA by haploinsufficiency. However, approximately 30% of RPS19 mutations are missense mutations that do not alter the stability of the RPS19 protein and are hypothesized to act by a dominant negative mechanism. To formally test this hypothesis, we generated a transgenic mouse model expressing an RPS19 mutation in which an arginine residue is replaced with a tryptophan residue at codon 62 (RPS19R62W). Constitutive expression of RPS19R62W in developing mice was lethal. Conditional expression of RPS19R62W resulted in growth retardation, a mild anemia with reduced numbers of erythroid progenitors, and significant inhibition of terminal erythroid maturation, similar to DBA. RNA profiling demonstrated more than 700 dysregulated genes belonging to the same pathways that are disrupted in RNA profiles of DBA patient cells. We conclude that RPS19R62W is a dominant negative DBA mutation.

Comparative evolution of the mitochondrial cytochrome b gene and nuclear S7 ribosomal protein gene intron 1 in sinipercid fishes and their relatives

The extant sinipercids are a group of freshwater percoid fishes endemic to East Asia. A recent mitochondrial cytochrome b phylogeny of sinipercids has challenged some aspects for their traditional taxonomy and molecular phylogeny, especially for the monophyly of Sinipercidae. In this study, we analyzed mitochondrial cytochrome b and nuclear encoded S7 ribosomal protein gene intron 1 for 10 sinipercid species and 11 related species to compare the phylogenetic signal and nucleotide substitution properties of these two gene sequences. The length of S7 intron 1 ranged from 461 to 719 bp, but alignment was not difficult, and the indels, the proportion of which in the total nucleotides ranged from 3.76 to 45.83%, were phylogenetically informative. Our results indicate that: (1) the relative rate presented by cyt b is five times that of S7 intron 1; (2) the proportion of phylogenetic information is higher in S7 than in cyt b; (3) S7 intron 1 has more base composition bias, but more uniform nucleotide substitution properties; (4) the overall ratio between transitions and transversions in S7 intron 1 is lower than in cyt b. Maximum parsimony and Bayesian analyses of aligned S7 intron 1 and the combined S7 and cyt b dataset resulted in phylogenies that contained the previously identified genera Siniperca and Coreoperca, whereas the monophyly of Coreoperca cannot be corroborated by separate cyt b analysis. The monophyly of Sinipercidae is not supported in separate and combined dataset analyses, although the alternative hypothesis cannot be significantly rejected based on approximately unbiased tests and Shimodaira–Hasegawa tests. Overall, maximum parsimony analyses result in trees with a lack of phylogenetic resolution in deep nodes, and the signal from S7 intron 1 conflicts the cyt b signal in the combined dataset analyses. The reasons for the poor performance of cyt b to S7 intron 1 in the phylogeny are discussed.

Phylogenetic studies of sinipercid fish (Perciformes: Sinipercidae) based on multiple genes, with first application of an immune-related gene, the virus-induced protein (viperin) gene

The sinipercid fish represent a group of 12 species of freshwater percoid fish endemic to East Asia. To date published morphological and molecular phylogenetics hypotheses of sinipercid fish are part congruent, and there are some areas of significant disagreement with respect to species relationships. The present study used separate and combined methods to analyze 7307 bp of data from three mitochondrial genes (cyt b, CO1 and 16S rRNA; ≈2312 bp) and three nuclear genes (viperin, the first two introns of S7 ribosomal protein gene; ≈4995 bp) for the attempts to estimate the relationships among sinipercids and to assess the phylogenetic utility of these markers. Phylogenetic trees were reconstructed using maximum parsimony, maximum likelihood and partitioned Bayesian analyses. Despite the detection of significant heterogeneity of phylogenetic signal between the mitochondrial and nuclear partitions, the combined data analysis represented the best-supported topology of all data. The sinipercid fish form a monophyletic group with two distinct clades, one corresponding to the genus Siniperca and the other to Coreoperca. Coreoperca whiteheadi is the sister taxon to Coreoperca herzi plus Coreoperca kawamebari. In the Siniperca, Siniperca undulata is the sister taxon to the other members of Siniperca, within the subclade containing the other members of the genus, Siniperca chuatsi and Siniperca kneri are sister species, next joined by Siniperca obscura, Siniperca roulei, Siniperca scherzeri and finally by Siniperca fortis. The potential utilities of six different genes for phylogenetic resolution of closely related sinipercid species were also evaluated, with special interest in that of the novel virus-induced protein (viperin) gene.

CDNA, genomic sequence cloning and overexpression of the ribosomal protein S13 gene in the giant panda (Ailuropoda melanoleuca)

The cDNA and the genomic sequence of ribosomal protein S13 (RPS13) of the giant panda (Ailuropoda melanoleuca) was cloned using reverse transcription-polymerase chain reaction (RT-PCR) and touchdown-PCR, respectively. These two sequences were sequenced and analyzed, and the cDNA of the RPS13 gene was overexpressed in Escherichia coli BL21. We compared the nucleotide sequences of the coding region and the amino acid sequences with those of seven other mammalian species retrieved from GenBank. The cDNA fragment of the RPS13 cloned from the giant panda is 496 bp in size, containing an open-reading frame of 456 bp, encoding 151 amino acids. The length of the genomic sequence is 2277 bp, with five exons and four introns. The coding sequence shows a high degree of homology to those of Homo sapiens, Bos taurus, Canis lupus familiaris, Macaca mulatta, Mus musculus, Rattus norvegicus, and Pan troglodytes; the degree of homology was 91.23, 94.30, 94.74, 92.11, 87.94, 87.72, and 91.45%, respectively. The homologies for the deduced amino acid sequences reached as high as 99%. Primary structure analysis revealed that the molecular weight of the putative RPS13 protein is 17.22325 kDa, with a theoretical pI of 10.42. Based on topology prediction, there is one protein kinase C phosphorylation site, one casein kinase II phosphorylation site, two N-myristoylation sites, and one ribosomal protein S15 signature in the RPS13 protein of the giant panda. The RPS13 gene can be expressed in E. coli and the RPS13 protein fused with the N-terminally GST-tagged form, which gave rise to the addition of an expected 43-kDa polypeptide.

Both p53-Dependent and -Independent Pathways Contribute to Erythroid Dysplasia in a Zebrafish Model for Diamond Blackfan Anemia.

Abstract 177Diamond Blackfan Anemia (DBA) is a congenital autosomal dominant bone marrow failure syndrome of childhood manifested as profound anemia. The disease is characterized by enhanced sensitivity of hematopoietic progenitors to apoptosis with evidence of stressed erythropoiesis. In addition to bone marrow defects, DBA patients often have craniofacial, genitourinary, cardiac and limb abnormalities and have an increased risk of developing hematopoietic malignancies and osteosarcoma. Twenty-five percent of patients with DBA have heterozygous mutations in the ribosomal protein S19 (RPS19) gene, which encodes a component of the 40S ribosomal subunit. Additionally, a growing percentage of DBA patients lacking a mutation in the RPS19 gene have been shown to have mutations in other ribosomal protein genes. These observations support the hypothesis that DBA is a disease of altered ribosome assembly and function. It is unclear how defects in ribosomal proteins have such a specific effect on erythroid maturation and cause increased apoptosis in the erythroid compartment. An attempt to model DBA by homozygous deletion of the Rps19 gene in mice proved to be embryonic lethal, and heterozygous mice appeared to fully compensate for the loss of one Rps19 allele, in contrast to the disease observed in humans. However, two groups have successfully modeled DBA in zebrafish using an antisense morpholino (MO) approach. These studies demonstrated that similar to the human disease, rps19 deficiency leads to defective erythropoiesis, increased apoptosis and to developmental abnormalities. A central role for the tumor suppressor p53 was suggested in one of these studies. It has previously been shown that any MO injection into zebrafish embryos can lead to the activation of the p53 pathway. Therefore, in order to clarify whether p53-independent effects also contributed to the DBA phenotype in zebrafish, we utilized the p53e7/e7 line that harbors a mutation within the p53 DNA-binding domain. Splice site and validated 5'UTR MOs targeting zebrafish rps19 were injected into one-cell stage embyros that were wildtype (WT) for p53 (AB) or mutated p53e7/e7. Staining for hemoglobin at 48 hours post fertilization showed a profound reduction in circulating blood in both p53 wild-type and p53 mutant embryos. Although p53 mutants injected with rps19 MO show a similar reduction in hemoglobin expression to WT morphants, they have a marked improvement in their developmental defects. A 20% decrease in expression of the transcription factor GATA-1 was observed in the rps19 morphants in the p53 mutant background compared to control MO injection. The implications of this finding are being further investigated and extended to include a panel of additional erythroid-specific factors. We have observed no increase in the levels of cell death, as measured by acridine orange (AO) staining or expression of the p53-regulated apoptosis associated gene PUMA, in the p53 mutant background. Taken together, our observations indicate that the phenotype observed in DBA has both a p53-dependent and a p53-independent component. We hypothesize that the p53-dependent component of DBA is likely responsible for the increased apoptosis associated with DBA while the erythroid maturation defect is associated, in large part, with a p53-independent component. Our studies are currently focused on identifying the players in the latter pathway. These investigations should shed light on thus far undefined pathways that will likely open new avenues for drug design and development for DBA. Disclosures:No relevant conflicts of interest to declare.

Genes within Genes: Multiple LAGLIDADG Homing Endonucleases Target the Ribosomal Protein S3 Gene Encoded within an rnl Group I Intron of Ophiostoma and Related Taxa

In some ascomycete fungi, ribosomal protein S3 (Rps3) is encoded within a group I intron (mL2449) that is inserted in the U11 region of the mitochondrial large subunit rDNA (rnl) gene. Previous characterization of the mL2449 intron in strains of Ophiostoma novo-ulmi subspecies americana (Dutch Elm Disease) revealed a complex genes-within-genes arrangement whereby a LAGLIDADG homing endonuclease gene (HEG) is inserted into the RPS3 gene near the 3' terminus, creating a hybrid Rps3-LAGLIDADG fusion protein. Here, we examined 119 additional strains of Ophiostoma and related taxa representing 85 different species by a polymerase chain reaction- based survey and detected both short (approximately 1.6 kb) and long (>2.2 kb) versions of the mL2449 intron in 88 and 31 strains, respectively. Among the long versions encountered, 21 were sequenced, revealing the presence of either intact or degenerated HEG-coding regions inserted within the RPS3 gene. Surprisingly, we identified two new HEG insertion sites in RPS3; one near the original C-terminal insertion site and one near the N-terminus of RPS3. In all instances, the HEGs are fused in-frame with the RPS3-coding sequences to create fusion proteins. However, comparative sequence analysis showed that upon insertion, the HEGs displaced a portion of the RPS3-coding region. Remarkably, the displaced RPS3-coding segments are duplicated and fused in-frame to the 3' end of RPS3, restoring a full-length RPS3 gene. We cloned and expressed the LAGLIDADG portion of two Rps3-HEG fusions, and showed that I-OnuI and I-LtrI generate 4 nucleotide (nt), 3' overhangs, and cleave at or 1 nt upstream of the HEG insertion site, respectively. Collectively, our data indicate that RPS3 genes are a refuge for distinct types of LAGLIDADG HEGs that are defined by the presence of duplicated segments of the host gene that restore the RPS3 gene, thus minimizing the impact of the HEG insertion on Rps3 function.

Targeted Resequencing and Analysis of the Diamond-Blackfan Anemia Disease Locus RPS19

BackgroundThe Ribosomal protein S19 gene locus (RPS19) has been linked to two kinds of red cell aplasia, Diamond-Blackfan Anemia (DBA) and Transient Erythroblastopenia in Childhood (TEC). Mutations in RPS19 coding sequences have been found in 25% of DBA patients, but not in TEC patients. It has been suggested that non-coding RPS19 sequence variants contribute to the considerable clinical variability in red cell aplasia. We therefore aimed at identifying non-coding variations associated with DBA or TEC phenotypes.Methodology/Principal FindingsWe targeted a region of 19'980 bp encompassing the RPS19 gene in a cohort of 89 DBA and TEC patients for resequencing. We provide here a catalog of the considerable, previously unrecognized degree of variation in this region. We identified 73 variations (65 SNPs, 8 indels) that all are located outside of the RPS19 open reading frame, and of which 67.1% are classified as novel. We hypothesize that specific alleles in non-coding regions of RPS19 could alter the binding of regulatory proteins or transcription factors. Therefore, we carried out an extensive analysis to identify transcription factor binding sites (TFBS). A series of putative interaction sites coincide with detected variants. Sixteen of the corresponding transcription factors are of particular interest, as they are housekeeping genes or show a direct link to hematopoiesis, tumorigenesis or leukemia (e.g. GATA-1/2, PU.1, MZF-1).ConclusionsSpecific alleles at predicted TFBSs may alter the expression of RPS19, modify an important interaction between transcription factors with overlapping TFBS or remove an important stimulus for hematopoiesis. We suggest that the detected interactions are of importance for hematopoiesis and could provide new insights into individual response to treatment.

A Mouse Model for Diamond-Blackfan Anemia Demonstrates a Dominant Negative Effect of a Point Mutation in the RPS19 Gene

Diamond-Blackfan Anemia (DBA) is associated with mutations in several ribosomal protein genes, including Ribosomal Protein S19 (RPS19), which is mutated in approximately 25% of patients. Most RPS19 mutations are deletions of all or part of the RPS19 gene and are predicted to cause DBA by a haploinsufficiency mechanism. However, approximately 30% of RPS19 mutations are missense mutations in the RPS19 coding sequence, which we hypothesize act through a dominant negative mechanism. To test for a dominant negative effect, we generated a transgenic mouse model expressing a common and penetrant mutation at codon 62 that replaces an Arginine with a Tryptophan (R62W). The constructs contain the ubiquitous actin promoter linked to the wild-type or R62W human RPS19 cDNA followed by the 3′ region of the Gamma globin gene to provide RNA stability and intron splicing to facilitate RNA transport to the cytoplasm. The constructs are flanked by chicken HS4 barrier elements to ensure transgene expression regardless of the location in the genome. Eight lines of wild-type RPS19 transgenic mice were fertile, expressed RPS19 in all tissues, and had normal hematology. Twelve RPS19R62W founder animals were generated, six of which died before they reached 2 months of age. Two of these animals were analyzed and found to have a macrocytic anemia. None of the other 6 founder animals transmitted the RPS19R62W transgene to F1 pups or d13.5 embryos, suggesting either that the RPS19R62W transgene was not present in the germ line and/or that expression of the RPS19R62W protein may cause early lethality. Supporting this hypothesis, embryonic stem cells (ES) expressing wild-type RPS19 were viable, while ES cells expressing RPS19R62W were not viable. To circumvent potential embryonic lethality, we generated conditional RPS19R62W transgenic mice with stop sequences flanked by lox P sites inserted between the promoter and the RPS19 gene. In the presence of Cre recombinase, lox P sites are combined, excising the sequences between them. Adult mice carrying the conditional RPS19R62W transgene and the interferon inducible Mx1-Cre gene were treated with poly (I:C) to induce excision of the stop sequence. Following poly (I:C) administration, hematocrits dropped significantly in RPS19R62W/Mx1-Cre animals compared to controls, but rebounded to normal within two weeks, due to incomplete stop sequence excision and expansion of unexcised cells in the bone marrow. Colony-forming cell assays indicate that RPS19R62W-expressing bone marrow contains 2 to 3 fold fewer BFU-E and CFU-E (p<0.05) and similar numbers of CFU-GM compared to wild-type animals. The decrease in erythroid progenitors was variable, indicating different levels of excision as well as penetrance. When RPS19R62W mice were crossed to Prion-Cre mice, which express Cre at the early embryonic stage, small, anemic d13.5 embryos and occasional small, adult animals with macrocytic anemia were observed. Day 13.5 RPS19R62W/Prion-Cre fetal livers had reduced overall numbers of erythroid cells, and reduced numbers of BFU-E and CFU-E. The decrease in erythroid progenitors was variable, especially in the line carrying 1 copy of the transgene compared to the line carrying 4 copies of the transgene. FACS analysis of d13.5 fetal liver and adult RPS19R62W/Prion-Cre erythroid cells revealed a relative accumulation of erythroid progenitor cells and a relative decrease in the number of terminally differentiating erythroid cells, suggesting that terminal erythroid differentiation is delayed. These findings are consistent with the reticulocytopenia observed in adult RPS19R62W/Prion-Cre mice. In summary we have successfully generated a mouse model of DBA caused by ectopic expression of mutant human RPS19R62W. The development of a severe anemia following conditional expression of mutant RPS19 suggests that the R62W missense mutation has a dominant negative effect that delays erythropoiesis causing an overall reduction in erythroid cells.