Cardiomyopathy is associated with ribosomal protein gene haplo-insufficiency in Drosophila melanogaster

The Minute syndrome in Drosophila melanogaster is characterized by delayed development, poor fertility, and short slender bristles. Many Minute loci correspond to disruptions of genes for cytoplasmic ribosomal proteins, and therefore the phenotype has been attributed to alterations in translational processes. Although protein translation is crucial for all cells in an organism, it is unclear why Minute mutations cause effects in specific tissues. To determine whether the heart is sensitive to haplo-insufficiency of genes encoding ribosomal proteins, we measured heart function of Minute mutants using optical coherence tomography. We found that cardiomyopathy is associated with the Minute syndrome caused by haplo-insufficiency of genes encoding cytoplasmic ribosomal proteins. While mutations of genes encoding non-Minute cytoplasmic ribosomal proteins are homozygous lethal, heterozygous deficiencies spanning these non-Minute genes did not cause a change in cardiac function. Deficiencies of genes for non-Minute mitochondrial ribosomal proteins also did not show abnormal cardiac function, with the exception of a heterozygous disruption of mRpS33. We demonstrate that cardiomyopathy is a common trait of the Minute syndrome caused by haplo-insufficiency of genes encoding cytoplasmic ribosomal proteins. In contrast, most cases of heterozygous deficiencies of genes encoding non-Minute ribosomal proteins have normal heart function in adult Drosophila.     

Disruption of an Arabidopsis cytoplasmic ribosomal protein S13-homologous gene by transposon-mediated mutagenesis causes aberrant growth and development

We identified a Dissociation (Ds) transposon-inserted Arabidopsis mutant of a gene (AtRPS13A) homologous to cytoplasmic ribosomal protein (RP) S13. We named our mutant pointed first leaf (pfl) 2 because of its similar phenotype to the pfl1 mutant of the RPS18 gene. This mutant caused multiple phenotypic changes, including aberrant leaf and trichome morphology, retarded root growth, and late flowering. Microscopic analysis showed that the first leaf blade of pfl2 contained a significantly reduced number of palisade cells, which suggests that the mutant phenotype was caused by reduced cell division. However, no phenotypic changes were observed during reproductive growth. In Arabidopsis, the RPS13 protein was encoded by a small expressed gene family including AtRPS13A. A pfl1 pfl2 double mutant showed no additive effect. These results suggest that RPS13 functions in quantitative and pleiotropic ways during growth and development, and that mutations at different kinds of RP gene loci are accumulatable without serious growth defects because they belong to small gene families.     

粘虫核糖体蛋白S7 cDNA的克隆和表达分析

运用RT-PCR和RACE技术克隆了粘虫Mythimna separata(Walker)核糖体蛋白S7基因(RPS7)的全长cDNA序列(GenBank登录号:JN582331),并对其进行生物信息学分析。结果表明,粘虫RPS7全长cDNA序列为762 bp,包括5'非编码区32 bp和3'非编码区67 bp。其开放阅读框(573 bp)编码190氨基酸肽链,具有核糖蛋白S7e蛋白家族典型特征。该肽链理论分子量为21.924 ku,等电点为9.82,富含4种类型的特定功能位点。该蛋白序列与其他动物RPS蛋白序列具有96.8%～98.2%高度同源性。应用荧光实时定量技术建立了粘虫脑部胚后发育RPS7表达模式。RPS7表达量随胚后发育脑部重建呈现出动态变化,这一结果显示RPS7是在转录水平上呈现发育性调节。     

A Drosophila ribosomal protein functions in mammalian cells.

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.     

Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.     

Molecular Genetics of Cryptopleurine Resistance in Saccharomyces Cerevisiae: Expression of a Ribosomal Protein Gene Family

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-δ1::URA3 null allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage λ, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-δ2::TRP1 cry2-δ1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transformation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cry1-δ2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-δ1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of β-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cry1-δ null allele.     

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D

The Minute phenotype results from mutations at?>50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P{lacW}M(3)66D ¹ , that maps to region 66D on chromosome 3L. Flies heterozygous for P{lacW}M(3)66D ¹ have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P{lacW}M(3)66D ¹ joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency.     

粘虫核糖体蛋白S7cDNA的克隆和表达分析

运用RT—PCR和RACE技术克隆了粘虫Mythimnaseparata（Walker）核糖体蛋白s7基因（RPS7）的全长eDNA序列（GenBank登录号：JN582331）,并对其进行生物信息学分析。结果表明,粘虫RPS7全长eDNA序列为762bp,包括5’非编码区32bp和3’非编码区67bp。其开放阅读框（573bp）编码190氨基酸肽链,具有核糖蛋白S7e蛋白家族典型特征。该肽链理论分子量为21．924ku,等电点为9．82,富含4种类型的特定功能位点。该蛋白序列与其他动物RPS蛋白序列具有96．8％-98．2％高度同源性。应用荧光实时定量技术建立了粘虫脑部胚后发育RPS7表达模式。RPS7表达量随胚后发育脑部重建呈现出动态变化,这一结果显示RPS7是在转录水平上呈现发育性调节。     

Ribosomal protein S24 gene is mutated in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare congenital red-cell aplasia characterized by anemia, bone-marrow erythroblastopenia, and congenital anomalies and is associated with heterozygous mutations in the ribosomal protein (RP) S19 gene (RPS19) in approximately 25% of probands. We report identification of de novo nonsense and splice-site mutations in another RP, RPS24 (encoded by RPS24 [10q22-q23]) in approximately 2% of RPS19 mutation-negative probands. This finding strongly suggests that DBA is a disorder of ribosome synthesis and that mutations in other RP or associated genes that lead to disrupted ribosomal biogenesis and/or function may also cause DBA.     

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.     

The Drosophila ribosomal protein L14-encoding gene, identified by a novel Minute mutation in a dense cluster of previously undescribed genes in cytogenetic region 66D

The Minute phenotype results from mutations at >50 loci scattered throughout the genome of Drosophila. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. Here, we report a novel P-element induced Minute mutation, P{lacW}M(3)66D ¹ , that maps to region 66D on chromosome 3L. Flies heterozygous for P{lacW}M(3)66D ¹ have a strong Minute phenotype. Molecular characterisation of the chromosomal region revealed three previously undescribed Drosophila genes clustered within a 5-kb genomic fragment. Two of the genes have significant sequence homology to genes for the mammalian ribosomal proteins L14 and RD, respectively, and share a joint 240-bp promoter region harbouring the P-element insert. Quantitative Northern blot analyses showed the mutation to affect RPL14 mRNA levels only. Interestingly, the reduction in abundance of RPL14 mRNA is not constitutive, indicating that the promoter function abolished by the inserted P-element is utilised with different efficiencies in different developmental situations. Remobilisation of the P element produced wild-type flies with normal levels of RPL14 mRNA, demonstrating that the mutant phenotype is caused by the insertion. P{lacW}M(3)66D ¹ joins a growing list of Minute mutations associated with ribosomal protein-haploinsufficiency. Received: 20 January 1997 / Accepted: March 3 1997     

Generation of Minute phenotypes by a transformed antisense ribosomal protein gene.

Antisense RNAs have been used for gene interference experiments in many cell types and organisms. However, relatively few experiments have been conducted with antisense genes integrated into the germ line. In Drosophila reduced ribosomal protein (r-protein) gene function has been hypothesized to result in a Minute phenotype. In this report we examine the effects of antisense r-protein 49 expression, a gene known to correspond to a Minute mutation An antisense rp49 gene driven by a strong and inducible promoter was transformed into the Drosophila germ line. Induction of this gene led to the development of flies with weak Minute phenotypes and to the transient arrest of oogenesis. Parameters that may affect the success of antisense gene inactivation are discussed.     

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.     

Molecular basis of Diamond-Blackfan anemia: structure and function analysis of RPS19

Diamond-Blackfan anemia (DBA) is a rare congenital disease linked to mutations in the ribosomal protein genes rps19, rps24 and rps17. It belongs to the emerging class of ribosomal disorders. To understand the impact of DBA mutations on RPS19 function, we have solved the crystal structure of RPS19 from Pyrococcus abyssi. The protein forms a five alpha-helix bundle organized around a central amphipathic alpha-helix, which corresponds to the DBA mutation hot spot. From the structure, we classify DBA mutations relative to their respective impact on protein folding (class I) or on surface properties (class II). Class II mutations cluster into two conserved basic patches. In vivo analysis in yeast demonstrates an essential role for class II residues in the incorporation into pre-40S ribosomal particles. This data indicate that missense mutations in DBA primarily affect the capacity of the protein to be incorporated into pre-ribosomes, thus blocking maturation of the pre-40S particles.