Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages

Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.     

Mosquito ribosomal protein S3 lacks a critical glutamine residue associated with DNA repair activity in homologous Drosophila proteins

In Drosophila melanogaster, ribosomal protein RpS3 has extra-ribosomal activities including apurinic/apyrimidinic lyase activity and N-glycosylase activity that participate in DNA repair. It has been suggested that these activities couple DNA repair to the translational machinery. To establish a basis for participation of RpS3 in DNA repair in mosquitoes, we cloned RpS3 cDNAs from Aedes aegypti and Aedes albopictus mosquito cell lines. The sequence data were used to reconstruct the homologous gene from the Anopheles gambiae database. Mosquito RpS3 is a single copy gene, which in Aedes albopictus, lacks introns in the amino acid coding region. Although RpS3 proteins are well-conserved among eukaryotes, a critical glutamine residue, Q59, essential to robust DNA repair activity in the Drosophila protein, is replaced by an asparagine (N) in all three mosquito RpS3 proteins. In this respect, the mosquito protein resembles human RpS3, which has relatively modest DNA repair activity. None of the insect RpS3 proteins available in the database, other than those from Drosophila, contain glutamine at position 59. However, in the Lepidoptera, N59 is consistently replaced by serine (S), and the putative interactive site at position 134 is replaced by arginine (R). These data suggest that in the case of RpS3, the Drosophila protein may be uniquely unusual in having robust DNA repair activities that are unlikely to be common to RpS3 from other insects.     

The chloroplast gene encoding ribosomal protein S4 in Chlamydomonas reinhardtii spans an inverted repeat — unique sequence junction and can be mutated to suppress a streptomycin dependence mutation in ribosomal protein S12

The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51–59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg₆₈ (CGT) to Len (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln₅₃ to Len ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S 12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu₆₈ mutations in rps4. Instead, six of 15 contained a Gln₇₃ (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln₇₃ to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

A novel nucleolar protein interacts with ribosomal protein S19

The gene encoding ribosomal protein S19 (RPS19) is mutated in approximately 25% of patients with Diamond-Blackfan anemia (DBA), which is a rare congenital erythroblastopenia. DBA patients have a variety of clinical characteristics, and the role of the RPS19 gene in the pathogenesis of the disease is presently unknown. To investigate a possible role for RPS19 in erythropoiesis, we looked for proteins associated with mouse RPS19 using a yeast two-hybrid system and identified a novel protein, which we named S19 binding protein (S19BP). The deduced amino acid sequence of S19BP derived from cDNA defines a calculated mass of 15,849 and an isoelectric point of 11.3. No known functional motifs were found in S19BP except a short polylysine tract embedded in a putative nucleolar localization signal. Immunolocalization experiments revealed that S19BP was highly concentrated in nucleoli after 6 h of transfection in Cos-7 cells. S19BP was expressed ubiquitously at a basal level but a significantly high level of expression was observed in some tissues.     

Regulation of global and specific mRNA translation by oral administration of branched-chain amino acids

The importance of branched-chain amino acids as nutrient regulators of protein synthesis in skeletal muscle was recognized more than 20 years ago. Of the branched-chain amino acids, leucine in particular was shown to play a central role in promoting muscle protein synthesis. However, it was only recently that the mechanism(s) involved in the stimulation of protein synthesis by leucine has begun to be defined. Studies performed in our laboratory during the past few years have revealed that oral administration of leucine to fasted rats enhances protein synthesis in association with increased phosphorylation of two proteins downstream of the protein kinase referred to as the mammalian target of rapamycin (mTOR). These proteins, eukaryotic initiation factor eIF4E binding protein (4E-BP)1 and ribosomal protein S6 kinase S6K1, control in part the step in translation initiation involving the binding of mRNA to the 40S ribosomal subunit. In theory the translation of all mRNAs can be regulated through such mechanisms, however, some mRNAs are more sensitive to the changes than others, resulting in modulation of gene expression through altered patterns of translation of specific mRNAs. Moreover, although a basal amount of plasma insulin is required for leucine to enhance signaling downstream of mTOR, the concentration observed in plasma of fasted rats is sufficient to observe maximal changes in phosphorylation of 4E-BP1 and S6K1.     

Phosphorylation of ribosomal proteins S3, L1 and L24 during spherulation in Physarum polycephalum

The phosphate content of ribosomal proteins S3, L1 and L24 has been determined in the course of spherulation of Physarum polycephalum. The major phosphoprotein, S3, was completely dephosphorylated after 4 h of differentiation. The phosphate content of L1 and L24 was not altered during the differentiation. The cellular level of ATP remained constant for at least 5 h. A 3-fold reduction of cyclic AMP concentration occurred in the first hour, followed by a slow increase to a final value of twice the level observed in growing cells. The results showed that the phosphorylation of ribosomal proteins is regulated by at least two different mechanisms and that the dephosphorylation of S3 is not induced by a lack of cellular ATP. Although cyclic AMP might trigger the dephosphorylation of S3, the phosphate content of this protein remained at a very low value even when the cellular concentration of cyclic AMP rose significantly. Since the polysome level remains constant during the first 24 h of spherulation, the phosphorylation of S3 is not necessary for active protein synthesis and the phosphorylation of L1 and L24 is not involved in ribosome inactivation, which occurs after 24 h.     

Organisation of the S10, spc and alpha ribosomal protein gene clusters in prokaryotic genomes

Although it is well known that there is no long range colinearity in gene order in bacterial genomes, it is thought that there are several regions that are under strong structural constraints during evolution, in which gene order is extremely conserved. One such region is the str locus, containing the S10-spc-alpha operons. These operons contain genes coding for ribosomal proteins and for a number of housekeeping genes. We compared the organisation of these gene clusters in 111 sequenced prokaryotic genomes (99 bacterial and 12 archaeal genomes). We also compared the organisation to the phylogeny based on 16S ribosomal RNA gene sequences and the sequences of the ribosomal proteins L22, L16 and S14. Our data indicate that there is much variation in gene order and content in these gene clusters, both in bacterial as well as in archaeal genomes. Our data indicate that differential gene loss has occurred on multiple occasions during evolution. We also noted several discrepancies between phylogenetic trees based on 16S rRNA gene sequences and sequences of ribosomal proteins L16, L22 and S14, suggesting that horizontal gene transfer did play a significant role in the evolution of the S10-spc-alpha gene clusters.     

Ribosomal protein S1 induces a conformational change of the 30S ribosomal subunit

A comparative study of the 30S ribosomal subunit in the complex with protein S1 and the subunit depleted of this protein has been carried out by the hot tritium bombardment method. Differences in exposure of some ribosomal proteins within the 30S subunit depleted of S1 and within the 30S–S1 complex were found. It was concluded that protein S1 binds in the region of the neck of the 30S ribosomal subunit inducing a conformational change of its structure.     

A novel nucleic acid-binding protein in the cyanobacterium Synechococcus sp. PCC6301: a soluble 33-kDa polypeptide with high sequence similarity to ribosomal protein S1

Cyanobacteria are prokaryotes that carry out plant-type photosynthesis and contain several eukaryotic-type RNA-binding proteins. Using a single-stranded DNA column, a 33-kDa protein was isolated and characterized from Synechococcus sp. PCC6301. This protein of 293 amino acids is similar in overall structure to the ribosomal protein S1 found in the same species, and contains three repeated units that are highly similar to the S1 motif originally found in the ribosomal protein S1 of Escherichia coli. However, the 33-kDa protein was found not to be associated with ribosomes and its nucleic acid binding specificity is distinct from that of the ribosomal protein S1. As this protein has high affinity for both single- and double-stranded DNA, as well as for poly(G) and poly(A), we tentatively named it nucleic acid-binding protein 1 (Nbp1). Received: 8 October 1999 / Accepted: 24 January 2000     

Functional interaction in establishment of ribosomal integrity between small subunit protein rpS6 and translational regulator rpL10/Grc5p

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.     

Arginine methylation of ribosomal protein S3 affects ribosome assembly

The human ribosomal protein S3 (rpS3), a component of the 40S small subunit in the ribosome, is a known multi-functional protein with roles in DNA repair and apoptosis. We recently found that the arginine residue(s) of rpS3 are methylated by protein arginine methyltransferase 1 (PRMT1). In this paper, we confirmed the arginine methylation of rpS3 protein both in vitro and in vivo. The sites of arginine methylation are located at amino acids 64, 65 and 67. However, mutant rpS3 (3RA), which cannot be methylated at these sites, cannot be transported into the nucleolus and subsequently incorporated into the ribosome. Our results clearly show that arginine methylation of rpS3 plays a critical role in its import into the nucleolus, as well as in small subunit assembly of the ribosome.     

Organization,sequence, and phylogenetic analysis of the ribosomal protein S3 gene from Drosophila virilis

Ribosomal protein S3 (RPS3) is a multifunctional ribosomal protein: it is a structural and functional component of the ribosome, and also a DNA repair enzyme involved in the DNA base excision repair pathway. Here we cloned and characterized the genomic organization of the ribosomal protein S3 gene (RpS3) homolog in Drosophila virilis. We then compared gene structure and protein sequences of RpS3 from vertebrates, invertebrates, and plants. These comparisons revealed that RpS3 genes from plants to mammals have highly conserved coding and amino acid sequences, and also protein size. Further comparisons of the protein sequences show that important domains are well conserved in both localization and sequence. In contrast, comparison of gene size and organization reveals differing patterns and levels of conservation. Whereas invertebrate RpS3 genes are small in size and gene organization is variable (from zero to four introns), vertebrates have a considerably larger (but variable) gene size and a uniform gene organization. The larger gene size in vertebrates is due to increased number and expansion of introns. Although the plant RpS3 genes are relatively small ( approximately 1.8 kb), their organization resembles that seen in vertebrates. The high conservation through different phyla may suggest that RPS3 might be under great functional constraints, both in its capacity as a component of the ribosome and as a component of a DNA repair system. Finally, electrophoretic mobility shift assays indicate that an upstream element binds a nuclear protein(s).     

Histone H1-like, lysine-rich low complexity amino acid extensions in mosquito ribosomal proteins RpL23a and RpS6 have evolved independently

Histone H1-like amino acid extensions have been described at the amino terminus of Drosophila RpL22 and RpL23a, and at the carboxyl terminus of mosquito ribosomal protein RpS6. An in silico search suggested that RpL23a, but not RpL22, in Anopheles gambiae has an amino-terminal extension. Because low complexity amino acid extensions are not common on eukaryotic ribosomal proteins, and their functions are unknown, we cloned cDNAs encoding RpL23a from Aedes albopictus and Anopheles stephensi mosquito cell lines. RpL23a proteins in Aedes and Anopheles mosquitoes are rich in lysine (approximately 25%), alanine (approximately 21%), and proline (approximately 8%), have a mass of approximately 40 kDa, a pI of 11.4 to 11.5, and contain an N-terminal extension of approximately 260 amino acid residues. The N-terminal extension in mosquito RpL23a is about 100 amino acids longer than that in the Drosophila RpL23a homolog, and contains several repeated amino acid motifs. Analysis of exon-intron organization in the An. gambiae and in D. melanogaster genes suggests that a short first exon encodes a series of 11 amino acid residues conserved in RpL23a proteins from Drosophila, mosquitoes, and the moth, Bombyx mori. The histone H1-like sequence in RpL23a is encoded entirely within the second exon. The C-terminal 126 amino acid residues of the RpL23a protein, encoded by exon 3 in Drosophila, and by exons 3 and 4 in Anopheles gambiae, are well conserved, and correspond to Escherichia coli RpL23 with the addition of the eukaryotic N-terminal nuclear localization sequence. Sequence comparisons indicate that the histone H1-like extensions on mosquito RpS6 and RpL23a have evolved independently of each other, and of histone H1 proteins.     

豌豆核糖体小亚基蛋白S21的cDNA克隆、序列分析及在G2豌豆中的表达研究

以G2豌豆幼苗为材料,构建了滴度为6.5×106pfu的cDNA文库,用同源序列筛选法从该文库中得到一个全长465bp的cDNA。杂交分析认为它是一完整的cDNA序列。DNA序列分析表明,它拥有一个282bp的开放读码框,编码94个氨基酸。计算机同源序列比较发现,它可能编码豌豆核糖体小亚基蛋白S21(RS21-PEA),因为该序列与已知的玉米、水稻S21在氨基酸全序列水平上有32%～35%的同源性,并含有与RNA结合的特征结构域。进化树分析显示S21蛋白可在一定程度上反映生物的进化及遗传变异趋势。