Cloning and characterization of SLTI98 encoding ribosomal protein s6 in soybean

We isolated and characterized a stress-inducible gene designated as SLTI98 encoding ribosomal protein S6 in soybean. The derived amino acid sequence of SLTI98 showed the highest identity of 93% with ribosomal protein S6 from Medicago truncatula (ABD32373). The size of the full-length genomic clone of SLTI98 is 2701 bp containing 6 exons and 5 introns, of which structure is similar to that of Arabidopsis ribosomal protein S6. Genomic southern-blot analysis confirmed that soybean genome has multiple copies of the SLTI98 gene. SLTI98 RNA expression was slightly induced by salt, ABA, or wounding stress, but not by dehydration stress. The present study implies that the nuclear SLTI98 plays an important role in translational control during abiotic stresses. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 1, pp. 131–138. The text was submitted by the authors in English. These authors contributed equally to the work.     

The ribosomal protein P0 of soybean (Glycine max L. Merr.) has antigenic cross-reactivity to soybean seed lectin

Soybean (Glycine max L. Merr.) mutants lacking the ability to produce the lectin normally found in soybean seeds (SBL) are designated Le-. A protein of higher molecular weight that cross-reacts with antibodies raised to SBL was found at nearly equivalent levels in roots, hypocotyls, and leaves, and at lower levels in cotyledons and dry seeds of both Le+ and Le- soybean cultivars. Earlier work suggested that this protein was a novel lectin. Clones isolated from a Le- soybean root cDNA library produced a cross-reacting protein of the same size in Escherichia coli. Sequence analysis of these clones revealed a high degree of similarity to the ribosomal protein P0. The cross-reacting protein co-purified with ribosomes, and a monoclonal antibody raised to purified brine shrimp P0 cross-reacted to the same protein. The protein showed no lectin activity in a hemagglutination assay, nor did it bind to an N-acetyl-D-galactosamine affinity column. On the basis of this evidence, we conclude that the SBL-cross-reacting protein is not a lectin but a homologue of the ribosomal protein P0. Consequently, Le- soybeans must produce a lectin that is dissimilar to SBL at both the DNA and amino acid levels and we suggest that it is this lectin which is involved in nodulation.     

Epitopes of Escherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

Nucleolar binding sequences of the ribosomal protein S6e family reside in evolutionary highly conserved peptide clusters

Proteomic analyses of the nucleolus have revealed almost 700 functionally diverse proteins implicated in ribosome biogenesis, nucleolar assembly, and regulation of vital cellular processes. However, this nucleolar inventory has not unveiled a specific consensus motif necessary for nucleolar binding. The ribosomal protein family characterized by their basic nature should exhibit distinct binding sequences that enable interactions with the rRNA precursor molecules facilitating subunit assembly. We succeeded in delineating 2 minimal nucleolar binding sequences of human ribosomal protein S6 by fusing S6 cDNA fragments to the 5' end of the LacZ gene and subsequently detecting the intracellular localization of the beta-galactosidase fusion proteins. Nobis1 (nucleolar binding sequence 1), comprising of 4 highly conserved amino acid clusters separated by glycine or proline, functions independently of the 3 authentic nuclear localization signals (NLSs). Nobis2 consists of 2 conserved peptide clusters and requires the authentic NLS2 in its native context. Similarly, we deduced from previous publications that the single Nobis of ribosomal protein S25 is also highly conserved. The functional protein domain organization of the ribosomal protein S6e family consists of 3 modules: NLS, Nobis, and the C-terminal serine cluster of the phosphorylation sites. This modular structure is evolutionary conserved in vertebrates, invertebrates, and fungi. Remarkably, nucleolar binding sequences of small and large ribosomal proteins reside in peptide clusters conserved over millions of years.     

Identification of ribosomal protein S3a as a candidate for a novel PI 3-kinase target in the nucleus

Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) is an important lipid second messenger that mediates various cell responses. We have searched for the nuclear PIP₃ binding proteins using PIP₃ analogue beads. A 33 kD protein was detected in this method, which was identified as ribosomal protein S3a by the mass spectrometric analysis. The recombinant S3a protein bound specifically to PIP₃. S3a localized not only in the cytosol but also in the nucleus. Interestingly, not cytosolic but nuclear S3a bound to PIP₃, suggesting different roles of S3a in the cytosol and the nucleus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular characterization,immunohistochemical localization and expression of a ribosomal protein L17 gene from Apis cerana cerana

Ribosomal protein L17 (RPL17) is a core protein of the large (60S) ribosomal subunit and plays an important role in protein synthesis. In this report, a RPL17 gene was isolated from Apis cerana cerana, designated as AccRPL17. Alignment analysis showed that AccRPL17 exhibits high homology to other known RPL17s. Moreover, genomic sequence analysis revealed that five exons are splitted by four introns, and the position of the first intron is comparatively conservative, being localized in the 5′ untranslated region. Partial putative cis‐acting elements related to development were also examined. Quantitative real‐time PCR showed that the highest mRNA level was detected in larvae on the fifth day. Simultaneously, immunohistochemical localization showed that AccRPL17 is primarily concentrated in muscular tissues, stigma, body wall, and the surrounding of the eye in the fifth‐instar larvae. Further studies suggested that AccRPL17 might be involved in responses to abiotic stresses. This is a report attempting to analyze the expression and distribution of RPL17 in A. cerana cerana. These results indicated that AccRPL17 might play an important role in insect development, and the importance of AccRPL17 in participating in abiotic stresses is discussed. © 2010 Wiley Periodicals, Inc.     

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P₂). Its activity is inhibited by Ca²⁺ and Zn²⁺ and is activated by Mg²⁺, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.     

Roles of the negatively charged N-terminal extension of Saccharomyces cerevisiae ribosomal protein S5 revealed by characterization of a yeast strain containing human ribosomal protein S5

Ribosomal protein (rp) S5 belongs to a family of ribosomal proteins that includes bacterial rpS7. rpS5 forms part of the exit (E) site on the 40S ribosomal subunit and is essential for yeast viability. Human rpS5 is 67% identical and 79% similar to Saccharomyces cerevisiae rpS5 but lacks a negatively charged (pI approximately 3.27) 21 amino acid long N-terminal extension that is present in fungi. Here we report that replacement of yeast rpS5 with its human homolog yielded a viable yeast strain with a 20%-25% decrease in growth rate. This replacement also resulted in a moderate increase in the heavy polyribosomal components in the mutant strain, suggesting either translation elongation or termination defects, and in a reduction in the polyribosomal association of the elongation factors eEF3 and eEF1A. In addition, the mutant strain was characterized by moderate increases in +1 and -1 programmed frameshifting and hyperaccurate recognition of the UAA stop codon. The activities of the cricket paralysis virus (CrPV) IRES and two mammalian cellular IRESs (CAT-1 and SNAT-2) were also increased in the mutant strain. Consistently, the rpS5 replacement led to enhanced direct interaction between the CrPV IRES and the mutant yeast ribosomes. Taken together, these data indicate that rpS5 plays an important role in maintaining the accuracy of translation in eukaryotes and suggest that the negatively charged N-terminal extension of yeast rpS5 might affect the ribosomal recruitment of specific mRNAs.     

Mapping the essential structures of human ribosomal protein L7 for nuclear entry, ribosome assembly and function

Human large subunit protein L7 carries multiple nuclear localization signals (NLS) in its structure: there are three monobasic partite NLSs at the NH2-region of the first 54 amino acid residues and a bipartite in the middle section at position of 156-167. The C-region of the last 50 amino acid residues displays membrane binding nature, and might involve in forming a nuclear microbody for pre-nucleolar ribosome assembly. The middle section covers 144 amino acid residues which are essential for the structure and function of ribosome. This is evident from findings that truncated L7 without the NH2-region or the C-region, or missing both regions, is capable of reaching nucleolus and incorporating in ribosome, however, only ribosomes bearing truncated L7 without the NH2-region is capable of engaging in polysome formation. Combining with the phylogenic findings from homologous sequence alignment, the NH2-region of L7, besides being as a eukaryotic expansion segment, can be excluded from building a functional eukaryotic ribosome.     

Site-specific cleavage of the 40S ribosomal subunit reveals eukaryote-specific ribosomal protein S28 in the subunit head

After resolving the crystal structure of the prokaryotic ribosome, mapping the proteins in the eukaryotic ribosome is a challenging task. We applied RNase H digestion to split the human 40S ribosomal subunit into head and body parts. Mass spectrometry of the proteins in the 40S subunit head revealed the presence of eukaryote-specific ribosomal protein S28e. Recombinant S28e was capable of specific binding to the 3′ major domain of the 18S rRNA (K_a = 8.0 ± 0.5 × 10⁹ M⁻¹). We conclude that S28e has a binding site on the 18S rRNA within the 40S subunit head.

Structured summary

MINT-8044084: S8 (uniprotkb:P62241) and S19 (uniprotkb:P39019) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044095: S8 (uniprotkb:P62241), S19 (uniprotkb:P39019) and S13 (uniprotkb:P62277) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044024: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S21 (uniprotkb:P63220), S20 (uniprotkb:P60866), S26 (uniprotkb:P62854), S25 (uniprotkb:P62851), S12 (uniprotkb:P25398), S17 (uniprotkb:P08708), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263), S16 (uniprotkb:P62249) and S11 (uniprotkb:P62280) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044065: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263) and S16 (uniprotkb:P62249) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)     

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10‐R98S). Our data support that RPL10‐R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock‐in of RPL10‐R98S was associated with a 1.7‐ to 2.5‐fold increased production of four transiently expressed recombinant proteins and 1.7‐fold for one out of two stably expressed proteins. In CHO‐S cells, eGFP reached a 2‐fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10‐R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10‐R98S phenotypes can maximize its exploitability for the mammalian protein production industry.     

cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella （Lepidoptera： Plutellidae）

Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.     

Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.     

The S7 ribosomal protein gene is truncated and overlaps a cytochrome c biogenesis gene in pea mitochondria

The pea mitochondrial genome contains a truncated rps7 gene lacking ca. 40 codons at its 5 terminus. This single-copy sequence is immediately downstream of and slightly overlapping an actively transcribed and edited reading frame of 744 bp (designated ccb248) homologous to the bacterial helC gene which encodes a subunit of the ABC-type heme transporter involved in cytochrome c biogenesis. This region of mitochondrial DNA appears recombinogenic, and the carboxy-termini of helC-type proteins are predicted to vary in sequence and length among plants. Sequences corresponding to the 5 coding region of rps7 were not detected elsewhere in the pea mitochondrial genome using wheat rps7 probes, and only a very short internal rps7 segment was observed in soybean mitochondrial DNA. The presence of rps7-homologous sequences in the nuclear genomes of pea and soybean is consistent with the recent transfer of a functional mitochondrial rps7 gene to the nucleus in certain plant lineages.     

Role of N-terminal helix in interaction of ribosomal protein S15 with 16S rRNA

The position and conformation of the N-terminal helix of free ribosomal protein S15 was earlier found to be modified under various conditions. This variability was supposed to provide the recognition by the protein of its specific site on 16S rRNA. To test this hypothesis, we substituted some amino acid residues in this helix and assessed effects of these substitutions on the affinity of the protein for 16S rRNA. The crystal structure of the complex of one of these mutants (Thr3Cys S15) with the 16S rRNA fragment was determined, and a computer model of the complex containing another mutant (Gln8Met S15) was designed. The available and new information was analyzed in detail, and the N-terminal helix was concluded to play no significant role in the specific binding of the S15 protein to its target on 16S rRNA.     

Evolutionary conservation of nuclear and nucleolar targeting sequences in yeast ribosomal protein S6A

Over 1 billion years ago, the animal kingdom diverged from the fungi. Nevertheless, a high sequence homology of 62% exists between human ribosomal protein S6 and S6A of Saccharomyces cerevisiae. To investigate whether this similarity in primary structure is mirrored in corresponding functional protein domains, the nuclear and nucleolar targeting signals were delineated in yeast S6A and compared to the known human S6 signals. The complete sequence of S6A and cDNA fragments was fused to the 5'-end of the LacZ gene, the constructs were transiently expressed in COS cells, and the subcellular localization of the fusion proteins was detected by indirect immunofluorescence. One bipartite and two monopartite nuclear localization signals as well as two nucleolar binding domains were identified in yeast S6A, which are located at homologous regions in human S6 protein. Remarkably, the number, nature, and position of these targeting signals have been conserved, albeit their amino acid sequences have presumably undergone a process of co-evolution with their corresponding rRNAs.     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.     

Human ribosomal protein S13 inhibits splicing of its own pre-mRNA

Recombinant human ribosomal protein (rp) S13 was shown to specifically bind with its own pre-mRNA fragment containing the first exon, first intron, second exon, and a part of the second intron and to inhibit its splicing in vitro. The binding of rpS13 was specific: recombinant human rpS10 and rpS16 bound with the fragment to a lower extent. Moreover, rpS13 binding with the rpS13 pre-mRNA fragment was inhibited by non-labeled poly(AU) and an adenovirus pre-mRNA fragment to a lower extent than by the nonlabeled rpS13 pre-mRNA fragment. The specificity of splicing inhibition was inferred from the finding that, in contrast to rpS13, recombinant rpS10 and rpS16 did not affect the efficiency of first intron excision from the rpS13 pre-mRNA fragment. Enzymatic footprinting was used to determine the rpS13 pre-mRNA nucleotides whose accessibility to RNases T1, T2, and V1 changed in the presence of rpS13. Such nucleotides were detected close to the 5′ and 3′ splicing sites of the first intron. Analysis with the EMBOS-Align program showed that the nucleotide sequence of the first intron of the mammalian rpS13 pre-mRNA is conserved to a greater extent as compared with the other introns. It was assumed that the first intron plays an important role in regulating the expression of the rpS13 gene at the splicing level in all mammals.