Arabidopsis L10 ribosomal proteins in UV-B responses

Ribosomal protein L10 (RPL10) is a ubiquitous protein that participates in joining the 40S and 60S ribosomal subunits into a functional 80S ribosome; however, increasing evidence indicates that RPL10 from various organisms has multiple extra-ribosomal functions, besides being a constituent of ribosome and its role in translation. Arabidopsis thaliana contains in its genome three genes encoding RPL10, named RPL10A, RPL10B and RPL10C. Previously, we found that in maize and in A. thaliana, UV-B induces a reduction in protein biosynthesis, probably as a consequence of ribosomal damage; however, cellular recovery occurs in the absence of UV-B. Here, we show that RPL10s are differentially regulated by UV-B in a dosage and time dependent manner: RPL10C is induced, RPL10B is downregulated at high UV-B intensity and RPL10A is not UV-B regulated. In addition, by co-immunoprecipitation studies using RPL10 antibodies and proteins from control and UV-B irradiated Arabidopsis plants, we demonstrate that RPL10 associates with different proteins under the two different conditions, including nuclear proteins, suggesting that at least one isoform may have extra-ribosomal roles.Key words: UV-B exposure, translation, ribosomal protein, co-immunoprecipitation     

Protein synthesis defects in temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

The ribosomes from four temperature-sensitive mutants of Escherichia coli have been examined for defects in cell-free protein synthesis. The mutants examined had alterations in ribosomal proteins S10, S15, or L22 (two strains). Ribosomes from each mutant showed a reduced activity in the translation of phage MS2 RNA at 44 degrees C and were more rapidly inactivated by heating at this temperature compared to control ribosomes. Ribosomal subunits from three of the mutants demonstrated a partial or complete inability to reassociate at 44 degrees C. 70-S ribosomes from two strains showed a reducton in messenger RNA binding. tRNA binding to the 30 S subunit was reduced in the strains with altered 30-S proteins and binding to the 50 S subunit was affected in the mutants with a change in 50 S protein L22. The relation between ribosomal protein structure and function in protein synthesis in these mutants is discussed.     

Ribosome topography in baby hamster kidney cells infected with Sindbis and vesicular stomatitis viruses

The topography of polysomal ribosomes in mock-infected and in Sindbis virus- and vesicular stomatitis virus-infected BHK cells was investigated using a double, radioactive labelling technique. Ribosomal proteins in intact polysomes were surface labelled by reductive methylation using [14C]formaldehyde. Following removal of ribosomal RNA, proteins were denatured in 6 M guanidine and labelled with [3H]borohydride. Labelled ribosomal proteins were separated by electrophoresis in two-dimensional gels and the 3H/14C ratio for each ribosomal protein was taken as an index of its relative surface exposure in intact ribosomes. Comparison of the ratios for individual ribosomal proteins in Sindbis virus-infected vs. control polysomes indicated that proteins L7, L8, L17, L26 and S19 became more 'buried' and others such as L4, L29, L36, S2 and S26 became more 'exposed' in infected cells. Most of the topographical alterations occurred in the large ribosomal subunit. In contrast, infection of BHK cells with vesicular stomatitis virus induced little or no topographical alteration.     

Control of ribosomal protein synthesis during wheat embryo imbibition

Dry wheat embryos contain large quantities of ribosomes, synthesized and assembled during embryogenesis. When messenger RNA isolated from dry embryos is translated, in vitro, a significant proportion of the total translation products (approx. 10%) is identifiable as ribosomal proteins, by electrophoresis in two distinct two-dimensional polyacrylamide gel electrophoretic systems. When germinating embryos are labelled with [³⁵S]methionine, during the first 24 h of imbibition, the appearance of newly synthesized ribosomal proteins in the cytosolic fraction is barely detectable. However, this low level (< 1% of total cytosolic protein synthesis) of observed ribosomal protein synthesis is not correlated with a correspondingly low level of ribosomal protein mRNA. Ribosomal proteins constitute at least 10% of the products of translation, in vitro, of mRNA isolated from germinating wheat embryos. Ribosomal proteins are also conspicuous products of translation when polyribosomes isolated from imbibing embryos are used to direct protein synthesis in a cell-free ‘run-off’ system, and newly synthesized ribosomal proteins can be detected in the nuclei isolated from germinating embryos. It is proposed that their absence from the cytosolic fraction is a consequence of post-translational regulatory events.     

Differential expression of ribosomal proteins in human normal and neoplastic colorectum.

Ribosomal proteins are a major component of ribosomes and play critical roles in protein biosynthesis. Recently it has been shown that the ribosomal proteins also function during various cellular processes that are independent of protein biosynthesis therefore called extraribosomal functions. In this study we have, for the first time, determined the expression profile of 12 ribosomal proteins (Sa, S8, S11, S12, S18, S24, L7, L13a, L18, L28, L32, and L35a) in normal epithelia of human colorectal mucosa using immunohistochemistry (IHC) and then compared their expression patterns with those of colorectal cancer. In the normal mucosa, ribosomal proteins were largely associated with the ribosomes of mucosal epithelia, and the expression level of ribosomal proteins, except for S11 and L7 proteins, was markedly increased in associated with maturation of the mucosal cells. On the other hand, these ribosomal proteins were markedly decreased in colorectal cancer compared with the normal mucosa. By contrast, S11 and L7 ribosomal proteins were rarely associated with the ribosomes of colorectal epithilia except immature mucosal cells, whereas their expression levels were significantly enhanced in colorectal cancer cells. In addition, L7 ribosomal protein was detected in the secretory granules of the enterochromaffin cells in the colorectal mucosa and in carcinoma cells expressing chromogranin A. These results indicate that the expression of ribosomal proteins is differentially regulated not only in normal mucosa but also in carcinoma of human colorectum, and suggest an extraribosomal function of L7 ribosomal protein in neuroendocrine function.     

Identification of the ribosomal proteins phosphorylated by the ribosome-associated casein kinase type II from cryptobiotic gastrulae of the brine shrimp Artemia sp

Phosphorylation of the ribosomal proteins by the extra-ribosomal protein kinase was investigated "in situ" and with purified 40 S or 60 S ribosomal proteins from cryptobiotic embryos of Artemia sp. Ribosomal proteins that were most readily phosphorylated in 80 S ribosomes included S6 and S8 of the 40 S subunit and proteins L9, L13 and L18 of the 60 S subunit. Several additional polypeptides were phosphorylated when purified 40 S or 60 S ribosomal proteins were separately incubated in the reconstituted system. The possible functions of ribosomal phosphorylation in protein synthesis will be discussed.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.     

Changes in ribosomal proteins in developing Artemia salina embryos

Ribosomal proteins from cysts and nauplii of Artemia salina were analyzed by three kinds of two-dimensional polyacrylamide gel electrophoresis. The basic-acidic and basic-SDS gel systems were used to compare the basic ribosomal proteins, and some changes were observed between the cysts and nauplii in proteins S6, S14, and L24. The phosphorylation of protein S6 was increased in the nauplii. Basic proteins S14 and L24 in the cysts changed and none of the corresponding proteins in the nauplii were detected at the same positions on two-dimensional gels as in the cysts. The acidic-SDS gel system was used to compare the acidic proteins in ribosomes and it was revealed that an acidic protein, AX (Mr = 24,000), in the cysts was not present in the ribosomes from the nauplii. The ribosomal activities as to the formation of an 80S initiation complex with globin mRNA and poly(U)-directed polyphenylalanine synthesis were compared. There was no significant difference between the cyst and nauplius ribosomes.     

Ribosome reactivation by replacement of damaged proteins

Ribosomal functions are vital for all organisms. Bacterial ribosomes are stable 2.4 MDa particles composed of three RNAs and over 50 different proteins. Accumulating damage to ribosomal RNA or proteins can disturb ribosome functioning. Organisms could benefit from degrading or possibly repairing inactive or partially active ribosomes. Reactivation of chemically damaged ribosomes by a process of protein replacement was studied in vitro. Ribosomes were inactivated by chemical modification of Cys residues. Incubation of modified ribosomes with total ribosomal proteins led to reactivation of translational activity. Intriguingly, ribosomal proteins extracted by LiCl are equally active in the restoration of ribosome function. Incubation of 70S ribosomes with isotopically labelled r‐proteins followed by separation of ribosomes was used to identify exchangeable proteins. A similar set of proteins was found to be exchanged in vivo under stress conditions in the stationary phase. We propose that repair of damaged ribosomes might be an important mechanism for maintaining protein synthesis activity following chemical damage.     

Zinc finger-like motifs in rat ribosomal proteins S27 and S29.

The primary structures of the rat 40S ribosomal subunit proteins S27 and S29 were deduced from the sequences of nucleotides in recombinant cDNAs and confirmed by determination of amino acid sequences in the proteins. Ribosomal protein S27 has 83 amino acids and the molecular weight is 9,339. Hybridization of cDNA to digests of nuclear DNA suggests that there are 4-6 copies of the S27 gene; the mRNA for the protein is about 620 nucleotides in length. Ribosomal protein S29 has 55 amino acids and the molecular weight is 6,541. There are 14-17 copies of the S29 gene and its mRNA is about 500 nucleotides in length. Rat ribosomal protein S29 is related to several members of the archaebacterial and eubacterial S14 family of ribosomal proteins. S27 and S29 have zinc finger-like motifs as do other proteins from eukaryotic, archaebacterial, eubacterial, and mitochondrial ribosomes. Moreover, ribosomes and ribosomal subunits appear to contain zinc and iron as well.     

Ribosomal Heterogeneity of Maize Tissues: Insights of Biological Relevance

In recent years, the selective role of ribosomes in the translational process of eukaryotes has been suggested. Evidence indicates that ribosomal heterogeneity at the level of protein stoichiometry and phosphorylation status differs among organisms, suggesting ribosomal specialization according to the state of development and the surrounding environment. During germination, protein synthesis is an active process that begins with the translation of the mRNAs stored in quiescent seeds and continues with the newly synthesized mRNAs. In this study, we identified differences in the abundance of ribosomal proteins (RPs) in maize embryos at different developmental stages. The relative quantification of RPs during germination revealed changes in six small subunit proteins, S3 (uS3), S5 (uS7), S7 (eS7), two isoforms of S17 (eS17), and S18 (uS13), and nine large subunit proteins, L1 (uL1), L5 (uL18), two isoforms of P0 (uL10), L11 (uL5), L14 (eL14), L15 (eL15), L19 (eL19), and L27 (eL27). Further analysis of ribosomal protein phosphorylation during germination revealed that the phosphorylation of PRP0 (uL10) and P1 increased and that of PRS3 (uS3) decreased in germinated versus quiescent embryos. The addition of insulin during germination increased the phosphorylation of the P1 protein, suggesting that its phosphorylation is controlled by the TOR pathway. Our results indicate that a heterogeneous ribosomal population provides to maize ribosomes during germination a different ability to translate mRNAs, suggesting another level of regulation by the ribosomes.     

Regulated heterogeneity in 12-kDa P-protein phosphorylation and composition of ribosomes in maize (Zea mays L.)

Maize (Zea mays L.) possesses four distinct approximately 12-kDa P-proteins (P1, P2a, P2b, P3) that form the tip of a lateral stalk on the 60 S ribosomal subunit. RNA blot analyses suggested that the expression of these proteins was developmentally regulated. Western blot analysis of ribosomal proteins isolated from various organs, kernel tissues during seed development, and root tips deprived of oxygen (anoxia) revealed significant heterogeneity in the levels of these proteins. P1 and P3 were detected in ribosomes of all samples at similar levels relative to ribosomal protein S6, whereas P2a and P2b levels showed considerable developmental regulation. Both forms of P2 were present in ribosomes of some organs, whereas only one form was detected in other organs. Considerable tissue-specific variation was observed in levels of monomeric and multimeric forms of P2a. P2b was not detected in root tips, accumulated late in seed embryo and endosperm development, and was detected in soluble ribosomes but not in membrane-associated ribosomes that copurified with zein protein bodies of the kernel endosperm. The phosphorylation of the 12-kDa P-proteins was also developmentally and environmentally regulated. The potential role of P2 heterogeneity in P-protein composition in the regulation of translation is discussed.     

Soluble expression of aggregating proteins by covalent coupling to the ribosome

Ribosomes are extremely soluble ribonucleoprotein complexes. Heterologous target proteins were fused to ribosomal protein L23 (rpL23) and expressed in an rpL23 deficient Escherichia coli strain. This enabled the isolation of 70S ribosomes with covalently bound target protein. Isolation of recombinant proteins from 70S ribosomes was achieved by specific proteolytic cleavage followed by efficient removal of ribosomes by centrifugation. By this procedure we isolated active green fluorescent protein, streptavidin (SA), and murine interleukin-6 (mIL-6). Approximately 500microg of each protein was isolated per gram cellular wet weight. By pull-down assays we demonstrate that SA covalently bound to the ribosome binds d-biotin. Ribosomal coupling is therefore suggested as a method for the investigation of protein interactions. The presented strategy is in particular efficient for the expression, purification, and investigation of proteins forming inclusion bodies in the E. coli cytoplasm.     

The primary structure of rat ribosomal protein L8.

The amino acid sequence of the rat 60S ribosomal subunit protein L8 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L8 has 257 amino acids and has a molecular weight of 28,007. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 4 or 5 copies of the L8 gene. The mRNA for the protein is about 950 nucleotides in length. Rat L8 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes.     

Influence of the state of ribosome association on the phosphorylation of ribosomal proteins in isolated ribosome--protein kinase systems from rat cerebral cortex.

Ribosomal protein phosphorylation was investigated in isolated ribosomal subunits and polyribosomes from rat cerebral cortex in the presence of [gamma-32P]ATP and purified catalytic subunit of cyclic AMP-dependent protein kinase from the same tissue. Ribosomal proteins that were most readily phosphorylated in isolated cerebral ribosomal subunits included proteins S2, S3a, S6 and S10 of the 40 S subunit and proteins L6, L13, L14, L19 and L29 of the 60 S subunit. These proteins were also phosphorylated in cellular preparations of rat cerebral cortex in situ or in vitro [Roberts & Ashby (1978) J. Biol. Chem. 253, 288-296; Roberts & Morelos (1979) Biochem. J. 184, 233-244]. However, several additional ribosomal proteins were phosphorylated when isolated 40 S or 60 S subunits were separately incubated in the reconstituted system. Analogous results were obtained with an equimolar mixture of cerebral 40 S and 60 S subunits under comparable conditions. In contrast, extensive exposure of purified cerebral polyribosomes to the catalytic subunit resulted in phosphorylation of only those ribosomal proteins of the 40 S subunit that were most highly labelled after the administration of [32P]Pi in vivo: proteins S2, S6 and S10. Ribosomal proteins of 60 S subunits that were readily phosphorylated in isolated cerebral polyribosomes included proteins L6, L13 and L29. These results indicate that polyribosome formation markedly decreases the number of ribosomal protein sites available for phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. Moreover, the findings suggest that, of the ribosomal protein phosphorylations observed in rat cerebral cortex in vivo, proteins S2, S6, S10, L6, L13 and L29 can be phosphorylated in polyribosomes, whereas proteins S3a, S5, L14 and L19 may become phosphorylated only in free ribosomal subunits.     

The primary structure of rat ribosomal protein L3.

The amino acid sequence of the rat 60S ribosomal subunit protein L3 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L3 has 403 amino acids and has a molecular weight of 46,106. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7 to 9 copies of the L3 gene. The mRNA for the protein is about 1,400 nucleotides in length. Rat L3 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes.     

Decreased activity of Blastocladiella emersonii zoospore ribosomes: correlation with developmental changes in ribosome-associated proteins

Ribosomal proteins isolated from dormant zoospores were compared to the ribosomal proteins found in the active growth phase by two-dimensional polyacrylamide gel electrophoresis. Zoospore ribosomes were found to contain a set of five proteins, designated Z1 to Z5, which were not present in growth phase ribosomes. The Z1-Z5 proteins were not removed by high-salt washes using either 1 M KCl or 1 M NH4 Cl. The Z1 protein is found associated with zoospore 60 S subunits while Z2-Z5 are bound to 40 S subunits. Zoospore monoribosomes and polyribosomes contain comparable levels of each of the five proteins. Approximately 60 min. after sporulation is induced, the Z1-Z5 proteins begin to accumulate on the ribosomes with the highest levels of these proteins found associated with ribosomes at the zoospore stage. During germination, the proteins gradually disappear and are not detectable on the ribosomes after 4 hr of germination. The presence of the Z1-Z5 proteins correlates with a decrease in in vitro protein synthetic activity of the fungal ribosomes. The data are consistent with the hypothesis that the proteins regulate translation by completely blocking protein synthesis on a subset of ribosomes while the remainder of the ribosomes function at normal rates.     

Covalent cross-linking of ribosomal RNA and proteins by methylene blue-sensitized photooxidation.

Ribosomal proteins are covalently cross-linked to ribosomal RNA by irradiation with visible light in the presence of methylene blue and O2. Proteins S3, S4, S5 and S7 from the 30 S subunit of Escherichia coli ribosomes and L2 and L3 from the 50 S subunit are among the cross-linked proteins. S3 and S5 had not previously been identified as RNA-binding proteins.