Showing papers on "Ribosomal protein published in 1995"
TL;DR: The relationship of the rat ribosomal proteins to the proteins in ribosomes from humans, yeast, archaebacteria, and Escherichia coli is collated.
Abstract: Mammalian (rat) ribosomes have 80 proteins; the sequence of amino acids in 75 have been determined. What has been learned of the structure of the rat ribosomal proteins is reviewed with particular ...
329 citations
TL;DR: Molecular dissection of this pathway should further unravel cytokine-mediated signaling processes and help devise new immunosuppressants.
314 citations
Journal Article•
TL;DR: Previous data on the localization of ribosomal proteins with respect to structural features of 16S rRNA are reinforced and extended, and many new constraints for three-dimensional modeling of the 30S Ribosomal subunit are offered.
Abstract: Complexes between 16S rRNA and purified ribosomal proteins, either singly or in combination, were assembled in vitro and probed with hydroxyl radicals generated from free Fe(II)-EDTA. The broad specificity of hydroxyl radicals for attack at the ribose moiety in both single- and double-stranded contexts permitted probing of nearly all of the nucleotides in the 16S rRNA chain. Specific protection of localized regions of the RNA was observed in response to assembly of most of the ribosomal proteins. The locations of the protected regions were in good general agreement with the footprints previously reported for base-specific chemical probes, and with sites of RNA-protein crosslinking. New information was obtained about interaction of ribosomal proteins with 16S rRNA, especially with helical elements of the RNA. In some cases, 5' or 3' stagger in the protection pattern on complementary strands suggests interaction of proteins with the major or minor groove, respectively, of the RNA. These results reinforce and extend previous data on the localization of ribosomal proteins with respect to structural features of 16S rRNA, and offer many new constraints for three-dimensional modeling of the 30S ribosomal subunit.
235 citations
TL;DR: It is observed that another nuclease activity, UV endonuclease III, is similarly not detected in XP-D cells and is inseparable from the AP end onuclease I activity.
186 citations
TL;DR: It is proposed that the putative sub-sites of the peptidyl transferase centre are physically separated, that some drugs bind to more than one of them, and that they are conformationally interdependent.
159 citations
TL;DR: The complete primary sequence of A. castellanii mtDNA is determined and it is found that the circular mtDNA encodes two rRNAs (small subunit and large subunit), 16 tRNAs and 33 proteins (17 subunits of the respiratory chain and 16 ribosomal proteins) and contains eight open reading frames larger than 60 codons and of undefined function.
158 citations
TL;DR: It is shown that UPF1 and ribosomal protein L1 co-localize in the cytoplasm and that UPFs co-sediments with polyribosomes, and the results suggest thatUPF1-3EP associates with poly Ribosome, suggesting that there are far fewer UPf1 molecules than ribosomes per cell.
Abstract: In Saccharomyces cerevisiae the UPF1 protein is required for nonsense-mediated mRNA decay, the accelerated turnover of mRNAs containing a nonsense mutation. Several lines of evidence suggest that translation plays an important role in the mechanism of nonsense mRNA decay, including a previous report that nonsense mRNAs assemble in polyribosomes. In this study we show that UPF1 and ribosomal protein L1 co-localize in the cytoplasm and that UPF1 co-sediments with polyribosomes. To detect UPF1, three copies of the influenza hemagglutinin epitope were placed at the C-terminus. The tagged protein, UPF1-3EP, retains 86% (+/- 5%) of function. Using immunological detection, we found that UPF1-3EP is primarily cytoplasmic and was not detected either in the nucleus or in the mitochondrion. UPF1-3EP and L1 co-distributed with polyribosomes fractionated in a 7-47% sucrose gradient. The sucrose sedimentation profiles for UPF1-3EP and L1 exhibited similar changes using three different sets of conditions that altered the polyribosome profile. When polyribosomes were disaggregated, UPF1-3EP and L1 accumulated in fractions coincident with 80S ribosomal particles. These results suggest that UPF1-3EP associates with polyribosomes. L3 and S3 mRNAs, which code for ribosomal proteins of the 60S and 40S ribosomal subunits, respectively, were on average about 100-fold more abundant than UPF1 mRNA. Assuming that translation rates for L3, S3, and UPF1 mRNA are similar, this result suggests that there are far fewer UPF1 molecules than ribosomes per cell. Constraints imposed by the low UPF1 abundance on the functional relationships between UPF1, polyribosomes, and nonsense mRNA turnover are discussed.
128 citations
TL;DR: The complete nucleotide sequence of the circular mitochondrial (mt) DNA from the red alga Chondrus crispus was determined and the main structural features of this genome are compared with the overall organization of mtDNAs and are discussed in view of the evolution of mitochondria.
121 citations
TL;DR: The cross‐linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three‐dimensional models, and a comparison of the structural data with mutations in ribOSomal proteins that lead to antibiotic resistance, and with those from protein‐antibiotic cross‐linking experiments, reveals functional implications for ribosome proteins that interact with rRNA.
Abstract: We have investigated protein-rRNA cross-links formed in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus at the molecular level using UV and 2-iminothiolane as cross-linking agents. We identified amino acids cross-linked to rRNA for 13 ribosomal proteins from these organisms, namely derived from S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29 and L36. Several other peptide stretches cross-linked to rRNA have been sequenced in which no direct cross-linked amino acid could be detected. The cross-linked amino acids are positioned within loop domains carrying RNA binding features such as conserved basic and aromatic residues. One of the cross-linked peptides in ribosomal protein S3 shows a common primary sequence motif--the KH motif--directly involved in interaction with rRNA, and the cross-linked amino acid in ribosomal protein L36 lies within the zinc finger-like motif of this protein. The cross-linked amino acids in ribosomal proteins S17 and L6 prove the proposed RNA interacting site derived from three-dimensional models. A comparison of our structural data with mutations in ribosomal proteins that lead to antibiotic resistance, and with those from protein-antibiotic cross-linking experiments, reveals functional implications for ribosomal proteins that interact with rRNA.
121 citations
TL;DR: High-affinity RNA ligand exhibited specificity for their respective targets, as well as conserved sequence and secondary structure reminiscent of naturally occurring, cis-acting mRNA elements.
Abstract: High-affinity RNA ligands were generated against intact 30S ribosomes, S1-depleted 30S ribosomes, and purified ribosomal protein S1. Sequence analysis indicated two classes of ligand: unstructured RNAs containing a Shine-Dalgarno sequence and structured RNAs containing a pseudoknot. The Shine-Dalgarno-containing ligands were generated against S1-depleted 30S ribosomes but, surprisingly, not against intact 30S ribosomes or ribosomal protein S1. In contrast, pseudoknot-containing ligands were generated against intact ribosomes as well as purified S1 protein. The two classes of ligand exhibited specificity for their respective targets, as well as conserved sequence and secondary structure reminiscent of naturally occurring, cis-acting mRNA elements.
117 citations
TL;DR: These results define additional critical targets of the cAMP-dependent protein kinase, suggest a mechanism to couple ribosome production to the metabolic activity of the cell, and emphasize that nutrient regulation is independent of the RAS/cAMP pathway.
Abstract: By differential hybridization, we identified a number of genes in Saccharomyces cerevisiae that are activated by addition of cyclic AMP (cAMP) to cAMP-depleted cells. A majority, but not all, of these genes encode ribosomal proteins. While expression of these genes is also induced by addition of the appropriate nutrient to cells starved for a nitrogen source or for a sulfur source, the pathway for nutrient activation of ribosomal protein gene transcription is distinct from that of cAMP activation: (i) cAMP-mediated transcriptional activation was blocked by prior addition of an inhibitor of protein synthesis whereas nutrient-mediated activation was not, and (ii) cAMP-mediated induction of expression occurred through transcriptional activation whereas nutrient-mediated induction was predominantly a posttranscriptional response. Transcriptional activation of the ribosomal protein gene RPL16A by cAMP is mediated through a upstream activation sequence element consisting of a pair of RAP1 binding sites and sequences between them, suggesting that RAP1 participates in the cAMP activation process. Since RAP1 protein decays during starvation for cAMP, regulation of ribosomal protein genes under these conditions may directly relate to RAP1 protein availability. These results define additional critical targets of the cAMP-dependent protein kinase, suggest a mechanism to couple ribosome production to the metabolic activity of the cell, and emphasize that nutrient regulation is independent of the RAS/cAMP pathway.
TL;DR: The data suggest that most mak mutations affect M1 propagation by their effects on the supply of proteins from the L-A virus and that the translation of the non-poly(A) L-a mRNA depends critically on the amount of free 60S ribosomal subunits.
Abstract: Over 30 MAK (maintenance of killer) genes are necessary for propagation of the killer toxin-encoding M1 satellite double-stranded RNA of the L-A virus. Sequence analysis revealed that MAK7 is RPL4A, one of the two genes encoding ribosomal protein L4 of the 60S subunit. We further found that mutants with mutations in 18 MAK genes (including mak1 [top1], mak7 [rpl4A], mak8 [rpl3], mak11, and mak16) had decreased free 60S subunits. Mutants with another three mak mutations had half-mer polysomes, indicative of poor association of 60S and 40S subunits. The rest of the mak mutants, including the mak3 (N-acetyltransferase) mutant, showed a normal profile. The free 60S subunits, L-A copy number, and the amount of L-A coat protein in the mak1, mak7, mak11, and mak16 mutants were raised to the normal level by the respective normal single-copy gene. Our data suggest that most mak mutations affect M1 propagation by their effects on the supply of proteins from the L-A virus and that the translation of the non-poly(A) L-A mRNA depends critically on the amount of free 60S ribosomal subunits, probably because 60S association with the 40S subunit waiting at the initiator AUG is facilitated by the 3' poly(A).
TL;DR: The experimental data available support the implication of the eukaryotic stalk components in some regulatory process that modulates the ribosomal activity and indicate that the P1 and P2 proteins are not essential for ribosome activity but are able to affect the translation of some specific mRNAs.
Abstract: The eukaryoic ribosomal stalk is thought to consist of the phosphoproteins P1 and P2, which form a complex with protein P0. This complex interacts at the GTPase domain in the large subunit rRNA, ov...
TL;DR: Trees constructed from an alignment of 225 complete large subunit (LSU) rRNA sequences representing Eucarya, Archaea, Bacteria, plastids, and mitochondria showed considerable differences according to the particular species set selected, which might imply that LSU rRNA is a better molecule for studying wide-range relationships.
Abstract: Evolutionary trees were constructed, by distance methods, from an alignment of 225 complete large subunit (LSU) rRNA sequences, representing Eucarya, Archaea, Bacteria, plastids, and mitochondria. A comparison was made with trees based on sets of small subunit (SSU) rRNA sequences. Trees constructed on the set of 172 species and organelles for which the sequences of both molecules are known had a very similar topology, at least with respect to the divergence order of large taxa such as the eukaryotic kingdoms and the bacterial divisions. However, since there are more than ten times as many SSU as LSU rRNA sequences, it is possible to select many SSU rRNA sequence sets of equivalent size but different species composition. The topologies of these trees showed considerable differences according to the particular species set selected. The effect of the dataset and of different distance correction methods on tree topology was tested for both LSU and SSU rRNA by repetitive random sampling of a single species from each large taxon. The impact of the species set on the topology of the resulting consensus trees is much lower using LSU than using SSU rRNA. This might imply that LSU rRNA is a better molecule for studying wide-range relationships. The mitochondria behave clearly as a monophyletic group, clustering with the Proteobacteria. Gram-positive bacteria appear as two distinct groups, which are found clustered together in very few cases. Archaea behave as if monophyletic in most cases, but with a low confidence.
TL;DR: In situ hybridizations show that expression of two genes encoding L16 during the early phase of lateral root initiation and in developing pollen may serve to increase levels of ribosomal proteins during the rapid growth of these tissues.
Abstract: Lateral roots can be synchronously induced in Arabidopsis by a brief auxin treatment. An early event in the development of a lateral root primordium is the accumulation of mRNAs encoding ribosomal proteins. In situ hybridizations show that mRNA encoding one ribosomal protein, L16, accumulates in all rapidly proliferating tissues including the shoot and root apical meristems and lateral root primordia. To understand further the mechanisms by which ribosomal proteins are coordinately synthesized, two genes encoding the ribosomal protein L16 were isolated from Arabidopsis thaliana. Promoter sequences from each RPL16A and RPL16B were fused to the beta-glucuronidase reporter gene GUS. The promoter of RPL16B(from -848 to -19) conferred X-Gluc staining in proliferating tissues including the shoot and root apical meristems. When GUS was expressed from the RPL16A promoter (from -875 to -22), X-Gluc staining was observed in cells in the root stele and in anthers. When seedlings transformed with either promoter construct were treated with auxin to induce lateral roots, X-Gluc staining accumulated in the lateral root primordia by 16 h after induction. Transcription of the RPL16B promoter appears to be correlated with cell division, while transcription of the RPL16A promoter is very cell specific. Expression of two genes encoding L16 during the early phase of lateral root initiation and in developing pollen may serve to increase levels of ribosomal proteins during the rapid growth of these tissues.
TL;DR: The results suggest that psi formation in vivo occurs at an intermediate stage of 30S assembly, and Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and psi sequence analysis established that B. subtILis has the equivalent of psi 516 in its small subunit rRNA.
Abstract: Pseudouridine (psi) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes. In Escherichia coli small subunit RNA, there is only one psi, at position 516, in a region of the RNA known to be involved in codon recognition [Bakin et al. (1994) Nucleic Acids Res. 22, 3681-3684]. To assess the function of this single psi residue, the enzyme catalyzing its formation was purified and cloned. The enzyme contains 231 amino acids and has a calculated molecular mass of 25,836 Da. It converts U516 in E. coli 16S RNA transcripts into psi but does not modify any other position in this RNA. It does not react with free unmodified 16S RNA at all, and only poorly with 30S particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins. The yield varied from 0.6 to 1.0 mol of psi/mol of RNA, depending on the preparation. Free RNA(1-678) was inactive, as was RNA(1-526) and the RNP particle made from it. 23S RNA and tRNAVal transcripts were also inactive. These results suggest that psi formation in vivo occurs at an intermediate stage of 30S assembly. The gene is located at 47.1 min immediately 5' to, and oriented in the same direction as, the bicyclomycin resistance gene. The gene was cloned behind a (His)6 leader for affinity purification. Virtually all of the overexpressed protein was found in inclusion bodies but could be purified to homogeneity on a Ni2+(-) containing resin. Over 200 mg of pure protein could be obtained from a liter of cell culture. Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and psi sequence analysis established that B. subtilis has the equivalent of psi 516 in its small subunit rRNA. On the other hand, no common sequence motifs could be detected among this enzyme and the two tRNA psi synthases which have been cloned up to now.
TL;DR: Results indicate that erythromycin has two effects on translation, preventing elongation of the polypeptide chain and also inhibiting the formation of the large ribosomal subunit.
Abstract: Erythromycin and other macrolide antibiotics have been examined for their effects on ribosome assembly in growing Escherichia coli cells. Formation of the 50S ribosomal subunit was specifically inhibited by erythromycin and azithromycin. Other related compounds tested, including oleandomycin, clarithromycin, spiramycin, and virginiamycin M1, did not influence assembly. Erythromycin did not promote the breakdown of ribosomes formed in the absence of the drug. Two erythromycin-resistant mutants with alterations in ribosomal proteins L4 and L22 were also examined for an effect on assembly. Subunit assembly was affected in the mutant containing the L22 alteration only at erythromycin concentrations fourfold greater than those needed to stop assembly in wild-type cells. Ribosomal subunit assembly was only marginally affected at the highest drug concentration tested in the cells that contained the altered L4 protein. These novel results indicate that erythromycin has two effects on translation, preventing elongation of the polypeptide chain and also inhibiting the formation of the large ribosomal subunit.
TL;DR: Results indicate that at least one out of the five protein P-like carboxyl termini present in the ribosome has to be firmly bound to the particle for protein synthesis and cell viability, and this structure can be provided by protein P0.
TL;DR: Sequence comparison of Arabidopsis rps10 to the plant mitochondrial‐counterparts shows that the edited version is present in the nucleus ofArabidopsis, corroborates RNA as an intermediate of a functional gene transfer between mitochondria and the nucleus.
Journal Article•
TL;DR: It is concluded that RAP primarily induced a G1 prolongation without blocking cell cycle progression of mitogen-activated resting T lymphocytes, and inhibition of protein synthesis caused by the primary inhibition of ribosomal protein mRNA translation probably explains the effect of RAP.
Abstract: We investigated the effects of rapamycin (RAP) on cell cycle progression and protein synthesis in mitogen-activated primary T lymphocytes. Stimulation of resting human T lymphocytes with phorbol ester and calcium ionophore rendered cells capable of initiating DNA synthesis within 30 h; roughly 60% of the cells entered the first G2/M phase of the cell cycle within 96 h. Addition of RAP delayed the entry into S phase by 9 h, although a similar percentage (approximately 50%) of cells entered the first G2/M phase and proliferated. On this basis, we concluded that RAP primarily induced a G1 prolongation without blocking cell cycle progression. Addition of the co-mitogens to resting T lymphocytes up-regulated the translation of ribosomal protein mRNA concurrent with activation of p70s6k. RAP inhibited this translational up-regulation of ribosomal protein mRNA as well as the activation of p70s6k without affecting translation of nonribosomal protein mRNA. RAP also prevented the synthesis and accumulation of ribosomal proteins. Further, this failure to increase ribosomal proteins, which probably reflects the failure to increase numbers of ribosomes, resulted in suppression of the synthesis of total cellular protein and a delay in the escalation of cell size. RAP-treated cells eventually initiated DNA synthesis when cell size became equivalent to that of the control cells entering S phase of the cell cycle. Thus, inhibition of protein synthesis caused by the primary inhibition of ribosomal protein mRNA translation probably explains the effect of RAP on cell cycle progression of mitogen-activated resting T lymphocytes.
Journal Article•
TL;DR: The identification of the specific sites of both mRNA and rRNA and individual ribosomal proteins involved in the crosslinking provide direct evidence for the occurrence of a partial relocation of the mRNA on the 30S ribosome subunits under the influence of the factors.
Abstract: The hypothesis of an adjustment of the mRNA in its ribosomal channel under the influence of the initiation factors has been tested by site-directed crosslinking experiments. Complexes containing 30S subunits with bound mRNA having 4-thio-uracil at specific positions were prepared in the presence or absence of initiation factors and/or fMet-tRNA and subjected to UV irradiation to obtain specific crosslinks of the radioactively labeled mRNA with bases of the 16S rRNA and with ribosomal proteins. The subsequent identification of the specific sites of both mRNA and rRNA and individual ribosomal proteins involved in the crosslinking, obtained under different conditions of complex formation, provide direct evidence for the occurrence of a partial relocation of the mRNA on the 30S ribosomal subunits under the influence of the factors. The nature of this mRNA relocation is compatible with our previous proposal of a shift of the template from an initial ribosomal "stand-by site" to a second site closer to that occupied when the initiation triplet of the mRNA is decoded in the P-site.
TL;DR: A novel strategy was applied to construct the transformation system, since auxotrophic mutants which could be used as hosts for transformation are not available, and it was indicated that this method is applicable to the transformation of other C. utilis strains, even though there is significant heterogeneity in chromosomal karyotypes among these strains.
Abstract: We have developed a transformation system for the yeast Candida utilis. A novel strategy was applied to construct the transformation system, since auxotrophic mutants which could be used as hosts for transformation are not available. A gene encoding the ribosomal protein L41 was cloned from C. utilis, which is sensitive to cycloheximide, and used as a marker gene conferring cycloheximide resistance after modification of its amino acid sequence. The marker gene was constructed by substitution of the proline codon at position 56 with the glutamine codon by in vitro mutagenesis, as it had been reported previously that the 56th amino acid residue of L41 is responsible for the cycloheximide sensitivity of various organisms (S. Kawai, S. Murao, M. Mochizuki, I. Shibuya, K. Yano, and M. Takagi, J. Bacteriol. 174:254-262 1992). The ribosomal DNA (i.e., DNA coding for rRNA) of C. utilis was also cloned and used as a multiple-copy target for the integration of vector DNA into the genome, which resulted in a high transformation efficiency. Transformants were obtained by electroporation with a maximum efficiency of approximately 1,400 transformants per 1 microgram of linearized DNA carrying the gene for cycloheximide resistance and part of the ribosomal DNA. No transformants were obtained with intact plasmids. Multiple copies of the linearized plasmid were integrated into the host chromosome by homologous recombination. Southern analysis of the transformants in which vector DNA was integrated at the L41 gene locus indicated that there are two copies of gene for the L41 protein per cell, suggesting that C. utilis is diploid. Transformants were obtained from a variety of C. utilis strains, indicating that this method is applicable to the transformation of other C. utilis strains, even though there is significant heterogeneity in chromosomal karyotypes among these strains.
TL;DR: L11, NH4+, and Mg2+ all bind and stabilize the same rRNA tertiary interactions, which are conserved and presumably important for ribosome function.
TL;DR: Northern blot analysis of RNAs extracted from colorectal cancer and ajdacent normal tissue from 6 patients revealed in all of them perturbations of expression in cancer, compared to normal, no correlation could however be made between the level of expression and the severity of the disease.
TL;DR: Results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4.
Abstract: Localized hydroxyl radical probing has been used to explore the rRNA neighborhood around a unique position in the structure of the Escherichia coli 30S ribosomal subunit Fe(II) was attached to ribosomal protein S4 at Cys-31 via the reagent 1-(p-bromoacetamidobenzyl)-EDTA [Fe-Cys31]S4 was then complexed with 16S rRNA or incorporated into active 30S ribosomal subunits by in vitro reconstitution with 16S rRNA and a mixture of the remaining 30S subunit proteins Hydroxyl radicals generated from the tethered Fe resulted in cleavage of the 16S rRNA chain in two localized regions of its 5' domain One region spans positions 419-432 and is close to the multihelix junction previously placed at the RNA binding site of S4 by chemical and enzymatic protection (footprinting) and crosslinking studies A second site of directed cleavage includes nucleotides 297-303, which overlap a site that is protected from chemical modification by protein S16, a near neighbor of S4 in the ribosome These results provide useful information about the three-dimensional organization of 16S rRNA and indicate that these two regions of its 5' domain are in close spatial proximity to Cys-31 of protein S4
TL;DR: The gene encoding the β subunit of Bacillus subtilis RNA polymerase was isolated from a γgt11 expression library using an antibody probe and Gene identity was confirmed by the similarity of its predicted product to the Escherichia coli β sub unit and by mapping an alteration conferring rifampicin resistance within the conserved rif coding region.
TL;DR: A molecular basis for the insensitivity of eukaryotic ribosomes to the antibiotic thiostrepton was investigated using synthetic 100-nucleotide-long fragments covering the GTPase domain of 23/28 S rRNA, and preserved three-dimensional conformation of the RNA backbone between prokaryotes and higher eUKaryotes is discussed.
TL;DR: Transgenic tobacco plants constitutively expressing Escherichia coli pyrophosphatase behind the 35S CaMV promoter accumulate high levels of soluble sugars in their leaves and these plants were considered a tool to study adaptation of leaves to photoassimilate accumulation at the molecular level.
Abstract: Transgenic tobacco plants (ppa-1) constitutively expressing Escherichia coli pyrophosphatase behind the 35S CaMV promoter accumulate high levels of soluble sugars in their leaves [27]. These plants were considered a tool to study adaptation of leaves to photoassimilate accumulation at the molecular level. By differential hybridization of a subtractive library enriched for transcripts present in the transgenic plants 12 different cDNAs were isolated. By sequence analysis four cDNAs could be identified as 1-aminocyclopropane-1-carboxylate-oxidase and as three different pathogenesis-related proteins (PR-1b, PR-Q and SAR 8.2). Two cDNAs were homologous to a calmodulin-like protein from Arabidopsis and a human ribosomal protein L19 while six cDNA clones remained unknown. One of these clones (termed PAR-1 for photoassimilate-responsive) displayed features similar to pathogenesis-related proteins: Hybridizing transcripts, 1.2 and 1.0 kb in length, were strongly inducible by salicylate and accumulated in tobacco plants after infection with potato virus Y (PVY) both in infected and uninfected systemic leaves. PAR-1 transcripts also accumulated in wildtype leaves upon floating on glucose and sucrose whereas sorbitol and polyethylene glycol had no effect. Rescreening of the ppa-1 cDNA library with the PAR-1 cDNA as probe resulted in 25 hybridizing cDNAs which by homology were found to fall into three classes (PAR-1a, b, c). The cDNAs coding for PAR-1a and b were 90.6% homologous on the DNA level while both were less related to the PAR-1c cDNA (70.5% and 75.2% homologous, respectively). One open reading frame was identified in all three PAR-1 cDNA classes. Translation would result in proteins with a theoretical molecular mass of about 20 kDa. The N-terminal amino acid sequences resemble a signal peptide which would direct the proteins to the secretory pathway. Using selective 3' hybridization probes of the three PAR-1 cDNAs it was possible to discriminate the different transcripts. Both PAR-1a and PAR-1c mRNAs are induced in plants treated with PVY.
TL;DR: It is proposed that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA and is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog ofNSR1.
Abstract: Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA.
TL;DR: Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6, and a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus.
Abstract: Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6.