Nuclear Ribosomes Research Articles

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 NH 2-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 NH 2-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 NH 2-region is capable of engaging in polysome formation. Combining with the phylogenic findings from homologous sequence alignment, the NH 2-region of L7, besides being as a eukaryotic expansion segment, can be excluded from building a functional eukaryotic ribosome.

Medusozoan Phylogeny and Character Evolution Clarified by New Large and Small Subunit rDNA Data and an Assessment of the Utility of Phylogenetic Mixture Models

A newly compiled data set of nearly complete sequences of the large subunit of the nuclear ribosome (LSU or 28S) sampled from 31 diverse medusozoans greatly clarifies the phylogenetic history of Cnidaria. These data have substantial power to discern among many of the competing hypotheses of relationship derived from prior work. Moreover, LSU data provide strong support at key nodes that were equivocal based on other molecular markers. Combining LSU sequences with those of the small subunit of the nuclear ribosome (SSU or 18S), we present a detailed working hypothesis of medusozoan relationships and discuss character evolution within this diverse clade. Stauromedusae, comprising the benthic, so-called stalked jellyfish, appears to be the sister group of all other medusozoans, implying that the free-swimming medusa stage, the motor nerve net, and statocysts of ecto-endodermal origin are features derived within Medusozoa. Cubozoans, which have had uncertain phylogenetic affinities since the elucidation of their life cycles, form a clade-named Acraspeda-with the scyphozoan groups Coronatae, Rhizostomeae, and Semaeostomeae. The polyps of both cubozoans and hydrozoans appear to be secondarily simplified. Hydrozoa is comprised by two well-supported clades, Trachylina and Hydroidolina. The position of Limnomedusae within Trachylina indicates that the ancestral hydrozoan had a biphasic life cycle and that the medusa was formed via an entocodon. Recently hypothesized homologies between the entocodon and bilaterian mesoderm are therefore suspect. Laingiomedusae, which has often been viewed as a close ally of the trachyline group Narcomedusae, is instead shown to be unambiguously a member of Hydroidolina. The important model organisms of the Hydra species complex are part of a clade, Aplanulata, with other hydrozoans possessing direct development not involving a ciliated planula stage. Finally, applying phylogenetic mixture models to our data proved to be of little additional value over a more traditional phylogenetic approach involving explicit hypothesis testing and bootstrap analyses under multiple optimality criteria. [18S; 28S; Cubozoa; Hydrozoa; medusa; molecular systematics; polyp; Scyphozoa; Staurozoa.].

Solution to the phylogenetic enigma of Tetraplatia , a worm-shaped cnidarian

Tetraplatia is a genus containing two species of pelagic cnidarians of curious morphology. Their vermiform shape and four swimming flaps are difficult to relate to the features of other cnidarians, thus obscuring their phylogenetic affinities. Since their discovery in the mid-1800s, a number of prominent cnidarian workers have weighed in on this conundrum, some arguing that they are aberrant hydrozoans and others concluding that they are unusual scyphozoans. Current taxonomic practice conforms to the latter view. However, data presented here from the large and small subunits of the nuclear ribosome leave little doubt that Tetraplatia is in fact a hydrozoan genus. Indeed, its precise phylogenetic position is within Narcomedusae, as some authors had previously deduced based on structural characters. The distinctive body plan of Tetraplatia is remarkable because it appears to have a recent origin, in contrast to the prevailing pattern of metazoan history.

The case for nuclear translation

Although it is frequently assumed that translation does not occur in eukaryotic nuclei, recent evidence suggests that some translation can take place and that it is closely coupled to transcription. The first evidence concerns the destruction of nuclear mRNAs containing premature termination codons by nonsense-mediated decay (NMD). Only ribosomes can detect termination codons, and as some NMD occurs within the nuclear fraction, active nuclear ribosomes could perform the required detection. The second evidence is the demonstration that tagged amino acids are incorporated into nascent polypeptides in a nuclear process coupled to transcription. The third evidence is that components involved in translation, NMD and transcription colocalize, coimmunoprecipitate and co-purify. All these results are simply explained if nuclear ribosomes scan nascent transcripts for premature termination codons at the site of transcription. Alternatively, the scanning needed for NMD might take place at the nuclear membrane, and contaminating cytoplasmic ribosomes might give the appearance of some nuclear translation. We argue, however, that the balance of evidence favours bona fide nuclear translation.

Premature termination codons do not affect the rate of splicing of neighboring introns.

Introduction of a premature termination codon (PTC) into an exon of a gene can lead to nonsense-mediated decay of the mRNA, which is best characterized as a cytoplasmic event. However, increasing evidence has suggested that PTCs may also influence the nuclear processing of an RNA transcript, leading to models of nuclear surveillance perhaps involving translating nuclear ribosomes. We used quantitative RT-PCR to measure the in vivo steady-state levels of every exon-intron junction in wild-type, PTC-containing, and missense-containing precursor mRNAs of both the nonrearranging dihydrofolate reductase (DHFR) and the somatically rearranging Ig- micro genes. We find that each exon-intron junction's abundance and, therefore, the rate of intron removal, is not significantly affected by the presence of a PTC in a neighboring exon in either the DHFR or Ig- micro pre-mRNA. Similarly, the abundance of the uncleaved Ig- micro polyadenylation sites does not differ between wild-type and PTC-containing Ig- micro pre-mRNAs. Our Ig- micro data were confirmed by RNase protection analyses, and multiple cell isolates were examined to resolve differences with previously published data on steady-state pre-mRNA levels. We conclude that the presence of a PTC affects the rate of neither splicing nor the cleavage step of 3' end formation during pre-mRNA processing in the nucleus. Our results are discussed with respect to existing evidence for nuclear surveillance mechanisms.

Molecular cross-talk between the transcription, translation, and nonsense-mediated decay machineries.

It is widely believed that translation occurs only in the cytoplasm of eukaryotes, but recent results suggest some takes place in nuclei, coupled to transcription. Support for this heterodoxy comes from studies of the nonsense-mediated decay (NMD) pathway; this pathway probably uses ribosomes to proofread messenger RNAs. We find components of the machineries involved in transcription, translation and NMD colocalise, interact and copurify, and that interactions between them are probably mediated by the C-terminal domain of the catalytic subunit of RNA polymerase II. These results are simply explained if the NMD machinery uses nuclear ribosomes to translate - and so proofread - newly made transcripts; then, faulty transcripts and any truncated peptides produced by nuclear translation would be degraded.

Modeling a Minimal Ribosome Based on Comparative Sequence Analysis

We have determined the three-dimensional organization of ribosomal RNAs and proteins essential for minimal ribosome function. Comparative sequence analysis identifies regions of the ribosome that have been evolutionarily conserved, and the spatial organization of conserved domains is determined by mapping these onto structures of the 30 S and 50 S subunits determined by X-ray crystallography. Several functional domains of the ribosome are conserved in their three-dimensional organization in the Archaea, Bacteria, Eucaryotic nuclear, mitochondria and chloroplast ribosomes. In contrast, other regions from both subunits have shifted their position in three-dimensional space during evolution, including the L11 binding domain and the α-sarcin–ricin loop (SRL). We examined conserved bridge interactions between the two ribosomal subunits, giving an indication of which contacts are more significant. The tRNA contacts that are conserved were also determined, highlighting functional interactions as the tRNA moves through the ribosome during protein synthesis. To augment these studies of a large collection of comparative structural models sampled from all major branches on the phylogenetic tree, Caenorhabditis elegans mitochondrial rRNA is considered individually because it is among the smallest rRNA sequences known. The C. elegans model supports the large collection of comparative structure models while providing insight into the evolution of mitochondrial ribosomes.

Intranuclear degradation of nonsense codon-containing mRNA.

Most vertebrate mRNAs with premature termination codons (PTCs) are specifically recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD) while still associated with the nucleus. However, it is still a matter of debate whether PTCs can be identified by intranuclear scanning or only by ribosomes on the cytoplasmic side of the nuclear envelope. Here we show that inhibition of mRNA export by two independent approaches does not affect the downregulation of PTC-containing T-cell receptor beta transcripts in the nuclear fraction of mammalian cells, providing strong evidence for intranuclear NMD. Our results are fully consistent with recently reported evidence for nuclear translation and suggest that an important biological role for nuclear ribosomes is the early elimination of nonsense mRNA during a pioneer round of translation.

Coupled transcription and translation within nuclei of mammalian cells.

It is widely assumed that the vital processes of transcription and translation are spatially separated in eukaryotes and that no translation occurs in nuclei. We localized translation sites by incubating permeabilized mammalian cells with [3H]lysine or lysyl-transfer RNA tagged with biotin or BODIPY; although most nascent polypeptides were cytoplasmic, some were found in discrete nuclear sites known as transcription "factories." Some of this nuclear translation also depends on concurrent transcription by RNA polymerase II. This coupling is simply explained if nuclear ribosomes translate nascent transcripts as those transcripts emerge from still-engaged RNA polymerases, much as they do in bacteria.

The Nucleolar Phosphoprotein B23 Interacts with Hepatitis Delta Antigens and Modulates the Hepatitis Delta Virus RNA Replication

Hepatitis delta virus (HDV) encodes two isoforms of delta antigens (HDAgs). The small form of HDAg is required for HDV RNA replication, while the large form of HDAg inhibits the viral replication and is required for virion assembly. In this study, we found that the expression of B23, a nucleolar phosphoprotein involved in disparate functions including nuclear transport, cellular proliferation, and ribosome biogenesis, is up-regulated by these two HDAgs. Using in vivo and in vitro experimental approaches, we have demonstrated that both isoforms of HDAg can interact with B23 and their interaction domains were identified as the NH(2)-terminal fragment of each molecule encompassing the nuclear localization signal but not the coiled-coil region of HDAg. Sucrose gradient centrifugation analysis indicated that the majority of small HDAg, but a lesser amount of the large HDAg, co-sedimented with B23 and nucleolin in the large nuclear complex. Transient transfection experiments also indicated that introducing exogenous full-length B23, but not a mutated B23 defective in HDAg binding, enhanced HDV RNA replication. All together, our results reveal that HDAg has two distinct effects on nucleolar B23, up-regulation of its gene expression and the complex formation, which in turn regulates HDV RNA replication. Therefore, this work demonstrates the important role of nucleolar protein in regulating the HDV RNA replication through the complex formation with the key positive regulator being small HDAg.

Coupling of transcription and translation in Dictyostelium discoideum nuclei.

The nuclei of Dictyostelium discoideum cells have been found to contain polyribosomes active in protein synthesis. mRNA molecules enter nuclear polyribosomes while they are still being synthesized. "Non sense mediated mRNA decay" occurs in the nucleus, through the interaction of the mRNAs containing a nonsense codon with newly formed nuclear ribosomes, rather than with cytoplasmic ribosomes, as previously generally supposed.

Nucleolar targeting of 5S RNA in Xenopus laevis oocytes: somatic-type nucleotide substitutions enhance nucleolar localization.

In Xenopus laevis oocytes, 5S RNA is stored in the cytoplasm until vitellogenesis, at which time it is imported into the nucleus and targeted to nucleoli for ribosome assembly. This article shows that throughout oogenesis there is a pool of nuclear 5S RNA which is not nucleolar-associated. This distribution reflects that of oocyte-type 5S RNA, which is the major 5S RNA species in oocytes; only small amounts of somatic-type, which differs by six nucleotides, are synthesized. Indeed, 32P-labeled oocyte-type 5S RNA showed a degree of nucleolar localization similar to endogenous 5S RNA (33%) after microinjection. In contrast, 32P-labeled somatic-type 5S RNA showed significantly enhanced localization, whereby 70% of nuclear RNA was associated with nucleoli. A chimeric RNA molecule containing only one somatic-specific nucleotide substitution also showed enhanced localization, in addition to other somatic-specific phenotypes, including enhanced nuclear import and ribosome incorporation. The distribution of 35S-labeled ribosomal protein L5 was similar to that of oocyte-type 5S RNA, even when preassembled with somatic-type 5S RNA. The distribution of a series of 5S RNA mutants was also analyzed. These mutants showed various degrees of localization, suggesting that the efficiency of nucleolar targeting can be influenced by many discrete regions of the 5S RNA molecule.

Calcium distribution in high-pressure frozen bone cells by electron energy loss spectroscopy and electron spectroscopic imaging.

Subcellular localization of total calcium requires tissue processing that preserves the chemical composition of the samples and a highly sensitive microanalytical technique. In this study rat fetal bone samples were submitted to high-pressure freezing and freeze substitution. Ultrastructural preservation was good in the superficial sections: osteoblasts near the bone mineral had clearly defined plasma and nuclear membranes, dense mitochondria, and numerous ribosomes. Electron energy loss spectroscopy allowed high-resolution calcium-sensitive images to be obtained using ionization edge loss electrons. In biological samples, the Ca-L2,3 signal is superimposed on the carbon edge and artifacts may result from thickness and scattering effects. Therefore the relative thickness was established for each area analyzed (t/lambda <0.5). Background was subtracted using the three-images method, allowing high resolution calcium-sensitive images of intramitochondrial granules and of intracellular compartments, and semiquantitative data from the granules to be obtained. Calcium maps were confirmed by spectra collected on defined areas of the images and the shape of the net Ca-L2,3 edges was compared to the characteristic Ca-L2,3 edge of bone crystals. These procedures will provide new information about total calcium localization in bone cells and the possibility of examining the distribution of other elements.

ITS1 sequences of nuclear ribosome DNA in rice species from China and their phylogenetic implications

This study assessed the capability of using internal transcribed spacer (ITS1) sequences for reconstructing the phylogeny of rice species from China. Three accessions of wild and two accessions of cultivated rice were used (Table 1). Total DNA was extracted from leaf samples following the procedures described by Tai and Tanksley (1990). Polymerase chain reaction (PCR) amplifications were performed in 50 μL volumes containing 100 ng DNA template and 2.5 μL Taq polymerase. A PE-480 thermal cycler was used under the following conditions: 35 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min. PCR-amplified DNA products were recovered, complemented, and then cloned into the vector pUC 118 with standard protocols. Sequencing of ITS1 was conducted on an AB1 373A automated sequencer using a TaqDye Primer Cycle Sequencing Kit (ABI). The ITS1 sequences of the five taxa together with a published rice sequence (Takaiwa et al 1985) were aligned with the Clustal V program. The data matrix was analyzed using UPGMA with Jukes-Cantor distance and Neighbor-Joining with Kimura 2-parameter distance of the MEGA version 1.02 (Kura et al 1993).

RESTRICTION SITE MAPPING OF RIBES NUCLEAR RIBOSOMAL DNA.

RESTRICTION SITE MAPPING OF RIBES NUCLEAR RIBOSOMAL DNA.

NUCLEAR RIBOSOMAL DNA VARIATION AMONG RAMETS AND GENETS OF WHITE CLOVER.

Central to population and evolutionary biology is understanding the sources and consequences of genetic variation revealed at various levels of organization: within and among individuals, populations, and taxa. Classical evolutionary theory emphasizes the role that variability plays in influencing the evolutionary potential of a population or taxon. Consequently, there are hundreds of studies for plants alone published on population level sources of genetic variation (Hamrick and Godt, 1990). In contrast there is a dearth of studies in population biology that have examined the occurrences or consequences of within individual genetic (i.e., somatic) variation (King and Schaal, 1990; Rogers et al., 1986; Schaal and Learn, 1988; Silander, 1985). Even fewer have attempted to place such studies in the context of genetic variation at various levels of organization (Schaal, 1985). The evolutionary significance of somatic variation continues to be a source of considerable speculation in plant population biology (Antolin and Strobeck, 1985; Klekowski, 1988; Silander, 1985; Walbot and Cullis, 1985; Whitham et al., 1984). The little documented information available is often ambiguous or difficult to interpret evolutionarily, and is met with skepticism (see review by Klekowski, 1988). If somatic variation is likely to have some evolutionary significance, vegetatively propagating plant populations are a likely place to begin a search (see Klekowski, 1988). Advantageous somatic variants can be perpetuated indefinitely in clonal plants even if they are not immediately incorporated into the germ line. Indeed some of the best direct evidence for the occurrence of somatic variation and its potential evolutionary significance comes from studies of natural, clonal populations of plants (Klekowski, 1988; Schaal, 1985; Schaal and Learn, 1988; Silander, 1985). This study focusses on genetic variation in nuclear ribosomal DNA (rDNA) from accessions of natural clover populations. Genetic variation in rDNA was found to be common among genets (i.e., genetic individuals) of white clover (Trifolium repens), as well as among different species of clover. This was due to both restriction site and restriction fragment length

Intracellular localization of titanium within xenografted sensitive human tumors after treatment with the antitumor agent titanocene dichloride

In the present study, the intracellular localization of titanium was analyzed in three xenografted human adenocarcinomas of the colon sigmoideum (S 90), the stomach (M-Stg 4), and the lung (L 261) in dependence on the time after application of a single therapeutic dose (80 mg/kg) of the organometallic antitumor agent titanocene dichloride (C 5H 5) 2TiCl 2. The investigations were performed by use of electron energy loss spectroscopy (EELS), a method which allows microanalysis in ultrathin sections, in combination with electron spectroscopic imaging (ESI), which offers the possibility to image the two-dimensional localization and distribution of light- and medium-weight elements in animal tissues. In all three tumors which were studied, titanium was at first detected within the nucleus and, some hours and days later, it was additionally found in cytoplasmic lysosomes. In the colon and lung tumors S 90 and L 261, already 12 hr after treatment, titanium was traceable as tender granules in the nuclear chromatin. During the following days, it was then accumulated in certain areas of the nuclear heterochromatin and, in the case of the L 261 tumor, also in the nucleolus. Maximum concentrations were attained in the nuclei and nucleoli at 48 hr after substance application. Thereafter, titanium was increasingly incorporated into cytoplasmic lysosomes which are known to be involved in intracellular degrading and digesting processes and which occurred in increased numbers in treated tumor cells. Regarding the stomach carcinoma M-Stg 4, titanium was recognized in the nuclear heterochromatin only 1 and 2 days after application of titanocene dichloride. At 48 hr, it was additionally detected in cytoplasmic lysosomes. In all cases where titanium was found accumulated in the nucleus and in lysosomes, phosphorus was simultaneously enriched in a similar local distribution and a concentration which even exceeded that within phosphorus-rich areas, e.g., the nuclear heterochromatin and cytoplasmic ribosomes. These results confirm a primary interaction of titanium-containing metabolites deriving from titanocene complexes with nucleic acid molecules, especially with nuclear DNA. They suggest the formation of aggregates between nucleic acids and titanium-containing metabolites which are obviously extruded out of the nuclei and incorporated into cytoplasmic lysosomes, known to be involved in intracellular digesting processes.

Ultrastructural localization of nucleic acids in plant tissues following the use of malachite green or neutral red in the fixative solution

SUMMARYMalachite green and neutral red, when added to glutaraldehyde for fixation of various tissues, yielded high‐contrast images of cell ultrastructure. Malachite green, in acid conditions, appeared to increase contrast of heterochromatin material in the nucleus whereas neutral red gave greater clarity to the nucleolus and to cytoplasmic ribosomes. Control tissue fixed under acid conditions showed little damage but there were ‘crystalline’ areas at the periphery of the nucleolus. RNase did not digest cytoplasmic ribosomes from tissue after neutral red glutaraldehyde fixation. These results suggested that neutral red became bound to RNA in the tissues. Fixation with malachite green, at a pH below 6, did not affect the digestion of RNA by RNase but did protect chromatin against the bleaching action of the chelating agent EDTA. The addition of malachite green (pH &lt; 6) or neutral red to glutaraldehyde are useful techniques for the investigation of the ultrastructure of nuclear material and cytoplasmic ribosomes.

Interaction of drugs with extranuclear genetic elements and its consequences

Bacterial ancestry of mitochondria and plastids is now generally accepted. Both organelles contain their own DNA and transcription-translation apparatus of a prokaryotic type. Due to this fact these systems carry bacteria-like properties. Thus organellar DNA and ribosomes are essentially different from nuclear DNA and cytoplasmic ribosomes in physical as well as in functional respects. Due to the bacterial character of both types of organelles they are susceptible to various antibacterial chemicals. Inhibitors of bacterial protein synthesis inhibit mitochondrial (plastidial) biogenesis. Therefore the cellular content of mitochondria (plastids)-made proteins decreases during cytoplasmic turnover or cell division in the presence of these drugs. Such drug activity consequently leads to a reduced capacity for oxidative phosphorylation or photosynthesis. Organellar genomes are less stable and more sensitive to mutagenesis as compared to nuclear genome. It means also that genotoxic agents induce various disorders of mitochondrial (plastidial) functions. Impairments in the respiratory chain are associated with structural as well as functional abnormalities of mitochondria. These are clinically expressed mostly in tissues with a high demand for ATP: brain, heart, skeletal muscle, and retina. On the other hand, some antibacterial inhibitors of mitochondrial biogenesis (e.g., tetracyclines) inhibit selectively tumor cell proliferation. Therefore they may be considered for use in anticancer therapy. The article summarizes the response of mitochondria and plastids in various organisms to drugs and environmental xenobiotics. Various model organisms suitable for detection of xenobiotic effect on mitochondria (plastids) are presented as well as the possible consequences of such interaction.

Morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus growing under nitrogen-fixing conditions

The morphological and ultrastructural characteristics of the cyanobacterium Mastigocladus laminosus growing under N2-fixing conditions were examined with light and electron microscopy. Vegetative cells in narrow filaments contained randomly arranged segments of thylakoid membrane, centrally located carboxysomes (polyhedral bodies), peripherally located lipid bodies, and large numbers of polysaccharide granules in addition to nuclear material and ribosomes. The ultrastructural characteristics of cells in wide filaments were similar, except for increased numbers of carboxysomes and lipid bodies. Heterocytes and proheterocysts developed at a variety of locations in narrow filaments, wide filaments, and the lateral branches off of wide filaments. Akinetes were not observed in any of the filaments. The morphological characteristics of heterocysts and proheterocysts were variable and depended on those of the vegative cells from which the heterocysts and proheterocysts developed. Mature M. laminosus heterocysts were somewhat similar to those formed in other cyanobacterial genera, but they possessed a number of distinct and unique ultrastructural characteristics, including (i) the absence of a fibrous and, possibly, a laminated wall layer, (ii) the presence many closely packed membranes throughout most of the cytoplasm, and (iii) the presence of unidentified, spherical inclusion bodies of variable electron density.