Ribosomal Precursor RNA Research Articles

In vivo release of mitotic silencing of ribosomal gene transcription does not give rise to precursor ribosomal RNA processing.

Nuclear RNA transcription is repressed when eukaryotic cells enter mitosis. Here, we found that the derepression of ribosomal gene (rDNA) transcription that normally takes place in telophase may be induced in prometaphase, metaphase, and anaphase mitotic HeLa cells, and therefore appears not to be dependent on completion of mitosis. We demonstrate for the first time that in vivo inhibition of the cdc2- cyclin B kinase activity is sufficient to give rise to okadaic acid-sensitive dephosphorylation of the mitotically phosphorylated forms of components of the rDNA transcription machinery, and consequently to restore rDNA transcription in mitotic cells. These results, showing that during mitosis the rDNA transcription machinery is maintained repressed by the cdc2-cyclin B kinase activity, provide an in vivo demonstration of the cell cycle-dependent regulation of rDNA transcription. Interestingly in mitotic cells, the newly synthesized 47S precursor ribosomal RNA (pre-rRNA) is not processed into the mature rRNAs, indicating that rDNA transcription and pre-rRNA processing may be uncoupled. Moreover this suggests that inhibition of the cdc2- cyclin B kinase is not sufficient to activate the 47S pre-rRNA processing machinery and/or to induce its relocalization at the level of newly synthesized 47S pre-rRNA. This in vivo approach provides new possibilities to investigate the correlation between pre-rRNA synthesis and pre-rRNA processing when the nucleolus reforms.

Secondary structure model for the ITS-2 precursor rRNA of strongyloid nematodes of equids: implications for phylogenetic inference

In order to maximise the positional homology in the primary sequence alignment of the second internal transcribed spacer for 30 species of equine strongyloid nematodes, the secondary structures of the precursor ribosomal RNA were predicted using an approach combining an energy minimisation method and comparative sequence analysis. The results indicated that a common secondary structure model of the second internal transcribed spacer of these nematodes was maintained, despite significant interspecific differences (2–56%) in primary sequences. The secondary structure model was then used to refine the primary second internal transcribed spacer sequence alignment. The “manual” and “structure” alignments were both subjected to phylogenetic analysis using three different tree-building methods to compare the effect of using different sequence alignments on phylogenetic inference. The topologies of the phylogenetic trees inferred from the manual second internal transcribed spacer alignment were usually different to those derived from the structure second internal transcribed spacer alignment. The results suggested that the positional homology in the second internal transcribed spacer primary sequence alignment was maximised when the secondary structure model was taken into consideration.

Small nucleolar RNAs.

Many small RNA species associate with the nucleolar structure. Some of these small nucleolar RNAs (snoRNAs) are required for cleavage processing of ribosomal RNA precursors. There are many pseudouridine residues and methylated riboses in mature ribosomal RNA. For most, if not all, of these modifications, each site is selected by base pairing with a specific snoRNA species. Some snoRNAs are needed for the 2'-O-ribose methylation of at least one spliceosomal small nuclear RNA. Many snoRNAs, particularly in yeast, are generated from independent transcription units. Most vertebrate snoRNAs are produced by processing of introns from protein-coding transcripts. Some snoRNAs are made by processing of introns from non-protein-coding transcripts.

Nucleolin functions in the first step of ribosomal RNA processing.

The first processing step of precursor ribosomal RNA (pre-rRNA) involves a cleavage within the 5' external transcribed spacer. This processing requires sequences downstream of the cleavage site which are perfectly conserved among human, mouse and Xenopus and also several small nucleolar RNAs (snoRNAs): U3, U14, U17 and E3. In this study, we show that nucleolin, one of the major RNA-binding proteins of the nucleolus, is involved in the early cleavage of pre-rRNA. Nucleolin interacts with the pre-rRNA substrate, and we demonstrate that this interaction is required for the processing reaction in vitro. Furthermore, we show that nucleolin interacts with the U3 snoRNP. Increased levels of nucleolin, in the presence of the U3 snoRNA, activate the processing activity of a S100 cell extract. Our results suggest that the interaction of nucleolin with the pre-rRNA substrate might be a limiting step in the primary processing reaction. Nucleolin is the first identified metazoan proteinaceous factor that interacts directly with the rRNA substrate and that is required for the processing reaction. Potential roles for nucleolin in the primary processing reaction and in ribosome biogenesis are discussed.

Base-pairing of 23 S rRNA ends is essential for ribosomal large subunit assembly

In ribosomal RNA precursors the spacer sequences bracketing mature 16 S and 23 S rRNA are base-paired to form long helices (processing stems). In pre-23 S rRNA, the processing stem is continued by eight base-pairs of mature 23 S rRNA known as helix 1. Recently, we have found that any part of 23 S rRNA between positions 40 and 2773 could be deleted without the loss of ribosome-like particle formation, while both end regions were indispensable. In this paper we have analyzed the role of the 5′ and 3′ end regions of 23 S rRNA during ribosomal 50 S assembly in vivo by using mutants of the 23 S rRNA gene. Deletions and substitutions in both strands of the helix 1 lead to the loss of plasmid derived 50 S formation. Compensatory mutations restoring helix 1 were assembled into functional 50 S subunits. We conclude that the helix 1 of 23 S rRNA is the main RNA determinant for ribosomal large-subunit assembly. Deletions in both the 5′ and 3′ strand of the processing stem reduced the ability of the 23 S rRNA to form ribosomal 50 S subunits. However, even the complete removal of either the 5′ or the 3′ strand of the processing stem did not abolish the 50 S assembly completely. Thus, processing stem facilitates, but is not essential for assembly.

A novel ribozyme with ester transferase activity

Background: The ‘RNA world’ hypothesis proposes that the early history of life on earth consisted of a period in which chemical transformations were catalyzed exclusively by ribozymes. Ribozymes that act as acyl transferases, or catalyze the formation of amide or peptide bonds seem particularly attractive candidates to link the RNA world to the modern protein-nucleic acid world. The central role played by aminoacylated RNAs in today's processes of translating RNA into protein suggests that aminoacyl transfer reactions catalyzed by RNA might have facilitated the development and optimization of the translation apparatus during early evolution. Results: We describe the isolation and characterization of a novel ribozyme that catalyzes the transfer of an amino-acid ester from an aminoacyl donor substrate onto the ribozyme itself. The site of aminoacylation was determined to be at an internal 2′-OH group of a cytosine residue. The aminoacylation depends on the presence of Mg 2+ and can be competitively inhibited by the AMP moiety of the aminoacyl donor substrate, suggesting that there is a specific binding pocket for this substrate. The originally selected ribozyme was engineered to act as an intermolecular catalyst that transfers the amino acid onto an external 28-residue oligonucleotide. The aminoacylated oligonucleotide was further used to quantify the reverse reaction catalyzed by the ribozyme. Conclusions: The ribozyme we have isolated is an example of a catalytic RNA with ester transferase activity which uses a substrate that is not templated by Watson-Crick-pairing hydrogen bonds. The reaction catalyzed by the ribozyme expands the scope of RNA catalysis to include acyl transferase activity from an RNA 3′ end to an internal 2′ position and the reverse. Ribozymes with such activity have been posulated to be evolutionary precursors of ribosomal RNA.

Changes in the rate of RNA synthesis during the cell cycle.

Although the rate of RNA synthesis is known to drop at mitosis, the recent identification of 11 stages in the cell cycle (El-Alfy et al., 1994) makes it possible to measure the rate of this synthesis at each one of the stages and thus find out how it varies throughout the cell cycle. Mice were injected intravenously with the RNA precursor, 3H-uridine; the duodenum was fixed 5-15 minutes later for embedment in Epon, and the duodenal crypts were cut in semithin serial sections for study of the rapidly dividing crypt columnar cells. Using Feulgen-stained sections, each cell nucleus was assigned to one of the 11 stages described in the cell cycle, and the same nucleus was identified in the next serial section that had been processed for radioautography, so that the overlying silver grains were enumerated. The count was taken as an index of the rate of RNA synthesis by this nucleus. Starting from stage I of the cell cycle (the period defined by the presence of a minimal amount of chromatin during which the S phase begins) and up to stage IV (when the S phase ends and the G2 phase begins), all or nearly all nuclei are synthesizing RNA with the rate peaking at stage III. During stages V to VIII (the period comprising the mitotic steps), the percentage of RNA-synthesizing nuclei decreases to over half at stage V (prophase), -10% at stages VIa (prometaphase) and VIb (metaphase) and none at stages VII (anaphase) and VIII (telophase). During stages IX-XI (which correspond to the G1 phase), the percentage rises sharply at stage XI to reach up to 100% at stages X and XI. Finally, on the average, 35% of nuclear silver grains are over the nucleolus (presumably representing ribosomal RNA precursors), whereas 65% are over the nucleoplasm (presumably representing mainly heterogeneous RNA precursors). Cells synthesize RNA during the interphase, but at a variable rate with a peak in S. The synthesis proceeds in a majority of the cells at prophase, but only in a few of them at prometaphase and metaphase, and in none at anaphase and telophase.

Changes in the rate of RNA synthesis during the cell cycle

Background Although the rate of RNA synthesis is known to drop at mitosis, the recent identification of 11 stages in the cell cycle (El-Alfy et al., 1994) makes it possible to measure the rate of this synthesis at each one of the stages and thus find out how it varies throughout the cell cycle. Methods Mice were injected intravenously with the RNA precursor, 3H-uridine; the duodenum was fixed 5-15 minutes later for embedment in Epon, and the duodenal crypts were cut in semithin serial sections for study of the rapidly dividing crypt columnar cells. Using Feulgen-stained sections, each cell nucleus was assigned to one of the 11 stages described in the cell cycle, and the same nucleus was identified in the next serial section that had been processed for radioautography, so that the overlying silver grains were enumerated. The count was taken as an index of the rate of RNA synthesis by this nucleus. Results Starting from stage I of the cell cycle (the period defined by the presence of a minimal amount of chromatin during which the S phase begins) and up to stage IV (when the S phase ends and the G2 phase begins), all or nearly all nuclei are synthesizing RNA with the rate peaking at stage III. During stages V to VIII (the period comprising the mitotic steps), the percentage of RNA-synthesizing nuclei decreases to over half at stage V (prophase), ≈10% at stages VIa (prometaphase) and VIb (metaphase) and none at stages VII (anaphase) and VIII (telophase). During stages IX-XI (which correspond to the G1 phase), the percentage rises sharply at stage XI to reach up to 100% at stages X and XI. Finally, on the average, 35% of nuclear silver grains are over the nucleolus (presumably representing ribosomal RNA precursors), wheras 65% are over the nucleoplasm (presumably representing mainly heterogeneous RNA precursors). Conclusions Cells synthesize RNA during the interphase, but at a variable rate with a peak in S. The synthesis proceeds in a majority of the cells at prophase, but only in a few of them at prometaphase and metaphase, and in none at anaphase and telophase. Anat. Rec. 250:6–12, 1998. © 1998 Wiley-Liss, Inc.

Reconstitution of Functional Eukaryotic Ribosomes fromDictyostelium discoideum Ribosomal Proteins and RNA

40 and 60 S ribosomal subunits have been reconstituted in vitro from purified ribosomal RNA and ribosomal proteins of Dictyostelium discoideum. The functionality of the reconstituted ribosomes was demonstrated in in vitro mRNA-directed protein synthesis. The reassembly proceeded well with immature precursors of ribosomal RNA but poorly if at all with mature cytoplasmic RNA species. Reassembly also required a preparation of small nuclear RNA(s), acting as morphopoietic factor(s).

Diversity of antinucelar antibody responses in hepatocellular carcinoma

Background/Aims: The development of antinuclear antibodies (ANA) in malignancies has been described but its mechanism is still not understood. The aim of this study was to examine ANA specificities in hepatocellular carcinoma to further understand autoimmunity in cancer. Methods: Two hundred and four hepatocellular carcinoma patients were compared with 68 chronic hepatitis C, with 126 chronic hepatitis B and with 30 alcoholic liver cirrhosis patients, as well as with 87 healthy donors. Indirect immunofluorescence, immunoblotting, and immunoprecipitation were used to study ANA reactivities. Results: Hepatocellular carcinoma had a significantly higher frequency of ANA using HEp-2 cells as substrate (31%) than chronic hepatitis C (10%), chronic hepatitis B (9.5%), alcoholic liver cirrhosis (10%) or healthy donors (4.5%). A great diversity of ANA specificities was found in hepatocellular carcinoma. Three hepatoma sera had antibodies that co-localized with non-snRNP splicing factor SC35, suggesting that the antigenic targets might be involved in mRNA splicing. We identified antibodies to two known nuclear autoantigens: fibrillarin and p330 d/CENP-F. These autoantigens are involved in the 5′ processing of precursor ribosomal RNA transcripts and in mitotic functions, respectively. Conclusions: Diversity was found in the autoantibody specificity, in contrast to the specific subsets of auto-antibodies seen in several systemic rheumatic autoimmune diseases. Our data suggest that immune response in hepatocellular carcinoma targets important proteins involved in cellular biosynthetic or proliferative functions.

U3 snoRNA may recycle through different compartments of the nucleolus.

A model is proposed in which U3 small nucleolar RNA (snoRNA) is recruited from an inactive, stored form in the dense fibrillar component (DFC) of the nucleolus to an active form that is associated with the initial ribosomal RNA (rRNA) precursor. The initial steps of rRNA processing occur in the DFC, and then it is proposed that the U3 snoRNA moves with intermediates in rRNA processing from the DFC to the granular component (GC) of the nucleolus. The nucleolar protein fibrillarin is located primarily in the DFC, and it is suggested that the complex of fibrillarin and U3 snoRNA dissociates when U3 snoRNA transits to the GC. Finally, when U3 snoRNA is released from the processed rRNA, the tether holding the rRNA in the nucleolus is broken and rRNA can then be exported from the nucleolus to the cytoplasm. U3 snoRNA is hypothesized to recycle back from the GC to the DFC where it is stored until future association with another initial rRNA precursor. Data supporting this model are summarized. U3 snoRNA is also stored in the coiled body of interphase cells and in the nucleolar remnants and prenucleolar bodies of mitotic cells, and there may be some similarity in the binding sites for stored U3 snoRNA in the DFC and in these structures.

Three small nucleolar RNAs that are involved in ribosomal RNA precursor processing.

Three small nucleolar RNAs (snoRNAs), E1, E2 and E3, have been described that have unique sequences and interact directly with unique segments of pre-rRNA in vivo. In this report, injection of antisense oligodeoxynucleotides into Xenopus laevis oocytes was used to target the specific degradation of these snoRNAs. Specific disruptions of pre-rRNA processing were then observed, which were reversed by injection of the corresponding in vitro-synthesized snoRNA. Degradation of each of these three snoRNAs produced a unique rRNA maturation phenotype. E1 RNA depletion shut down 18 rRNA formation, without overaccumulation of 20S pre-rRNA. After E2 RNA degradation, production of 18S rRNA and 36S pre-rRNA stopped, and 38S pre-rRNA accumulated, without overaccumulation of 20S pre-rRNA. E3 RNA depletion induced the accumulation of 36S pre-rRNA. This suggests that each of these snoRNAs plays a different role in pre-rRNA processing and indicates that E1 and E2 RNAs are essential for 18S rRNA formation. The available data support the proposal that these snoRNAs are at least involved in pre-rRNA processing at the following pre-rRNA cleavage sites: E1 at the 5' end and E2 at the 3' end of 18S rRNA, and E3 at or near the 5' end of 5.8S rRNA.

The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria.

The Cbp2 protein is encoded in the nucleus and is required for the splicing of the terminal intron of the mitochondrial COB gene in Saccharomyces cerevisiae . Using a yeast strain that lacks this intron but contains a related group I intron in the precursor of the large ribosomal RNA, we have determined that Cbp2 protein is also required for the normal accumulation of 21S ribosomal RNA in vivo . Such strains bearing a deletion of the CBP2 gene adapt slowly to growth in glycerol/ethanol media implying a defect in derepression. At physiologic concentrations of magnesium, Cbp2 stimulates the splicing of the ribosomal RNA intron in vitro . Nevertheless, Cbp2 is not essential for splicing of this intron in mitochondria nor is it required in vitro at magnesium concentrations >5 mM. A similar intron exists in the large ribosomal RNA (LSU) gene of Saccharomyces douglasii . This intron does need Cbp2 for catalytic activity in physiologic magnesium. Similarities between the LSU introns and COB intron 5 suggest that Cbp2 may recognize conserved elements of the these two introns, and protein-induced UV crosslinks occur in similar sites in the substrate and catalytic domains of the RNA precursors.

New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme.

Group I introns self-splice via two consecutive trans-esterification reactions in the presence of guanosine cofactor and magnesium ions. Comparative sequence analysis has established that a catalytic core of about 120 nucleotides is conserved in all known group I introns. This core is generally not sufficient for activity, however, and most self-splicing group I introns require non-conserved peripheral elements to stabilize the complete three-dimensional (3D) structure. The physico-chemical properties of group I introns make them excellent systems for unraveling the structural basis of the RNA-RNA interactions responsible for promoting the self-assembly of complex RNAs. We present phylogenetic and experimental evidence for the existence of three additional tertiary base pairings between hairpin loops within peripheral components of subgroup IC1 and ID introns. Each of these new long range interactions, called P13, P14 and P16, involves a terminal loop located in domain 2. Although domains 2 of IC and ID introns share very strong sequence similarity, their terminal loops interact with domains 5 and 9 (subgroup IC1) and domain 6 (subgroup ID). Based on these tertiary contacts, comparative sequence analysis, and published experimental results such as Fe(II)-EDTA protection patterns, we propose 3D models for two entire group I introns, the subgroup IC1 intron in the large ribosomal precursor RNA of Tetrahymena thermophila and the SdCob.1 subgroup ID intron found in the cytochrome b gene of Saccharomyces douglasii. Three-dimensional models of group I introns belonging to four different subgroups are now available. They all emphasize the modular and hierarchical organization of the architecture of group I introns and the widespread use of base-pairings between terminal hairpin loops for stabilizing the folded and active structures of large and complex RNA molecules.

Inhibition of nucleolar function and morphological change by adriamycin associated with heat shock protein 70 accumulation.

Adriamycin (ADR) has been considered to target mainly DNA metabolism in the nucleus. Recently, we observed the nuclear translocation of heat shock protein 70 (HSP70) after ADR treatment. We examined which intranuclear changes might be related to this alteration of HSP70 localization. We found considerable alternations in the nucleolar morphology and function in ADR‐treated tumor cells, i.e., a ring‐shaped segregation of granular components of almost all nucleoli and a dramatic reduction of nucleolar 45S ribosomal precursor RNA biosynthesis in HeLa cells exposed to 100 μM ADR for 2 h. Concomitantly with these changes, HSP70 was concentrated into the nucleoli, as in the case of heat shock treatment. These results indicate a novel anticancer effect of ADR via the suppression of cellular protein biosynthesis, in addition to its effect on DNA.

Accurate processing of a eukaryotic precursor ribosomal RNA by ribonuclease MRP in vitro.

Very few of the enzymes required for eukaryotic precursor ribosomal RNA (pre-rRNA) processing have been identified. Ribonuclease (RNase) MRP was characterized as a nuclease that cleaves mitochondrial replication primers, but it is predominantly nucleolar. Previous genetic evidence revealed that this ribonucleoprotein is required, directly or indirectly, for cleavage of the yeast pre-rRNA in vivo at site A3. Here, an in vitro processing system that accurately reproduces this cleavage is described. Biochemical purification and the use of extracts depleted of the MRP RNA demonstrate that endonucleolytic cleavage of the pre-rRNA is directly mediated by RNase MRP. This establishes a role for RNase MRP in the nucleolus.

Processing of pre‐ribosomal RNA in Saccharomyces cerevisiae

Post-transcriptional processing of precursor-ribosomal RNA comprises a complex pathway of endonucleolytic cleavages, exonucleolytic digestion and covalent modifications. The general order of the various processing steps is well conserved in eukaryotic cells, but the underlying mechanisms are largely unknown. Recent analysis of pre-rRNA processing, mainly in the yeast Saccharomyces cerevisiae, has significantly improved our understanding of this important cellular activity. Here we will review the data that have led to our current picture of yeast pre-rRNA processing.

Ribosomal RNA precursor processing by a eukaryotic U3 small nucleolar RNA-like molecule in an archaeon

An RNA-containing endonuclease that catalyzes the excision and maturation of the 16S ribosomal RNA (rRNA) from the rRNA primary transcript (pre-rRNA) in the hyperthermophilic archaeon Sulfolobus acidocaldarius has been characterized. The ribonucleoprotein was inactivated by micrococcal nuclease treatment and inactivation was reversed by reconstitution with bulk RNA. A 159-nucleotide RNA with sequence and structural similarity to U3 small nucleolar RNAs of eukaryotes copurified with the endonuclease activity. Oligonucleotide-targeted ribonuclease H inactivation of the U3-like RNA component also abolished processing activity. A motif within the U3 homolog is complementary to the region around the three cleavage sites in the pre-RNA substrate. Thus, U3-mediated processing of pre-rRNA is not specific to eukaryotes; its origin predates the divergence of archaea and eukaryotes.

Were RNA replication and translation directly coupled in the RNA (+protein?) world?

The ribosome is proposed to have evolved from an ancestor that simultaneously replicated and translated template RNA. At its decoding site, this ancestor to the ribosome carried a ribozyme that assembled product RNA by sequentially ligating anticodon triplets excised from tRNAs. This ribozyme was the ancestor of the Group I introns, which are still present on some ribosomal RNA precursors. Coupling of reversible RNA replication by transesterification with the thermodynamically favourable process of transpeptidation provides a rationale for the evolution of the complete ribosome as a replicase for large RNAs in the RNA (+protein?) world. A detailed and experimentally verifiable mechanism can be proposed for simultaneous replication and translation. Sequence requirements for recognition of the decoding complex as a substrate helix by these ribozymes are consistent with earlier models for the origin of the genetic code, but require an indirect mode for ribosomal self-replication. This proposal has the potential to explain the location of Group I introns in the anticodon loops of some tRNAs.

Detection of rifampin-resistant bacteria using DNA probes for precursor rRNA

Ribosomal RNA precursor (pre-rRNA) molecules have terminal domains (tails) which are removed during late steps in rRNA processing, to yield the mature rRNA subunits. Transcriptional inhibitors such as rifampin can deplete pre-rRNA in sensitive cells by inhibiting de novo pre-rRNA synthesis while allowing maturation to proceed. We developed direct DNA probe assays for pre-rRNA tail sequences of Escherichia coli, and evaluated their ability to rapidly distinguish rifampin-resistant from rifampin-sensitive strains in cultures treated with the drug. Pre-rRNA became undetectable in sensitive cells less than a generation time after rifampin exposure, but remained abundant in resistant cells. Resistant cells were detectable by this method against a 100-fold excess of sensitive cells, showing that this method can detect resistant mutants even when present as a small percentage of a pathogen population. Our data indicate that the response of pre-rRNA to antibiotic treatment is sufficient in rate and magnitude to make it a useful metabolic marker for antibiotic sensitivity.