rRNA Transcription Unit Research Articles

Ribosomal intergenic spacers are filled with transposon remnants.

Eukaryotic ribosomal DNA (rDNA) comprises tandem units of highly-conserved coding genes separated by rapidly-evolving spacer DNA. The spacers of all 12 species examined were filled with short direct repeats (DRs) and multiple long tandem repeats (TRs), completing the rDNA maps that previously contained unannotated and inadequately studied sequences. The external transcribed spacers also were filled with DRs and some contained TRs. We infer that the spacers arose from transposon insertion, followed by their imprecise excision, leaving short DRs characteristic of transposon visitation. The spacers provided a favored location for transposon insertion because they occupy loci containing hundreds to thousands of gene repeats. The spacers' primary cellular function may be to link one rRNA transcription unit to the next, whereas transposons flourish here because they have colonized the most frequently-used part of the genome.

Draft genomes of Perkinsus olseni and Perkinsus chesapeaki reveal polyploidy and regional differences in heterozygosity

Perkinsus spp. parasites have significant impact on aquaculture and wild mollusc populations. We sequenced the genomes of five monoclonal isolates of Perkinsus olseni and one Perkinsus chesapeaki from international sources. Sequence analysis revealed similar levels of repetitive sequence within species, a polyploid genome structure, and substantially higher heterozygosity in Oceanian-sourced isolates. We also identified tandem replication of the rRNA transcriptional unit, with high strain variation. Characterized gene content was broadly similar amongst all Perkinsus spp. but P. olseni Oceanian isolates contained an elevated number of genes compared to other P. olseni isolates and cox3 could not be identified in any Perkinsus spp. sequence. Phylogenetics and average nucleotide identity scans were consistent with all P. olseni isolates being within one species. These are the first genome sequences generated for both P. olseni and P. chesapeaki and will allow future advances in diagnostic design and population genomics of these important aquatic parasites.

Initial Steps in RNA Processing and Ribosome Assembly Occur at Mitochondrial DNA Nucleoids

Mammalian mitochondrial DNA (mtDNA) resides in compact nucleoids, where it is replicated and transcribed into long primary transcripts processed to generate rRNAs, tRNAs, and mRNAs encoding 13 proteins. This situation differs from bacteria and eukaryotic nucleoli, which have dedicated rRNA transcription units. The assembly of rRNAs into mitoribosomes has received little study. We show that mitochondrial RNA processing enzymes involved in tRNA excision, ribonuclease P (RNase P) and ELAC2, as well as a subset of nascent mitochondrial ribosomal proteins (MRPs) associate with nucleoids to initiate RNA processing and ribosome assembly. SILAC pulse-chase labeling experiments show that nascent MRPs recruited to the nucleoid fraction were highly labeled after the pulse in a transcription-dependent manner and decreased in labeling intensity during the chase. These results provide insight into the landscape of binding events required for mitochondrial ribosome assembly and firmly establish the mtDNA nucleoid as a control center for mitochondrial biogenesis.

Myb-binding Protein 1a (Mybbp1a) Regulates Levels and Processing of Pre-ribosomal RNA

Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes.

Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum

The amoeba Dictyostelium discoideum is a well established model organism for studying numerous aspects of cellular and developmental functions. Its ribosomal RNA (rRNA) is encoded in an extrachromosomal palindrome that exists in ∼100 copies in the cell. In this study, we have set out to investigate the sequence of the expressed rRNA. For this, we have ligated the rRNA ends and performed RT-PCR on these circular RNAs. Sequencing revealed that the mature 26 S, 17 S, 5.8 S, and 5 S rRNAs have sizes of 3741, 1871, 162, and 112 nucleotides, respectively. Unlike the published data, all mature rRNAs of the same type uniformly display the same start and end nucleotides in the analyzed AX2 strain. We show the existence of a short lived primary transcript covering the rRNA transcription unit of 17 S, 5.8 S, and 26 S rRNA. Northern blots and RT-PCR reveal that from this primary transcript two precursor molecules of the 17 S and two precursors of the 26 S rRNA are generated. We have also determined the sequences of these precursor molecules, and based on these data, we propose a model for the maturation of the rRNAs in Dictyostelium discoideum that we compare with the processing of the rRNA transcription unit of Saccharomyces cerevisiae.

Ribosomal RNA genes in eukaryotic microorganisms: witnesses of phylogeny?

The study of genomic organization and regulatory elements of rRNA genes in metazoan paradigmatic organisms has led to the most accepted model of rRNA gene organization in eukaryotes. Nevertheless, the rRNA genes of microbial eukaryotes have also been studied in considerable detail and their atypical structures have been considered as exceptions. However, it is likely that these organisms have preserved variations in the organization of a versatile gene that may be seen as living records of evolution. Here, we review the organization of the main rRNA transcription unit (rDNA) and the 5S rRNA genes (5S rDNA). These genes are reiterated in the genome of microbial eukaryotes and may be coded alone, in tandem repeats, linked to each other or linked to other genes. They may be found in the chromosome or extrachromosomally in linear or circular units. rDNA coding regions may contain introns, sequence insertions, protein-coding genes or additional spacers. The 5S rDNA can be found in tandem repeats or genetically linked to genes transcribed by RNA polymerases I, II or III. Available information from about a hundred microbial eukaryotes was used to review the unexpected diversity in the genomic organization of rRNA genes.

Epigenetic regulation of TTF-I-mediated promoter–terminator interactions of rRNA genes

Ribosomal RNA synthesis is the eukaryotic cell's main transcriptional activity, but little is known about the chromatin domain organization and epigenetics of actively transcribed rRNA genes. Here, we show epigenetic and spatial organization of mouse rRNA genes at the molecular level. TTF-I-binding sites subdivide the rRNA transcription unit into functional chromatin domains and sharply delimit transcription factor occupancy. H2A.Z-containing nucleosomes occupy the spacer promoter next to a newly characterized TTF-I-binding site. The spacer and the promoter proximal TTF-I-binding sites demarcate the enhancer. DNA from both the enhancer and the coding region is hypomethylated in actively transcribed repeats. 3C analysis revealed an interaction between promoter and terminator regions, which brings the beginning and end of active rRNA genes into close contact. Reporter assays show that TTF-I mediates this interaction, thereby linking topology and epigenetic regulation of the rRNA genes.

De novo ribosome biosynthesis is transcriptionally regulated in Eimeria tenella, dependent on its life cycle stage

Protozoan parasites go through various developmental stages during their parasitic life, which requires the expression of different genes. To identify stage specific gene products in Eimeria tenella, a differential screening was performed comparing the intracellular schizont stage with the extracellular oocyst stage. De novo transcripts of 18S–5.8S–26S rRNA transcription units and of two ribosomal proteins (RPL5 and RPL23) were specifically identified in schizonts and were undetectable in oocysts. The stage specific transcription of pre-rRNAs (prior to processing) was confirmed with Northern blot analysis. Since the E. tenella genome contains a repeated gene cluster with an estimated 140 large rRNA transcription units, they all might be similarly regulated. Specific expression of RPL5 and RPL23 in E. tenella schizonts was also confirmed by Northern blotting. Furthermore, an analysis of the E. tenella EST database with 26,705 ESTs showed that 9.5% of all merozoite ESTs and only 0.2% of the sporozoite ESTs encoded ribosomal proteins (RPs). These ESTs encoded 69 different RPs, suggesting that most and possibly all RPs are differentially transcribed in E. tenella. Analysis of EST data from other Coccidia, such as Toxoplasma gondii, indicated a similar stage dependent transcription of RP genes. We conclude that ribosome biosynthesis is transcriptionally regulated in E. tenella and other Coccidia, such that rapidly growing parasite stages utilize much of their resources to de novo biosynthesis of ribosomes, and that “dormant” oocyst stages do not synthesize new ribosomes. The 50- to 100-fold reduction in transcription of RPs together with the reduced rRNA transcription prevents that unnecessary new ribosomes are synthesized in oocysts.

Transcription termination factor reb1p causes two replication fork barriers at its cognate sites in fission yeast ribosomal DNA in vivo.

Polar replication fork barriers (RFBs) near the 3' end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe. S. pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3' to 5' order. The transcription termination protein reb1 and its two binding sites, present at the 3' end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo. On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, cis- and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also. S. pombe is suggested to be a transitional species in which both mechanisms coexist.

STEPHANODISCUS NEOASTRAEA AND STEPHANODISCUS HETEROSTYLUS (BACILLARIOPHYTA) ARE ONE AND THE SAME SPECIES

Stephanodiscus neoastraea Håkansson et Hickel is distinguished from S. heterostylus Håkansson et Meyer by the absence of valve face fultoportulae. Scanning electron microscopy analyses (SEM) revealed, that cultures established from a single cell of S. neoastraea and/or S. heterostylus persistently contained a mixture' of cells with and without valve face fultoportulae. Amplification of the ITS2 spacer region of the nuclear encoded rRNA transcription unit yielded a single band in an agarose gel electrophoresis for three “mixed” cultures tested. Furthermore, sequencing of the ITS2 region resulted in unambiguous sequences that were identical for each strain investigated. Ambiguous peaks which may originate from sequencing a mixture of different templates did not appear in the electropherogram. Thus, no, one or several valve face fultoportulae appear in the same species. Absence or presence of fultoportulae is not a useful character for differentiating S. neoastraea and S. heterostylus. Presence or absence of tubular processes on the valve face is a variable character, clearly expressed in clonal culture, and thus cannot be used for species discrimination. The species name S. heterostylus is a “nomen nudum”, and thus invalid.

Nucleotide sequences of ribosomal internal transcribed spacers and their utility in distinguishing closely related Perinereis polychaets (Annelida; Polychaeta; Nereididae).

Nucleotide sequences of a segment of the rRNA transcription unit spanning from the 3' end of the 18S rDNA to the 5' end of 28S rDNA were determined for four species of Perinereis polychaetes: P. aibuhitensis, P. floridana, and two undescribed species, Perinereis sp1 and sp2. The 5.8S rDNA sequences are identical among the four species. Intraspecific variability was low with the Kimura 2-parameter (K2P) distance, ranging from 0 to 0.0138 for ITS1 and 0 to 0.0247 for ITS2. The interspecific nucleotide difference was significantly higher than those within species, with a mean K2P of 0.172 for ITS1 and 0.204 for ITS2, suggesting that comparisons of ITS regions can be used to evaluate the phylogenetic relationships among Perinereis species. Both neighbor-joining and parsimony analyses of ITS variability indicate a close relationship between the two undescribed species of Perinereis. These findings highlight the utility of the ITS sequence in conjunction with other morphological and ecological characters to delineate species boundaries among closely related polychaetes.

Replication Initiates at Multiple Dispersed Sites in the Ribosomal DNA Plasmid of the Protozoan Parasite Entamoeba histolytica

In the protozoan parasite Entamoeba histolytica (which causes amoebiasis in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid. For strain HM-1:IMSS, the size of the rDNA plasmid is 24.5 kb, and 200 copies per genome are present. Each circle contains two rRNA transcription units as inverted repeats separated by upstream and downstream spacers. We have studied the replication of this molecule by neutral/neutral two-dimensional gel electrophoresis and by electron microscopy. All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to simple Y's and bubbles. This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid. On the basis of the intensity of the bubble arcs, initiations from the rRNA transcription units seemed to occur more frequently than those from intergenic spacers. Multiple, dispersed initiation sites were also seen in the rDNA plasmid of strain HK-9 when it was analyzed by two-dimensional gel electrophoresis. Electron microscopic visualization of replicating plasmid molecules in strain HM-1:IMISS showed multiple replication bubbles in the same molecule. The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which linearize the molecule, and with SacII, which cuts the circle twice. The distance of the bubbles from one end of the molecule was measured by electron microscopy. The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the molecule and that they appeared more frequently in rRNA transcription units. The same interpretation was drawn from electron microscopic analysis of the HK-9 plasmid. Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmid initiates at multiple sites. Potential replication origins are distributed throughout the plasmid. Such a mechanism is not known to operate in any naturally occurring prokaryotic or eukaryotic plasmid.

Effects of the Antiterminator BoxA on Transcription Elongation Kinetics and ppGpp Inhibition of Transcription Elongation in Escherichia coli

It has been shown previously that two different mRNA chains (lacZ and infB) are elongated at a rate of approximately 40 nucleotides (nt)/s during steady state growth on minimal medium and that the rate of mRNA chain elongation is inhibited by ppGpp in vivo. On the other hand, it was found that a truncated ribosomal RNA chain was elongated at a rate of approximately 80 nt/s, independent of growth condition (Vogel, U., and Jensen, K. F. (1994) J. Biol. Chem. 269, 16236-16241). We reasoned that the different transcriptional behavior of mRNA genes and rRNA operons might be caused by the antiterminator sequences present in the rRNA operons. To test this possibility, we have (a) inserted the minimal antiterminator boxA sequence between the promoter and the lacZ and infB genes and (b) deleted the antiterminator sequences from the rRNA transcription unit and measured transcription elongation rates in vivo on the resulting hybrid genes. We found that insertion of boxA in front of the coding region of lacZ increased the transcription elongation rate from 42 nt/s to 69 nt/s during steady state growth and that it eliminated the ppGpp-dependent decrease in the transcription elongation rate during the stringent response. On the other hand, deletion of the antiterminator sequences from the rRNA operon resulted in a reduced transcription elongation rate, but the elongation rate was still insensitive to changes in the ppGpp pool. These results are consistent with the hypothesis that the antiterminator boxA is a primary determinant of the rate of transcription elongation rate.

Effects of different growth conditions on the in vivo activity of the tandem Escherichia coli ribosomal RNA promoters P1 and P2.

We have analyzed the relative activities of the Escherichia coli ribosomal RNA promoters P1 and P2 in vivo under different physiological conditions. Promoter efficiencies were determined by quantitative comparison of the transcript-specific primer extension products obtained from total RNA preparations. Cells were analyzed at different stages of the growth cycle, at different growth rates, and under conditions of stringent control. In addition, the rRNA gene dosage was altered by transformation with plasmids containing additional rrnD or rrnB transcription units, or rRNA operons in which one of the tandem promoters (P1) had been deleted. Under conditions of amino acid starvation (stringent control) we observed the expected strong reduction in P1-directed transcription. In contrast to the previous assumption that the P2 promoter is not regulated, we simultaneously noticed a smaller but significant repression of P2-directed transcription. In strains in which the rRNA gene dosage was increased by transformation with plasmids bearing rRNA transcription units, a similar degree of repression was observed. Repression of the P1 promoter activity was increased, however, when cells contained extra rRNA operons with P2 promoters only. As demonstrated under stringent control conditions, changes in the growth cycle also affected the activity of promoters P1 and P2. A greater proportion of P2-derived transcripts was observed when cells changed from exponential to stationary growth or if cultures were grown in minimal medium. Under steady-state, slow growth conditions (minimal medium) we obtained evidence showing that the ratio of P1/P2 transcription products is much lower for cells with extra rrnB as compared to extra rrnD operons or cells lacking extra rRNA operons, implying an operon-specific regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural localization of rDNA and rRNA by in situ hybridization in the nucleolus of human spermatids

The ultrastructural localization of rDNA and rRNA within the nucleolus of human spermatids was investigated by in situ hybridization at steps 1 and 2. Two different digoxigenin-labeled human probes from the rRNA transcription unit were used. Identification of hybrids was performed with immunogold techniques. Comparative observations in the Sertoli cell nucleolus as controls revealed that rDNA was predominantly visualized in the threads of the dense fibrillar component, while rRNA was detected over both the fibrillar component and the granular component. Within the nucleolus of round spermatids in the same sections of seminiferous tubules, rDNA labeling was localized over the spherical or stranded dense fibrillar components. rRNA labeling was found not only over these components but also in the adjacent nucleoplasm rich in ribonucleoprotein particles. These results are consistent with the view that the round spermatid nucleolus is transcriptionally active.

Alternate pathways for processing in the internal transcribed spacer 1 in pre-rRNA of Saccharomyces cerevisiae.

We have extended the system of Nogi et al. (Proc. Natl. Acad. Sci. USA 88, 1991, 3962-3966) for transcription of rRNA from an RNA polymerase II promoter in strains lacking functional RNA polymerase I. In our strains two differentially marked rRNA transcription units can be expressed alternately. Using this system we have shown that the A2 processing site in the internal transcribed spacer 1 (ITS1) of the pre-rRNA is dispensable. According to the accepted processing scheme, the A2 site serves to separate the parts of the primary rRNA transcript that are destined for incorporation into the two ribosomal subunits. However, we have found that, when A2 is impaired, separation of the small and large subunit rRNAs occurs at a processing site further downstream in ITS1, indicating that alternate pathways for ITS1 processing exist. Short deletions in the A2 region still allow residual processing at the A2 site. Mapping of the cleavage sites in such deletion transcripts suggests that sequences downstream of the A2 site are used for determining the position of the cleavage.

The regulation of ribosomal RNA synthesis and bacterial cell growth.

The life style of bacteria such as Escherichia coli can be characterized as 'feast or famine'. As a consequence, bacteria have evolved mechanisms enabling efficient adaptation of their growth rates to the rapid changes in nutritional supply, but also to environmental alterations, like temperature, osmolarity or competition by other species. Bacteria are capable, for instance, in a very effective way, to change their protein synthesizing capacity according to the growth demands. However, there is a natural upper limit in the rate at which a single ribosome catalyzes polypeptide formation (ca. 20 amino acids/s) (Engbaek et al. 1973). Therefore, in fast-growing cells, the number of ribosomes has to increase in proportion to the cell mass (Gausing 1977). Since ribosomes are multi-component particles, composed of 3 different RNA molecules and at least 52 different proteins, coordination and balance in the synthesis of all components is a prerequisite for efficient ribosome formation. The key molecules for the regulation of ribosome biogenesis are the ribosomal RNAs (rRNAs). Their synthesis rates increase in proportion to the square of the cell growth rates, and the expression of many, if not all, of the ribosomal proteins is linked to the availability of the free rRNA fraction in the cell by a translational feedback mechanism (Nomura et al. 1984). Consequently, the regulation of rRNA synthesis is of prime importance for the rate of ribosome formation, and thus, for the establishment of conditions for rapid cell growth. It is only natural, therefore, that the synthesis of rRNAs is regulated in a very complex way, combining many different cellular parameters. Both, positive and negative regulatory mechanisms are acting in concert to maintain a balanced synthesis. Control is exerted at all different levels, including changes in the gene dosage during replication, factor-dependent and independent activation and repression of transcription, but also post-transcriptional mechanisms affecting the maturation and assembly of ribosomal particles. The multiplicity of the various different regulatory aspects is covered by several recent reviews (Lamond 1985; Lindahl and Zengel 1986; Nomura etal. 1984; Wagner 1989; Wagner et al. 1992). Exciting new findings have been discovered since then. This review is focused mainly on different facets of transcriptional regulation. In the first part some features of regulation related to the chromosomal organization and the tandem promoter arrangements of rRNA transcription units are summarized. Old and some new aspects of the mechanisms underlying the global regulation, known as stringent and growth rate control, will then be discussed. Finally, molecular mechanisms of transcription activation, the new identification of a specific repressor, and a model explaining the counteracting effects of transcription factors in the activation and repression of rRNA synthesis, according to the growth conditions, will be reported.

The terminal balls characteristic of eukaryotic rRNA transcription units in chromatin spreads are rRNA processing complexes.

When spread chromatin is visualized by electron microscopy, active rRNA genes have a characteristic Christmas tree appearance: From a DNA "trunk" extend closely packed "branches" of nascent transcripts whose ends are decorated with terminal "balls." These terminal balls have been known for more than two decades, are shown in most biology textbooks, and are reported in hundreds of papers, yet their nature has remained elusive. Here, we show that a rRNA-processing signal in the 5'-external transcribed spacer (ETS) of the Xenopus laevis ribosomal primary transcript forms a large, processing-related complex with factors of the Xenopus oocyte, analogous to 5' ETS processing complexes found in other vertebrate cell types. Using mutant rRNA genes, we find that the same rRNA residues are required for this biochemically defined complex formation and for terminal ball formation, analyzed electron microscopically after injection of these cloned genes into Xenopus oocytes. This, plus other presented evidence, implies that rRNA terminal balls in Xenopus, and by inference, also in the multitude of other species where they have been observed, are the ultrastructural visualization of an evolutionarily conserved 5' ETS processing complex that forms on the nascent rRNA.

Nucleotide sequence analysis of the rRNA transcription unit of a pathogenic Entamoeba histolytica strain HM-1:IMSS.

Nucleotide sequence analysis of the rRNA transcription unit of a pathogenic Entamoeba histolytica strain HM-1:IMSS.

Evidence that intergenic spacer repeats of Drosophila melanogaster rRNA genes function as X-Y pairing sites in male meiosis, and a general model for achiasmatic pairing.

In Drosophila melanogaster males, X-Y meiotic chromosome pairing is mediated by the nucleolus organizers (NOs) which are located in the X heterochromatin (Xh) and near the Y centromere. Deficiencies for Xh disrupt X-Y meiotic pairing and cause high frequencies of X-Y nondisjunction. Insertion of cloned rRNA genes on an Xh- chromosome partially restores normal X-Y pairing and disjunction. To map the sequences within an inserted, X-linked rRNA gene responsible for stimulating X-Y pairing, partial deletions were generated by P element-mediated destabilization of the insert. Complete deletions of the rRNA transcription unit did not interfere with the ability to stimulate X-Y pairing as long as most of the intergenic spacer (IGS) remained. Within groups of deletions that lacked the entire transcription unit and differed only in length of residual IGS material, pairing ability was proportional to the dose of 240-bp intergenic spacer repeats. Deletions of the complete rRNA transcription unit or the 28S sequences alone blocked nucleolus formation, as determined by binding of an antinucleolar antibody, yet did not interfere with pairing ability, suggesting that X-Y pairing may not be mechanistically related to nucleolus formation. A model for achiasmatic pairing in Drosophila males based upon the combined action of topoisomerase I and a strand transferase is proposed.