Arrangement of the genes coding for ribosomal ribonucleic acids in Neurospora crassa.

We have cloned and characterized Neurospora crassa ribosomal deoxyribonucleic acid (rDNA). The rDNA is found as a tandemly repeated 6.0-megadalton sequence. We have mapped a portion of the rDNA repeat unit with respect to its sites for 13 restriction endonucleases and defined those regions coding for the 5. 8S, 17S, and 26S ribosomal ribonucleic acids (rRNA's). We have also isolated several clones containing 5S rRNA sequences. The 5S rRNA coding sequences are not found within the rDNA repeat unit. We found that the sequences surrounding the 5S rRNA coding regions are highly heterogeneous.     

Saccharomyces cerevisiae nucleolar protein Nop7p is necessary for biogenesis of 60S ribosomal subunits

To identify new gene products that participate in ribosome biogenesis, we carried out a screen for mutations that result in lethality in combination with mutations in DRS1, a Saccharomyces cerevisiae nucleolar DEAD-box protein required for synthesis of 60S ribosomal subunits. We identified the gene NOP7that encodes an essential protein. The temperature-sensitive nop7-1 mutation or metabolic depletion of Nop7p results in a deficiency of 60S ribosomal subunits and accumulation of halfmer polyribosomes. Analysis of pre-rRNA processing indicates that nop7 mutants exhibit a delay in processing of 27S pre-rRNA to mature 25S rRNA and decreased accumulation of 25S rRNA. Thus Nop7p, like Drs1p, is required for essential steps leading to synthesis of 60S ribosomal subunits. In addition, inactivation or depletion of Nop7p also affects processing at the A0, A1, and A2 sites, which may result from the association of Nop7p with 35S pre-rRNA in 90S pre-rRNPs. Nop7p is localized primarily in the nucleolus, where most steps in ribosome assembly occur. Nop7p is homologous to the zebrafish pescadillo protein necessary for embryonic development. The Nop7 protein contains the BRCT motif, a protein-protein interaction domain through which, for example, the human BRCA1 protein interacts with RNA helicase A.     

gar2 is a nucleolar protein from Schizosaccharomyces pombe required for 18S rRNA and 40S ribosomal subunit accumulation.

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.     

The Saccharomyces cerevisiae TIF6 gene encoding translation initiation factor 6 is required for 60S ribosomal subunit biogenesis

Eukaryotic translation initiation factor 6 (eIF6), a monomeric protein of about 26 kDa, can bind to the 60S ribosomal subunit and prevent its association with the 40S ribosomal subunit. In Saccharomyces cerevisiae, eIF6 is encoded by a single-copy essential gene. To understand the function of eIF6 in yeast cells, we constructed a conditional mutant haploid yeast strain in which a functional but a rapidly degradable form of eIF6 fusion protein was synthesized from a repressible GAL10 promoter. Depletion of eIF6 from yeast cells resulted in a selective reduction in the level of 60S ribosomal subunits, causing a stoichiometric imbalance in 60S-to-40S subunit ratio and inhibition of the rate of in vivo protein synthesis. Further analysis indicated that eIF6 is not required for the stability of 60S ribosomal subunits. Rather, eIF6-depleted cells showed defective pre-rRNA processing, resulting in accumulation of 35S pre-rRNA precursor, formation of a 23S aberrant pre-rRNA, decreased 20S pre-rRNA levels, and accumulation of 27SB pre-rRNA. The defect in the processing of 27S pre-rRNA resulted in the reduced formation of mature 25S and 5.8S rRNAs relative to 18S rRNA, which may account for the selective deficit of 60S ribosomal subunits in these cells. Cell fractionation as well as indirect immunofluorescence studies showed that c-Myc or hemagglutinin epitope-tagged eIF6 was distributed throughout the cytoplasm and the nuclei of yeast cells.     

Probing the conformation of 26S rRNA in yeast 60S ribosomal subunits with kethoxal

The conformation and accessibility of 26S rRNA in yeast 60S ribosomal subunits were probed with kethoxal. Oligonucleotides originating from reactive sites were isolated by diagonal electrophoresis and sequenced. From over 70 oligonucleotide sequences, 26 kethoxal-reactive sites could be placed in the 26S rRNA sequence. These are in close agreement with a proposed secondary structure model for the RNA that is based on comparative sequence analysis. At least seven kethoxal-reactive sites in yeast 26S rRNA are in positions that are exactly homologous to reactive positions in E. coli 23S rRNA; each of these sites has previously been implicated in some aspect of ribosomal function.     

Molecular coevolution among cryptically simple expansion segments of eukaryotic 26S/28S rRNAs

The set of "expansion segments" of any eukaryotic 26S/28S ribosomal RNA (rRNA) gene is responsible for the bulk of the difference in length between the prokaryotic 23S rRNA gene and the eukaryotic 26S/28S rRNA gene. The expansion segments are also responsible for interspecific fluctuations in length during eukaryotic evolution. They show a consistent bias in base composition in any species; for example, they are AT rich in Drosophila melanogaster and GC rich in vertebrate species. Dot-matrix comparisons of sets of expansion segments reveal high similarities between members of a set within any 28S rRNA gene of a species, in contrast to the little or spurious similarity that exists between sets of expansion segments from distantly related species. Similarities among members of a set of expansion segments within any 28S rRNA gene cannot be accounted for by their base-compositional bias alone. In contrast, no significant similarity exists within a set of "core" segments (regions between expansion segments) of any 28S rRNA gene, although core segments are conserved between species. The set of expansion segments of a 26S/28S gene is coevolving as a unit in each species, at the same time as the family of 28S rRNA genes, as a whole, is undergoing continual homogenization, making all sets of expansion segments from all ribosomal DNA (rDNA) arrays in a species similar in sequence. Analysis of DNA simplicity of 26S/28S rRNA genes shows a direct correlation between significantly high relative simplicity factors (RSFs) and sequence similarity among a set of expansion segments. A similar correlation exists between RSF values, overall rDNA lengths, and the lengths of individual expansion segments. Such correlations suggest that most length fluctuations reflect the gain and loss of simple sequence motifs by slippage-like mechanisms. We discuss the molecular coevolution of expansion segments, which takes place against a background of slippage-like and unequal crossing-over mechanisms of turnover that are responsible for the accumulation of interspecific differences in rDNA sequences.      

Sequence analysis of the maize mitochondrial 26 S rRNA gene and flanking regions

A single copy of the large ribosomal 26 S rRNA gene is found in the maize mitochondrial genome. The sequence of this gene and the flanking regions has been determined using the M13 dideoxy sequencing method. The maize mt 26 S rDNA shares a high degree of homology with the Escherichia coli 23 S rDNA, and the approximate 5′ and 3′ ends of the maize 26 S rDNA have been located by comparison with the E. coli sequence. The maize mt 26 S rDNA has also been compared with the sequences of the maize chloroplast 23 S rDNA, the human mitochondrial 16 S rDNA, part of the yeast mitochondrial 21 S rDNA, and the yeast cytoplasmic 25 S rDNA. In all cases, there are numerous regions of 70% or higher homology.     

Repeated reunions and splits feature the highly dynamic evolution of 5S and 35S ribosomal RNA genes (rDNA) in the Asteraceae family

Background  

In flowering plants and animals the most common ribosomal RNA genes (rDNA) organisation is that in which 35S (encoding 18S-5.8S-26S rRNA) and 5S genes are physically separated occupying different chromosomal loci. However, recent observations established that both genes have been unified to a single 35S-5S unit in the genus Artemisia (Asteraceae), a genomic arrangement typical of primitive eukaryotes such as yeast, among others. Here we aim to reveal the origin, distribution and mechanisms leading to the linked organisation of rDNA in the Asteraceae by analysing unit structure (PCR, Southern blot, sequencing), gene copy number (quantitative PCR) and chromosomal position (FISH) of 5S and 35S rRNA genes in ~200 species representing the family diversity and other closely related groups.     

Ribosomal RNA genes ofEndomyces fibuliger: isolation, sequencing and the use of the 26S rRNA gene in integrative transformation ofSaccharomyces cerevisiae for efficient expression of the α-amylase gene ofEndomyces fibuliger

Endomyces fibuliger is a dimorphic yeast which is homothallic and exists predominantly in the diploid phase with a brief haploid phase. A repeat unit of the ribosomal RNA genes, or rDNA, from E. fibuliger 8014 met has been isolated, cloned and sequenced. In this report, the sequences of the 17S, 5.8S and 26S rRNA genes are presented. Homology between the sequenced rRNA genes and those of closely-related yeast strains, particularly Saccharomyes cerevisiae and Candida albicans, was observed. As a step towards the eventual development of a transformation system for the yeast E. fibuliger, an integrative plasmid containing the 5.8S and a part of the 26S rRNA gene, a selectable marker conferring resistance to the G418 antibiotic and a reporter gene, the α-amylase (ALP1) gene of E. fibuliger, was constructed. This plasmid was linearized at a unique restriction site within the 26S rRNA gene, and transformed into S. cerevisiae INVSC2 MATa his3 ura3 using the lithium acetate method to test the functionality of the vector system. Transformation into S. cerevisiae INVSC2 MATa his3 ura3 was by virtue of the extensive homology between the sequenced 26S rRNA gene of E. fibuliger 8014 met and that of S. cerevisiae, so that homologous pairing and integration into the recipient chromosome was possible. The G418-resistant S. cerevisiae transformants produced halos on starch medium due to hydrolysis by α-amylase, and they were further analysed by Southern hybridization with the ALP1 gene and the gene encoding the aminoglycoside 3′- phosphotransferase I enzyme which confers resistance to the G418 antibiotic. A band of 13.7 kb which corresponded to the linearized size of the transforming plasmid DNA was obtained on the autoradiogram, suggesting that tandem copies of the plasmid DNA are present in the chromosome. Finally, an assay of the α-amylase enzyme secreted extracellularly was performed on the transformants.     

The release and reassociation of 5.8 S rRNA with yeast ribosomes.

Yeast 5.8 S rRNA is released from purified 26 S rRNA when it is dissolved in water or low salt buffer (50 mM KCl, 10mM Tris-HCl, pH 7.5); it is not released from 60 S ribosomal subunits under similar conditions. The 5.8 S RNA component together with 5 S rRNA can be released from subunits or whole ribosomes by brief heat treatment or in 50% formamide; the Tm for the heat dissociation of 5.8 S RNA is 47 degrees C. This Tm is only slightly lower when 5 S rRNA is released first with EDTA treatment prior to heat treatment. No ribosomal proteins are released by the brief heat treatment. A significant portion of the 5.8 S RNA reassociates with the 60 S subunit when suspended in a higher salt buffer (e.g.0.4 m KCl, 25 mM Tris-HCl, pH 7.5, 6 mM magnesium acetate, 5 mM beta-mercaptoethanol). The Tm of this reassociated complex is also 47 degrees C. The results indicate that in yeast ribosomes the 5.8 S-26 S rRNA interaction is stabilized by ribosomal proteins but that the association is sufficiently loose to permit a reversible dissociation of the 5.8 S rRNA molecule.     

Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene.

The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

Specific Role for Yeast Homologs of the Diamond Blackfan Anemia-associated Rps19 Protein in Ribosome Synthesis

Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3'-end of 18 S rRNA. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells under-expressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemia-associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis.