Termination region in rRNA genes from a eucaryotic thermophile, Thermomyces lanuginosus.

S1 mapping of the termination region in the ribosomal DNA from a thermophilic fungus, Thermomyces lanuginosus, revealed three distinct termini corresponding to the mature 25S rRNA, a precursor that is 19 nucleotides longer and corresponds to the 37S precursor in yeast cells, and a putative termination site at +96 that bears a limited sequence homology with the SalI box of mammalian cells. An estimate of the secondary structure suggested that the three termini are in close proximity, a feature that may be essential to precursor termination and maturation. The results raise questions regarding recently reported relationships between ribosomal DNA termination and spacer enhancer elements in fungi.

Epistatic influence in tomato Ve1-mediated resistance.

Resistance to Verticillium wilt disease is associated with the tomato Ve-locus; however, the individual functional roles of Ve1 and Ve2 in host plants remain controversial. As a first step towards Ve mutational analyses in planta, the Ve1 coding region from a resistant tomato near-isoline (cv. Craigella GCR218) was introduced into a susceptible near-isoline (cv. Craigella GCR26). 35S:Ve1 plants segregated into two distinct classes; roughly half were resistant and half were susceptible. Ve1 transcript levels were up-regulated in both classes compared to wild-type plants, showing stable transgenic expression. Expression analysis of Ve2 revealed that mRNA levels were similar between 35S:Ve1 and wild-type tomatoes, demonstrating that Ve1 transgene introduction does not alter endogenous Ve2 expression. Overall, the results of this study confirm the functional role of Ve1 protein in resistance to the vascular fungal pathogen V. dahliae race 1 (Vd1), but suggest that a yet undefined factor exerts an epistatic influence on the Ve1 gene.

Structural equivalence in the transcribed spacers of pre-rRNA transcripts in Schizosaccharomyces pombe.

The structure of the internal transcribed spacer 2 (ITS2) in Schizosaccharomyces pombe was re-evaluated with respect to phylogenetically conserved features in yeasts, features in other transcribed spacer regions as well as the binding of transacting factors which potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a very simple core structure consisting of a single extended hairpin which includes the interacting termini of the mature 5.8S and 25S rRNAs. Comparisons with ITS2 sequences in greatly diverging organisms indicate that the same feature also can be recognized. This is especially clear in organisms that contain very short sequences in which the putative structures are much less ambiguous. Diversity between organisms is the result of changes in hairpin length as well as the addition of branched helices. Protein binding and gel retardation studies with the S.pombe ITS2 further indicate that, as observed in the 3" external transcribed spacer (ETS) and ITS1 regions, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data suggest that the spacer regions all may act in a similar fashion, not only to organize the maturing terminal sequences, but also serve to organize specific soluble factors possibly acting with snoRNAs or in a manner which is analogous with that of the free snoRNPs.

Methylation of ribosomal RNA as a possible factor in cell differentiation.

The mammalian 5.8S ribosomal RNA (rRNA) contains a 2'-O-methylated uridylic acid residue which is methylated in the cytoplasm of normal tissues but is highly undermethylated in the newly synthesized RNA of rapidly growing neoplastic tissues (R. N. Nazar, T. O. Sitz, and K. D. Somers, J. Mol. Biol., 142: 117-121, 1980). To further assess the significance of these differing levels of methylation, new assays have been developed and used to analyze the steady state levels of methylation in several tissues of varied mitotic activity and degree of cellular differentiation. The results indicate that the total population of 5.8S rRNA is also undermethylated in tumor tissues and the low levels of 2'-O-methyluridylic acid which were observed in previous studies are not simply due to a slow cytoplasmic methylation process. A substantial increase in mitotic activity, as observed in regenerating rat liver, appears not to result in a general demethylation of the 5.8S rRNA but, in contrast, when rapidly dividing cells such as muscle myoblasts differentiate in vitro into postmitotic multinucleated muscle fibers, the existing 5.8S RNA population is subject to a significantly increased level of 2'-O-methylation. These data provide further evidence for the potential importance of RNA methylation in cell differentiation and tumor development.

Significance of S-adenosylmethionine pools in the hypomethylation of ribosomal RNA during the propagation of tissue culture cells and oncogenesis.

The 5.8S rRNA of normal tissues contains a partially 2'-O-methylated uridylic acid residue which is methylated in the cytoplasm and undermethylated in rapidly growing neoplastic tissues (R. N. Nazar, T. O. Sitz, and K. D. Somers, J. Mol. Biol., 142: 117-121, 1980). This difference in methylation was further characterized by examining the effect of cell age or cell culture passage number on the level of methylation of 5.8S RNAs from normal and malignant cell lines and simultaneous changes in intracellular pools of S-adenosylmethionine and S-adenosylhomocysteine. The results indicate that the level of methylation decreases continuously with cell culture passage number as the cells become aneuploid, transformed, or tumorigenic, but there is no direct correlation with the intracellular pools of S-adenosylmethionine or S-adenosylhomocysteine. In contrast, there is a dramatic but inverse increase in the S-adenosylmethionine:S-adenosylhomocysteine ratio which correlates with the decreasing levels of 2'-O-methylation. The significance of these changes in substrate levels to the hypomethylation of 5.8S and other RNAs during oncogenesis is discussed.

Extended secondary structure as a basis of increased RNA stability in a thermophilic alga Cyanidium caldarium.

To identify important structural features in the intergenic sequences of ribosomal DNAs, the nucleotide sequence of the 18-25S rRNA intergenic region was determined in a thermophilic alga, Cyanidium caldarium. Although the mature 5.8S RNA is more stable to thermal denaturation, sequence comparisons reveal a longer molecule with a surprisingly low G/C nucleotide composition. Estimates of the structure further indicate that, unlike other thermophilic examples, thermostability in this organism results, at least in part, from an extended secondary structure.

An efficiently expressed 5.8S rRNA 'tag' for in vivo studies of yeast rRNA biosynthesis and function.

Inefficient expression or detrimental markers have limited mutational analyses of eukaryotic 5.8S rRNA and the associated rDNA transcribed spacers. We have found a neutral, 4-base insertion mutation that effectively tags the 5.8S rRNA for improved studies of rRNA expression, processing and function. Cells expressing the tagged rDNA plasmid contain 50-60% mutant 5.8S rRNA, but show a normal growth rate and polysomal profile and a constant distribution of tagged 5.8S rRNA. The high level of expression also demonstrates that plasmid-associated rDNA is preferentially transcribed over chromosomal copies.

An enhanced thermostability in thermophilic 5-S ribonucleic acids under physiological salt conditions.

The secondary structure of 5-S rRNAs of Thermus aquaticus (an extreme thermophile), Bacillus stearothermophilus (a moderate thermophile) and Escherichia coli (a mesophile) was compared using thermal denaturation techniques under varying ionic conditions. At a low ionic strength (10 mM K+), the Tm of T. aquaticus 5-S RNA differed by only 1 degrees C from that of E. coli RNA and the molecule was fully denatured well below the optimum growth temperature of the thermophile. The internal Na+, K+ and Mg2+ concentrations of T. aquaticus cells were determined to be 91 mM, 130 mM and 59 mM, respectively. Under these salt conditions, T. aquaticus 5-S RNA was significantly more stable than E. coli RNA and the 5-S RNA from B. stearothermophilus was intermediate as is its optimum growth temperature. The results suggest that the thermostability of macromolecules from thermophilic organisms may be specially dependent on the internal salt concentration. Furthermore, under these salt conditions, most of the secondary structure of the RNA remained stable at the optimum growth temperatures suggesting that ribosomal RNAs of thermophilic organisms contribute more to the thermostability of the ribosome than previously thought.

Conserved core structure in the internal transcribed spacer 1 of the Schizosaccharomyces pombe precursor ribosomal RNA.

The structure of the internal transcribed spacer 1 (ITS1) in Schizosaccharomyces pombe was examined with respect to phylogenetically conserved features in yeasts as well as the binding of transacting factors that potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a compact, more highly organized structure than previously proposed in Saccharomyces cerevisiae, with distinct structural features which can be recognized in S. cerevisiae. These include a central extended hairpin structure as well as smaller hairpins immediately adjacent to the maturing termini. Comparisons with ITS sequences in more diverse organisms indicate that the same features also can be recognized. This is especially clear in organisms which contain very short sequences in which the putative structures are much less ambiguous. Again nuclease protection analyses in one of these, Verticillium albo-atrum, confirm a central hairpin with additional hairpins linked to the maturing termini. Protein binding and gel retardation studies with the S. pombe ITS1 further indicate that, as observed in the 3' external transcripted spacer (ETS) region, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing, but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data provide evidence that the spacer regions act not only to organize the maturing terminal sequences but also may serve to organize specific soluble factors, possibly acting in a manner which is analogous with that of the free small nucleolar ribonucleo protein particles (snoRNPs).

Effect of sequence mutations on the higher order structure of the yeast 5 S rRNA.

Mutant yeast ribosomal 5 S RNAs were probed by enzymatic cleavage and chemical reactivity to define further the higher order structure. Mutations that destabilized helix IV resulted in an altered tertiary structure in which a reduced reactivity to ethylnitrosourea at U90 and G91 could be correlated with greater enzymatic and Fe(II)-EDTA cleavages in helices II and V. The results provide direct evidence for, and a further definition of, a structural juxtaposition between helix II and the end of helix IV and indicate that, in contrast to earlier suggestions, the remaining tertiary structure is sufficiently stable to prevent "pseudoknot-like" interactions between helices III and IV. The data are fully consistent with the "lollipop" model of the tertiary structure.

Structure of the ribosome-associated 5.8 S ribosomal RNA.

The structure of the 5.8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5.8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60 S subunit-associated 5.8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G + C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60 S subunit. Further comparisons with the ribosome-associated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, C56, C100, C127 and C128, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed.

Interdependence in the processing of ribosomal RNAs in Schizosaccharomyces pombe.

Eukaryotic rRNAs are produced by cleavage of a large 35 to 45 S pre-rRNA transcript which initially must be fully transcribed and assembled into an 80 to 90 S nucleolar ribonucleoprotein particle. Despite this need for a completed transcript, several investigations have reported a split processing scheme for independent maturation of the large and small subunit rRNAs. Here, an efficiently expressed rDNA plasmid was used to quantitatively analyze the effects of mutations in the internal transcribed spacer (ITS) region in the yeast, Schizosaccharomyces pombe. The results show that substitution of ITS regions inhibits the processing of distant external transcribed spacers (ETS) and that deletion of the ITS2 spacer not only prevents the maturation of the large subunit, but severely affects maturation of the small subunit rRNA. This indicates that the processing mechanisms are not fully split and, when taken together with other evidence of interdependences in rRNA maturation, the results suggest that the interdependences act as a quality control mechanism to help ensure that only functional rRNA is incorporated into ribosomes.

Essential structural features in the Schizosaccharomyces pombe pre-rRNA 5' external transcribed spacer.

The proximal region in the 5' external transcribed spacer (5'ETS) of the genes encoding ribosomal RNAs in Schizosaccharomyces pombe was examined with respect to structural features which underlie rRNA maturation. Computer analyses and partial digestion with nuclease probes indicate a crucifix-like structure composed primarily of three extended hairpins which are more highly ordered than previously proposed in Saccharomyces cerevisiae. A re-evaluation of the same region in S. cerevisiae indicates a conserved core structure, including the U3 snoRNA binding site within this higher-order structure. The sequences encoding the individual hairpins were deleted by PCR-mediated mutagenesis and the mutant rDNAs were expressed in vivo to determine the effect of these features on rRNA maturation. Quantitative hybridization analyses indicate that the first hairpin only has modest effects on 18 S rRNA maturation, but the other two regions are critical and no mature 18 S rRNA was observed. When smaller changes were systematically introduced into the critical regions, strong correlations were observed with known or putative events in rRNA maturation. Changes associated with an intermediate cleavage site in helix II and with the putative U3 snoRNA binding site were again critical to 18 S rRNA production. In each case, the effects were sequence dependent and not simply the result of disrupted structure. Further analyses of the 5.8 S rRNA indicate that the large ribosomal subunit RNA can be properly processed in each case but the efficiency is reduced by as much as 60 %, an observation which provides new evidence of interdependency in the maturation process. The results illustrate that rRNA processing is more critically dependent on the 5'ETS than previously believed.

Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1.

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed.

Intragenic processing in yeast rRNA is dependent on the 3' external transcribed spacer.

The nucleotide sequence of the 3' external transcribed spacer (3' ETS) region in Schizosaccharomyces pombe rDNA was determined to define structural features which mediate the termination of RNA transcription and subsequent rRNA maturation. S1 nuclease protection studies suggest three alternative termination sites and four cleavage sites in the processing of the 3' ETS sequence. Each of the termination sites precedes a "Sal box"-like sequence which has been demonstrated to mediate the termination of rRNA transcription in mammalian cells. A highly conserved extended hairpin structure in the ETS sequence was deleted by PCR-mediated mutagenesis and the mutant rDNA was expressed in vivo to determine its role in rRNA maturation. Despite an efficient expression of the mutant gene, mature 5.8 S or 25 S rRNA was not observed. Labelling kinetics and S1 nuclease protection analyses indicate that the deletion not only fully inhibits the removal of the 3' ETS but also fully inhibits the processive excision of the second internal transcribed spacer (ITS2). Instead, a relatively stable 27 S nRNA precursor remains easily detectable in the whole cell RNA population. The results demonstrate a critical dependence of ITS processing on the 3' ETS raising the possibility that these sequences interact in a common processing domain.

In vivo analyses of upstream promoter sequence elements in the 5 S rRNA gene from Saccharomyces cerevisiae.

Upstream promoter elements of the Saccharomyces cerevisiae 5 S rRNA gene have been characterized by genomic DNase I "footprinting" and by in vivo mutational analyses using base substitutions and deletions. A high copy shuttle-vector was used to efficiently express the mutant 5 S rRNA genes in vivo and a structural mutation in the 5 S rRNA, which was previously shown to be functionally neutral but easily detected by gel electrophoresis, allowed for an accurate measure of gene expression. The results provide direct evidence for upstream regulatory elements which confirms a start site element (sse) from -1 to -8 and identifies a new independent upstream promoter element (upe) centered from about -17 to -20. In contrast to previous reports with reconstituted systems, both elements dramatically affect the efficiency of gene expression and suggest that the saturated conditions which are used in reconstituted studies mask sequence dependence; a dependency that could be physiologically significant and play a role in the regulation of 5 S rRNA expression. The footprint analyses support an extended region of protein interaction as recently observed in reconstituted systems but again provide evidence of significant structural rearrangements when the upstream sequence is changed.

A widely distributed "CAT" family of repetitive DNA sequences.

The yeast genome contains a family of repetitive sequences consisting primarily of a tandemly arranged trinucleotide, CAT, or a closely related CGT sequence. To characterize similar sequences in divergent organisms, a previously isolated "CAT" sequence was used to isolate homologous genomic clones from a human cell line, an insect and a higher plant. Sequence analyses show that comparable repetitive sequences are widely distributed and may be present in all eukaryotic genomes. In situ hybridization analyses indicate that in yeast, the CAT elements are dispersed among all the chromosomes, and a more detailed analysis in Drosophila indicates that at least one of these sequences maps on the X chromosome between known genetic loci which are actively expressed. Repeated searches of yeast cDNA libraries indicate that these CAT clusters are not expressed but substantial effects on the expression of a cloned gene strongly suggest that they play an important role in gene regulation.

Structural features in the 3' external transcribed spacer affecting intragenic processing of yeast rRNA.

A highly conserved extended hairpin structure in the 3' external transcribed spacer (3' ETS) region of nascent eukaryotic rRNA transcripts is essential for the maturation of the large ribosomal subunit RNAs (5.8 S and 25 to 28 S rRNAs). Systematic changes were introduced into this structure by PCR-mediated mutagenesis and the mutant rDNAs were expressed in vivo to determine the structural features that are essential for rRNA maturation. Changes in the lower half of the stem or the large loop at the end had little or no effect on the maturation of either the 5.8 S or 25 S rRNA, but changes that disrupted secondary structure in the upper half of this stem had equal and dramatic effects on both RNAs. When the RNA stem was incubated with a cellular protein extract, gel retardation studies indicated that the stem forms a ribonucleoprotein complex, and a comparison with mutant RNA indicated that protein binding could be compromised by changes that were critical for rRNA maturation. Sequence comparisons with other spacer regions as well as snRNAs reveal some structural analogy, which, when taken together with the mutational studies, raise the possibility that this hairpin functions during RNA processing in a manner that may be analogous with that of free snRNPs.

Conserved upstream sequence elements in plant 5S ribosomal RNA-encoding genes.

As a basis for further comparative studies, nuclear 5S rRNA gene repeats from two plants of the Solanaceae family, tobacco (Nicotiana rustica) and tomato (Lycopersicon esculentum), were isolated and sequenced. The more abundant 5S rRNA gene repeat in tobacco is 430 bp long, while a second less common variant is 521 bp long. In contrast, the 5S rRNA gene repeat from tomato is only 355 bp long. The spacer sequences from these gene repeats, as well as from other published plant nuclear 5S rRNA genes, were compared for repeating or conserved sequence elements. The results indicate that often observed, but non-conserved, repeating sequence elements probably arise spontaneously by unequal crossover with no functional significance. However, three conserved sequence elements immediately upstream of the coding sequence; a C residue at -1, a G + C-rich element centered at -13, and an A + T-rich element centered at -26 resemble regulatory features which have been identified in other types of genes.

Cotranscription of 5S rRNA-tRNA(Arg)(ACG) from Brassica napus chloroplasts and processing of their intergenic spacer.

S1 mapping showed that at least a significant portion of the 5S rRNA and tRNA(Arg)(ACG) is co-transcribed in canola chloroplast, making trnR the last gene transcribed in an operon of which the final sequence is 5'-16S-tRNA(Ile)-tRNA(Ala)-23S-4.5S-5S-tRNA(Arg)-3'. Various RNA termini representing RNA processing sites at several parts of the 5S rRNA-tRNA(Arg) area were detected. This gene spacer is substantially conserved among various species compared here, and a secondary structure model for this chloroplast region in canola applies to other plant sequences. The conservation of this intergenic sequence suggests a functional role, possibly by providing recognition structures for endogenous RNases involved in its maturing process.