Eukaryotic Ribosomal RNA Research Articles

Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences.

Aminoglycoside antibiotics bind specifically to a conserved sequence of the 16S ribosomal RNA (rRNA) A site and interfere with protein synthesis. One model for the mechanism underlying the deleterious effects of aminoglycosides on protein synthesis invokes a drug-induced conformational change in the rRNA that involves the destacking of two adenine residues (A1492 and A1493 in Escherichia coli) at the A site. We describe here a fluorescence-based approach for detecting and characterizing this drug-induced conformational change in the target rRNA. In this approach, we insert the fluorescent base analogue 2-aminopurine in place of A1492 in an E. coli 16S rRNA A-site model oligonucleotide (EcWT) as well as in a mutant form of this oligomer (A1408G) in which A1408 has been replaced with a guanine. The presence of guanine at 1408 instead of adenine represents one of the major sequence differences between prokaryotic and eukaryotic A sites, with the latter A sites being resistant to the deleterious effects of aminoglycosides. Binding of the aminoglycoside paromomycin to the 2AP-substituted forms of EcWT and A1408G induced changes in fluorescence quantum yield consistent with drug-induced base destacking in EcWT but not A1408G. Isothermal titration calorimetry studies reveal that paromomycin binds to the EcWT duplex with a 31-fold higher affinity than the A1408G duplex, with this differential affinity being enthalpic in origin. In the aggregate, these observations are consistent with both rRNA binding affinity and drug-induced base destacking being important determinants in the prokaryotic specificity of aminoglycosides. Combining fluorescence quantum yield and lifetime data allows for quantification of the extent of drug-induced base destacking, thereby providing a convenient tool for evaluating the relative impacts of both novel and existing A-site targeting ligands on rRNA conformation and potentially for predicting relative antibiotic activities and specificities.

Unusually expanded SSU ribosomal DNA of primary osmotrophic euglenids: molecular evolution and phylogenetic inference.

Expansion segments within eukaryotic nuclear SSU ribosomal RNA have been characterized in many diverse organisms. So far, only a few studies have examined the evolutionary history of SSU rDNA variable regions for monophyletic groups. A euglenozoan SSU rDNA data set was analyzed combining phylogenetic inference and examination of expansion segment evolution. Although SSU rDNA length expansion could be ascribed to all Euglenozoa, most unusual length variation occurs within primary osmotrophic euglenids, particularly within the genus Distigma. The longest SSU rRNA gene reported to date can be found in D. sennii, comprising more than 4500 bases. RT-PCR analyses revealed that the complete gene is transcribed into RNA without posttranscriptional modifications. Further investigations uncovered that most of the length extension is due to elongated variable regions V2 and V4, but virtually all variable regions except for V3 are extended within primary osmotrophic euglenids. Analyses of secondary structure revealed several insertion points within variable regions, some of which are of phylogenetic importance. Varying GC content has been detected among species and between expansion segments and core regions. Nevertheless, individual expansion segments of one species as well as variable sequence positions within core regions tend to evolve in parallel concerning nucleotide frequencies. The presence of a large internal repeat within V2 of Distigma sennii hints at a possible mechanism for large-scale sequence length expansion.

Identification and characterization of a novel U14 small nucleolar RNA gene cluster in Oryza sativa

Small nucleolar RNAs (snoRNAs) are required for ribose 2′-O-methylation of eukaryotic ribosomal RNA. Through computer search in international rice genome database, a novel U14 snoRNA gene cluster, consisting of two U14 snoRNA gene candidates, was found on rice chromosome II. They both have box C/D sequences and a 14 nucleotides (nt)-long complementarity to rice 18S ribosomal RNA (rRNA). Functional analysis of this gene cluster indicated that both were transcribed in vivo and might guide the methylations of C418 in rice 18S rRNA. By using primer extension, 5′ and 3′ rapid amplification of cDNA ends, the 5′ and 3′ ends of two snoRNAs were determined. The 52 nt long intergenic spacer of the gene cluster is rich in uridine. The absence of a conserved promoter element in this spacer, the proximity of the genes and the detection of transcripts containing linked U14 snoRNAs by reverse transcript polymerase chain reaction suggest that the rice U14 snoRNAs encoded in the cluster are transcribed as a polycistron under an upstream promoter, and individual U14 snoRNAs are released after processing of the precursor RNAs.

DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription.

Eukaryotic ribosomal RNA promoters exhibit an unusual conservation of non-canonical DNA structure (curvature, twist angle and duplex stability) despite a lack of primary sequence conservation. This raises the possibility that rRNA transcription factors might utilize structural anomalies in their sequence recognition process. We have analyzed in detail the interaction of the polymerase I transcription factor TIF-IB from Acanthmoeba castellanii with the CORE promoter. TIF-IB interacts primarily with the minor groove of the promoter. By correlating the effects on transcription and on DNA structure of promoter point mutations, we show that the TIF-IB interaction is strongly inhibited by increases in minor groove width. This suggests that a particular DNA structure is required for interaction with the transcription factor. In addition, TIF-IB induces a small bend in the promoter upon binding. Modeling of this bend reveals that it requires an additional narrowing of the minor groove, which would favor binding to mutants with narrower grooves. We also discuss how this narrowing would induce a small destabilization of the helix upstream of the transcription start site. Telestability predicts this would result in destabilization of the sequence that melts during initiation, suggesting that TIF-IB may have a role in stimulating melting.

Characterization of a fission yeast subunit of an RNA polymerase I essential transcription initiation factor, SpRrn7h/TAFI68, that bridges yeast and mammals: association with SpRrn11h and the core ribosomal RNA gene promoter

Production of eukaryotic ribosomal RNAs (rRNAs) entails sequence-specific recognition of regulatory sequences in the rRNA gene promoter. A putative subunit of the Schizosaccharomycespombe essential transcription initiation factor for rRNA synthesis has been identified that shares homology with both murine TAFI68 and Saccharomycescerevisiae Rrn7p, subunits of their species' transcription initiation factor. Affinity purified putative SpRrn7h and associated factors, including a putative Rrn11p homolog, SpRrn11h, bear RNA polymerase I transcription initiation factor activity, and recombinant SpRrn7h associates with S.pombe core rDNA promoter sequences. In the first widespread search for putative homologs of SpRrn7h/murine TAFI68, and SpRrn11h/murine TAFI48, multiple ones were identified across eukaryotes. Analysis of residues conserved between the fission yeast and murine essential initiation factor subunits aided in these identifications. Sequences in the core rRNA gene promoter contributing to transcriptional activation were investigated, including a perfect TATAAA element located at −35.

Structural basis for selectivity and toxicity of ribosomal antibiotics.

Ribosomal antibiotics must discriminate between bacterial and eukaryotic ribosomes to various extents. Despite major differences in bacterial and eukaryotic ribosome structure, a single nucleotide or amino acid determines the selectivity of drugs affecting protein synthesis. Analysis of resistance mutations in bacteria allows the prediction of whether cytoplasmic or mitochondrial ribosomes in eukaryotic cells will be sensitive to the drug. This has important implications for drug specificity and toxicity. Together with recent data on the structure of ribosomal subunits these data provide the basis for development of new ribosomal antibiotics by rationale drug design.

Structural origins of aminoglycoside specificity for prokaryotic ribosomes

Aminoglycoside antibiotics, including paromomycin, neomycin and gentamicin, target a region of highly conserved nucleotides in the decoding region aminoacyl-tRNA site (A site) of 16 S rRNA on the 30 S subunit. Change of a single nucleotide, A1408 to G, reduces the affinity of many aminoglycosides for the ribosome; G1408 distinguishes between prokaryotic and eukaryotic ribosomes. The structures of a prokaryotic decoding region A-site oligonucleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1a were determined previously. Here, the structure of a eukaryotic decoding region A-site oligonucleotide bound to paromomycin has been determined using NMR spectroscopy and compared to the prokaryotic A-site-paromomycin structure. A conformational change in three adenosine residues of an internal loop, critical for high-affinity antibiotic binding, was observed in the prokaryotic RNA-paromomycin complex in comparison to its free form. This conformational change is not observed in the eukaryotic RNA-paromomycin complex, disrupting the binding pocket for ring I of the antibiotic. The lack of the conformational change supports footprinting and titration calorimetry data that demonstrate approximately 25-50-fold weaker binding of paromomycin to the eukaryotic decoding-site oligonucleotide. Neomycin, which is much less active against Escherichia coli ribosomes with an A1408G mutation, binds non-specifically to the oligonucleotide. These results suggest that eukaryotic ribosomal RNA has a shallow binding pocket for aminoglycosides, which accommodates only certain antibiotics.

An analysis of G-U base pair occurrence in eukaryotic 5S rRNAs.

The structure-function relationship in RNA molecules is a key to understanding of the expression of genetic information. Various types of RNA play crucial roles at almost every step of protein biosynthesis. In recent years, it has been shown that one of the most important structural elements in RNA is a wobble pair G-U. In this paper, we present for the first time an analysis of the distribution of G-U pairs in eukaryotic 5S ribosomal RNAs. Interestingly, the G-U pair in 5S rRNA species is predominantly found in two intrahelical regions of the stems I and V and at the junction of helix IV and loop A. The distribution of G-U pairs and the nature of adjacent bases suggests their possible role as a recognition site in interactions with other components of protein biosynthesis machinery.

Long branch attraction effects and the status of "basal eukaryotes": phylogeny and structural analysis of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis.

The three taxa emerging at the base of the eukaryotic ribosomal RNA phylogenetic tree (Diplomonadida, Microspora, and Parabasalia) include a wide array of parasitic species. and some free-living organisms that appear to be derived from a parasitic ancestry. The basal position of these taxa, which lack mitochondria, has recently been questioned. I sequenced most of the ribosomal RNA gene cluster of the free-living diplomonad Trepomonas agilis and a secondary structure model was reconstructed for the SSU rRNA. I conducted a RASA matrix analysis to identify, independently from tree reconstruction, putative long branch attraction effects in the data matrix. The results show that each of the basal clades and the euglenozoan clade act, indeed, as long branches and may have been engaged in a process of accelerated rate of evolution. A nucleotide signature analysis was conducted in the conserved regions for positions defining the three great domains of life (Eubacteria, Archea, and Eukaryota). For the three basal taxa, this analysis showed the presence of a significant number of different non-eukaryotic nucleotides. A precise study of the nature and location of these nucleotides led to conclusions supporting the results of the RASA analysis. Altogether, these findings suggest that the basal placement of these taxa in the SSU ribosomal RNA phylogenetic tree is artifactual, and flawed by long branch attraction effects.

Construction of a variability map for eukaryotic large subunit ribosomal RNA.

In this paper, we present a variability map of the eukaryotic large subunit ribosomal RNA, showing the distribution of variable and conserved sites in this molecule. The variability of each site in this map is indicated by means of a colored dot. Construction of the variability map was based on the substitution rate calibration (SRC) method, in which the substitution rate of each nucleotide site is computed by looking at the frequency with which sequence pairs differ at that site as a function of their evolutionary distance. Variability maps constructed by this method provide a much more accurate and objective description of site-to-site variability than visual inspection of sequence alignments.

A computational screen for methylation guide snoRNAs in yeast.

Small nucleolar RNAs (snoRNAs) are required for ribose 2'-O-methylation of eukaryotic ribosomal RNA. Many of the genes for this snoRNA family have remained unidentified in Saccharomyces cerevisiae, despite the availability of a complete genome sequence. Probabilistic modeling methods akin to those used in speech recognition and computational linguistics were used to computationally screen the yeast genome and identify 22 methylation guide snoRNAs, snR50 to snR71. Gene disruptions and other experimental characterization confirmed their methylation guide function. In total, 51 of the 55 ribose methylated sites in yeast ribosomal RNA were assigned to 41 different guide snoRNAs.

Termination of Mammalian rDNA Replication: Polar Arrest of Replication Fork Movement by Transcription Termination Factor TTF-I

A replication fork barrier (RFB) at the 3′ end of eukaryotic ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. We have reproduced the RFB in vitro in HeLa cell extracts using 3′ terminal murine rDNA fused to an SV40 origin-based vector. The RFB is polar and modularly organized, requiring both the Sal box transcription terminator and specific flanking sequences. Mutations within the terminator element, depletion of the RNA polymerase I–specific transcription termination factor TTF-I, or deletion of the termination domain of TTF-I abolishes RFB activity. Thus, the same factor that blocks elongating RNA polymerase I prevents head-on collision between the DNA replication apparatus and the transcription machinery.

A small nucleolar RNP protein is required for pseudouridylation of eukaryotic ribosomal RNAs.

Eukaryotic rRNAs possess numerous post-transcriptionally modified nucleotides. The most abundant modifications, 2'-O-ribose methylation and pseudouridylation, occur in the nucleolus during rRNA processing. The nucleolus contains a large number of small nucleolar RNAs (snoRNAs) most of which can be classified into two distinct families defined by conserved sequence boxes and common associated proteins. The C and D box-containing snoRNAs are associated with fibrillarin, and most of them function as guide RNAs in site-specific ribose methylation of rRNAs. The nucleolar function of the other class of snoRNAs, which share box H and ACA elements and are associated with a glycine- and arginine-rich nucleolar protein, Gar1p, remains elusive. Here we demonstrate that the yeast Saccharomyces cerevisiae Gar1 snoRNP protein plays an essential and specific role in the overall pseudouridylation of yeast rRNAs. These results establish a novel function for Gar1 protein and indicate that the box H/ACA snoRNAs, or at least a subset of these snoRNAs, function in the site-specific pseudouridylation of rRNAs.

Eukaryotic ribosomal RNA: the recent excitement in the nucleotide modification problem.

Eukaryotic ribosomal RNA (rRNA) contains numerous modified nucleotides: about 115 methyl groups and some 95 pseudouridines in vertebrates; about 65 methyl groups and some 45 pseudouridines in Saccharomyces cerevisiae. All but about ten of the methyl groups are ribose methylations. The remaining ten are on heterocyclic bases. The ribose methylations occur very rapidly upon the primary rRNA transcript in the nucleolus, probably on nascent chains, and they appear to play an important role in ribosome maturation, at least in vertebrates. All of the methyl groups occur in the conserved core of rRNA. However, there is no consensus feature of sequence or secondary structure for the methylation sites; thus the nature of the signal(s) for site-specific methylations had until recently remained a mystery. The situation changed dramatically with the discovery that many of the ribose methylation sites are in regions that are precisely complementary to small nucleolar RNA (snoRNA) species. Experimental evidence indicates that structural motifs within the snoRNA species do indeed pinpoint the precise nucleotides to be methylated by the putative 2'-O-methyl transferase(s). Regarding base methylations, the gene DIM1, responsible for modification of the conserved dimethyladenosines near the 3' end of 18S rRNA, has been shown to be essential for viability in S. cerevisiae and is suggested to play a role in the nucleocytoplasmic transport of the small ribosomal subunit. Recently nearly all of the pseudouridines have also been mapped in the rRNA of several eukaryotic species. As is the case for ribose methylations, most pseudouridine modifications occur rapidly upon precursor rRNA, within core sequences, and in a variety of local primary and secondary structure environments. In contrast to ribose methylation, no potentially unifying process has yet been identified for the enzymic recognition of the many pseudouridine modification sites. However, the new data afford the basis for a search for any potential involvement of snoRNAs in the recognition process.

Site-Specific Pseudouridine Formation in Preribosomal RNA Is Guided by Small Nucleolar RNAs

During the nucleolar maturation of eukaryotic ribosomal RNAs, many selected uridines are converted into pseudouridine by a thus far undefined mechanism. The nucleolus contains a large number of small RNAs (snoRNAs) that share two conserved sequence elements, box H and ACA. In this study, we demonstrate that site-specific pseudouridylation of rRNAs relies on short ribosomal signal sequences that are complementary to sequences in box H/ACA snoRNAs. Genetic depletion and reconstitution studies on yeast snR5 and snR36 snoRNAs demonstrate that box H/ACA snoRNAs function as guide RNAs in rRNA pseudouridylation. These results define a novel function for snoRNAs and further reinforce the idea that base pairing is the most common way to obtain specific substrate–“enzyme” interactions during rRNA maturation.

Synthesis of functional eukaryotic ribosomal RNAs in trans: development of a novel in vivo rDNA system for dissecting ribosome biogenesis.

Active 18S and 25S ribosomal RNAs were produced in trans in yeast, from plasmids containing RNA polymerase II transcription signals and rDNA fragments with unique hybridization tags. Analyses were carried out in cells with temperature-sensitive RNA polymerase I. Functional rRNAs were derived from separate 18S and 5.8/25S rRNA coding units, however, active 25S rRNA could be produced only by cotranscription with 5.8S rRNA. The results demonstrate that the polycistronic organization of the large rDNA operon is not required for successful processing of rRNA or assembly of functional ribosomes. The split operon system should facilitate future efforts to dissect eukaryotic ribosome biogenesis.

Targeted ribose methylation of RNA in vivo directed by tailored antisense RNA guides.

Eukaryotic ribosomal RNAs are post-transcriptionally modified by methylation at the ribose sugar of specific nucleotides. This takes place in the nucleolus and involves a family of small nucleolar RNAs (snoRNAs) with long regions (10-21 nucleotides) complementary to rRNA sequences spanning the methylation site--a complementary snoRNA is required for methylation at a specific site. Here we show that altering the sequence of the snoRNA is sufficient to change the specificity of methylation. Mammalian cells transfected with a snoRNA engineered to be complementary to an arbitrary rRNA sequence direct the methylation of the predicted nucleotide in that sequence. We have further identified structural features, both of the guide and substrate RNA, required for methylation and have used these to design an exogenous transcript, devoid of rRNA sequence, that is site-specifically methylated when coexpressed with an appropriate guide snoRNA. Endogenous non-ribosomal RNA can thus be targeted, possibly providing a highly selective tool for the alteration of gene expression at the post-transcriptional level.

RRP5 is required for formation of both 18S and 5.8S rRNA in yeast.

Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25-28S) are synthesized as a single precursor which is subsequently processed into the mature rRNAs by a complex series of cleavage and modification reactions. In the yeast Saccharomyces cerevisiae, the early pre-rRNA cleavages at sites A0, A1 and A2, required for the synthesis of 18S rRNA, are inhibited in strains lacking RNA or protein components of the U3, U14, snR10 and snR30 small nucleolar ribonucleoproteins (snoRNPs). The subsequent cleavage at site A3, required for formation of the major, short form of 5.8S rRNA, is carried out by another ribonucleoprotein, RNase MRP. A screen for mutations showing synthetic lethality with deletion of the non-essential snoRNA, snR10, identified a novel gene, RRP5, which is essential for viability and encodes a 193 kDa nucleolar protein. Genetic depletion of Rrp5p inhibits the synthesis of 18S rRNA and, unexpectedly, also of the major short form of 5.8S rRNA. Pre-rRNA processing is concomitantly impaired at sites A0, A1, A2 and A3. This distinctive phenotype makes Rrp5p the first cellular component simultaneously required for the snoRNP-dependent cleavage at sites A0, A1 and A2 and the RNase MRP-dependent cleavage at A3 and provides evidence for a close interconnection between these processing events. Putative RRP5 homologues from Caenorhabditis elegans and humans were also identified, suggesting that the critical function of Rrp5p is evolutionarily conserved.

Rat Ribosomal RNA Gene Can Utilize Primate RNA Polymerase I Transcription Machinery: Lack of Absolute Species Specificity in rDNA Transcription

The transcriptional activity of rodent ribosomal RNA gene (rDNA) in the primate cell was examined in the light of reported species specificity of eukaryotic ribosomal RNA synthesis. The present study showed that rat rDNA can be transcribed in HeLa nuclear extract whereas mouse rDNA was not transcribed in the heterologous extract. Rat and mouse rDNA transcription factors were interchangeable with respect to efficiency and accuracy of transcription. The initiation of rat ribosomal gene transcription by RNA polymerase I occurred at the +1 site in the heterologous extract. Initiation of transcription at the correct site also occurredin vivofollowing transfection of cloned rat rDNA into the primate (COS-7) cells. These data indicate that rat ribosomal RNA gene can be expressed in the primate systemin vitroandin vivo.The absolute lack of species specificity in rDNA transcription has been discussed based on the present data and other reports.

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.