Nucleolin functions in the first step of ribosomal RNA processing.

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

Primary and secondary structure of rat 28 S ribosomal RNA.

The primary structure of rat (Rattus norvegicus) 28 S rRNA is determined inferred from the sequence of cloned rDNA fragments. The rat 28 S rRNA contains 4802 nucleotides and has an estimated relative molecular mass (Mr, Na-salt) of 1.66 X 10(6). Several regions of high sequence homology with S. cerevisiae 25 S rRNA are present. These regions can be folded in characteristic base-paired structures homologous to those proposed for Saccharomyces and E. coli. The excess of about 1400 nucleotides in the rat 28 S rRNA (as compared to Saccharomyces 25 S rRNA) is accounted for mainly by the presence of eight distinct G+C-rich segments of different length inserted within the regions of high sequence homology. The G+C content of the four insertions, containing more than 200 nucleotides, is in the range of 78 to 85 percent. All G+C-rich segments appear to form strongly base-paired structures. The two largest G+C-rich segments (about 760 and 560 nucleotides, respectively) are located near the 5'-end and in the middle of the 28 S rRNA molecule. These two segments can be folded into long base-paired structures, corresponding to the ones observed previously by electron microscopy of partly denatured 28 S rRNA molecules.     

Higher order structure in ribosomal RNA

[This corrects the article on p. 1111 in vol. 5, PMID: 3720727.].     

Dynamics of ribosomal RNA structure

The structural dynamics of ribosomal 5S RNAs have been investigated by probing single strandedness through enzymatic cleavage and chemical modification. This comparative study includes 5S rRNAs from E. coli, B. stearothermophilus, T. thermophilus, H. cutirubrum, spinach chloroplast, spinach cytomplasm, and Artemia salina. The structural studies support a unique tertiary interaction in eubacterial 5S rRNAs, involving nucleotides around positions 43 and 75. In addition long range structural effects are demonstrated in E. coli 5S rRNA due to the conversion of C to U at position 92.     

Probing the secondary structure of expansion segment ES6 in 18S ribosomal RNA

Expansion segment ES6 in 18S ribosomal RNA is, unlike many other expansion segments, present in all eukaryotes. The available data suggest that ES6 is located on the surface of the small ribosomal subunit. Here we have analyzed the secondary structure of the complete ES6 sequence in intact ribosomes from three eukaryotes, wheat, yeast, and mouse, representing different eukaryotic kingdoms. The availability of the ES6 sequence for modification and cleavage by structure sensitive chemicals and enzymatic reagents was analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The experimental results were used to restrict the number of possible secondary structure models of ES6 generated by the folding software MFOLD. The modification data obtained from the three experimental organisms were very similar despite the sequence variation. Consequently, similar secondary structure models were obtained for the ES6 sequence in wheat, yeast, and mouse ribosomes. A comparison of sequence data from more than 6000 eukaryotes showed that similar structural elements could also be formed in other organisms. The comparative analysis also showed that the extent of compensatory base changes in the suggested helices was low. The in situ structure analysis was complemented by a secondary structure analysis of wheat ES6 transcribed and folded in vitro. The obtained modification data indicate that the secondary structure of the in vitro transcribed sequence differs from that observed in the intact ribosome. These results suggest that chaperones, ribosomal proteins, and/or tertiary rRNA interactions could be involved in the in vivo folding of ES6.     

Equilibria in 5-S ribosomal RNA secondary structure. Bulges and interior loops in 5-S RNA secondary structure may serve as articulations for a flexible molecule

The basic assumption in this paper is that the secondary structure of a 5-S ribosomal RNA cannot be represented by a single model. We propose that the molecule can adopt, at least within the ribosome, a series of slightly different structures of nearly equal stability. The different structures arise from the existence of ambiguous base-pairing opportunities in bulged helices and the adjacent interior loops. In eubacterial 5-S RNAs there is one such an area, in eukaryotic 5-S RNAs two such areas that can give rise to structural switches. We explain how a change in secondary structure in these areas may influence the relative orientation of the surrounding helices, in other words how bulges and interior loops may serve as articulations and give rise to a flexible tertiary structure.     

Metastable secondary structures in ribosomal RNA molecular hysteresis in the acid-base titration of Escherichia coli ribosomal RNA

The metastable conformational states which underlie the hysteresis displayed by Escherichia coli ribosomal RNA in its pH titration in the acid range have been analyzed in terms of acid-stable RNA secondary structures. Sedimentation measurements show that the phenomenon is intramolecular, so that analysis of the hysteresis loops can, in principle, reveal details of molecular architecture. Hysteresis cycles obtained spectrophotometrically and potentiometrically were compared for RNA in solutions of different ionic strengths and ionic compositions. The effect is much smaller at lower ionic strength and disappears in the absence of magnesium ions. The curve followed upon addition of acid appears to reflect the equilibrium state of the system at each pH value. On the “base branch” of the loop, a slow absorbance change (complete in hours) was observed after the pH was raised by addition of a portion of base. This slow process is attributed to the annealing of “mismatched” multihelical regions of the ribosomal RNA. Certain regions, however, remain in metastable configurations for days and it is these long-lived non-equilibrium structures that underlie the hysteresis. Titration at 35 °C gave hysteresis loops of the same size and shape as at 20 °C; indeed, we found that the metastabilities are not removed even at 80 °C. Ultraviolet light absorbance difference spectra at 80 °C between solutions at the same pH, but on different branches of the cycle, give insight into the nature of the metastable conformation(s).Our experimental observations lead us to propose that the hysteresis is due to the formation at acidic pH of double-helical structures involving protonated guanine and adenine base pairs. The G.G pairs seem especially important to account for the very high thermal stability, as well as for the fact that the structures formed at a given pH value as acid is added dissociate only at higher pH values when the solution is titrated with base. Titrations of transfer RNA, along with literature data on 16 S rRNA primary structure, imply that the metastable regions in rRNA may consist of perhaps 10 to 15 base pairs.     

Higher order structure in ribosomal RNA.

The only reliable general method currently available for determining precise higher order structure in the large ribosomal RNAs is comparative sequence analysis. The method is here applied to reveal 'tertiary' structure in the 16S-like rRNAs, i.e. structure more complex than simple double-helical, secondary structure. From a list of computer-generated potential higher order interactions within 16S rRNA one such interaction considered likely was selected for experimental test. The putative interaction involves a Watson-Crick one to one correspondence between positions 570 and 866 in the molecule (E. coli numbering). Using existing oligonucleotide catalog information several organisms were selected whose 16S rRNA sequences might test the proposed co-variation. In all of the (phylogenetically independent) cases selected, full sequence evidence confirms the predicted one to one (Watson-Crick) correspondence. An interaction between positions 570 and 866 is, therefore, considered proven phylogenetically.     

Nucleotide sequence of a crustacean 18S ribosomal RNA gene and secondary structure of eukaryotic small subunit ribosomal RNAs

The primary structure of the gene for 18 S rRNA of the crustacean Artemia salina was determined. The sequence has been aligned with 13 other small ribosomal subunit RNA sequences of eukaryotic, archaebacterial, eubacterial, chloroplastic and plant mitochondrial origin. Secondary structure models for these RNAs were derived on the basis of previously proposed models and additional comparative evidence found in the alignment. Although there is a general similarity in the secondary structure models for eukaryotes and prokaryotes, the evidence seems to indicate a different topology in a central area of the structures.     

Destabilization of secondary structure in 16S ribosomal RNA by dimethylation of two adjacent adenosines.

Fragments comprising the 49 nucleotides from the 3'-end have been purified from 16S ribosomal RNA of wild-type Escherichia coli and from a kasugamycin-resistant mutant that specifically lacks dimethylation of two adjacent adenines near the 3'-terminus. These fragments, obtained after treatment of ribosomes in vitro with the bacteriocin cloacin DF13, were used to study the effect of the methyl groups on the temperature dependent unfolding of double-stranded regions. Both fragments contain at least 3 independent melting transitions, of which the one with the highest Tm corresponds with the unfolding of a nine-basepair long central hairpin. Dimethylation of the adenines in the loop of this hairpin lowers the melting temperature (Tm) by approximately 2 degrees C at 0.2 M NaCl and by about 5 degrees C at 0.15 M NaCl. It is suggested that m6(2)Am6(2)A is more antagonistic to loop formation that ApA and that the function of the methyl groups is to help to destabilize the 3'-terminal hairpin in 16S rRNA in order to facilitate intermolecular interactions.     

Revolutions in RNA secondary structure prediction

RNA structure formation is hierarchical and, therefore, secondary structure, the sum of canonical base-pairs, can generally be predicted without knowledge of the three-dimensional structure. Secondary structure prediction algorithms evolved from predicting a single, lowest free energy structure to their current state where statistics can be determined from the thermodynamic ensemble. This article reviews the free energy minimization technique and the salient revolutions in the dynamic programming algorithm methods for secondary structure prediction. Emphasis is placed on highlighting the recently developed method, which statistically samples structures from the complete Boltzmann ensemble.     

Vienna RNA secondary structure server

The Vienna RNA secondary structure server provides a web interface to the most frequently used functions of the Vienna RNA software package for the analysis of RNA secondary structures. It currently offers prediction of secondary structure from a single sequence, prediction of the consensus secondary structure for a set of aligned sequences and the design of sequences that will fold into a predefined structure. All three services can be accessed via the Vienna RNA web server at http://rna.tbi.univie.ac.at/.     

An RNA secondary structure workbench

A multiple approach to the study of RNA secondary structure is described which provides for the independent drawing of structures using base-pairing lists, for the generation of local structures in the form of hairpins, and for the generation of global structures by both Monte Carlo and dynamic programming methodologies. User-adjustable parameters provide for limiting the size of hairpin loops, bulges and inner loops, and constraints can be imposed relative to position-dependent base pairing.     

Sequence and secondary structure variation in the Gyrodactylus (Platyhelminthes: Monogenea) ribosomal RNA gene array

Nucleotide sequences were determined for the rRNA internal transcribed spacers 1 and 2 (ITS1 and 2) and the 5' terminus of the large subunit rRNA in selected Gyrodactylus species. Examination of primary sequence variation and secondary structure models in ITS2 and variable region V4 of the small subunit rRNA revealed that structure was largely conserved despite significant variation in sequence. ITS1 sequences were highly variable, and models of structure were unreliable but, despite this, show some resemblance to structures predicted in Digenea. ITS2 models demonstrated binding of the 3' end of 5.8S rRNA to the 5' end of the large subunit rRNA and enabled the termini of these genes to be defined with greater confidence than previously. The structure model shown here may prove useful in future phylogenetic analyses.