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PubMed2006

Identification and classification of conserved RNA secondary structures in the human genome.

The discoveries of microRNAs and riboswitches, among others, have shown functional RNAs to be biologically more important and genomically more prevalent than previously anticipated. We have developed a general comparative genomics method based on phylogenetic stochastic context-free grammars for identifying functional RNAs encoded in the human genome and used it to survey an eight-way genome-wide alignment of the human, chimpanzee, mouse, rat, dog, chicken, zebra-fish, and puffer-fish genomes for deeply conserved functional RNAs. At a loose threshold for acceptance, this search resulted in a set of 48,479 candidate RNA structures. This screen finds a large number of known functional RNAs, including 195 miRNAs, 62 histone 3'UTR stem loops, and various types of known genetic recoding elements. Among the highest-scoring new predictions are 169 new miRNA candidates, as well as new candidate selenocysteine insertion sites, RNA editing hairpins, RNAs involved in transcript auto regulation, and many folds that form singletons or small functional RNA families of completely unknown function. While the rate of false positives in the overall set is difficult to estimate and is likely to be substantial, the results nevertheless provide evidence for many new human functional RNAs and present specific predictions to facilitate their further characterization.

3' Untranslated Regions↗
PubMed2003

A Mg2+-dependent RNA tertiary structure forms in the Bacillus subtilis trp operon leader transcript and appears to interfere with trpE translation control by inhibiting TRAP binding.

Expression of the trpEDCFBA operon of Bacillus subtilis is regulated by transcription attenuation and translation control mechanisms. In each case, binding of the trp RNA-binding attenuation protein (TRAP) to the untranslated trp leader transcript mediates conformational changes in the RNA secondary structure. We examined the structure of the trp leader readthrough RNA in the absence of TRAP. Using chemical and enzymatic probes, the secondary structure of the trp leader RNA was found to be similar to predicted models. In addition, this RNA was found to adopt a Mg(2+)-dependent, long-range tertiary interaction under physiological monovalent salt conditions. Formation of this tertiary structure does not require significant changes in the preformed secondary structure. Enzymatic probing of the RNA in the presence of competitor DNA oligonucleotides that were designed to disrupt the predicted tertiary structure allowed identification of the interacting partners as the single-stranded portion of the purine-rich TRAP binding target and a large downstream pyrimidine-rich internal loop. UV cross-linking experiments utilizing 5'-p-azidophenacyl-containing transcripts revealed a Mg(2+)-dependent cross-link. Mapping of this cross-link provided evidence that the single-stranded segment of the TRAP binding site is in close proximity to the internal loop. Results from UV melting experiments with wild-type and mutant trp leader transcripts suggested a likely base-pairing register for the tertiary structure. Filter-binding studies demonstrated that the addition of Mg(2+) inhibits TRAP binding, which may be partially due to the effect of Mg(2+) on RNA tertiary structure formation. Results from expression studies using trpE'-'lacZ translational fusions and RNA-directed cell-free translation experiments suggest that the Mg(2+)-dependent tertiary structure inhibits TRAP's ability to regulate translation of trpE.

Bacillus subtilis↗
PubMed2005

Mutually exclusive splicing of the insect Dscam pre-mRNA directed by competing intronic RNA secondary structures.

Drosophila Dscam encodes 38,016 distinct axon guidance receptors through the mutually exclusive alternative splicing of 95 variable exons. Importantly, known mechanisms that ensure the mutually exclusive splicing of pairs of exons cannot explain this phenomenon in Dscam. I have identified two classes of conserved elements in the Dscam exon 6 cluster, which contains 48 alternative exons--the docking site, located in the intron downstream of constitutive exon 5, and the selector sequences, which are located upstream of each exon 6 variant. Strikingly, each selector sequence is complementary to a portion of the docking site, and this pairing juxtaposes one, and only one, alternative exon to the upstream constitutive exon. The mutually exclusive nature of the docking site:selector sequence interactions suggests that the formation of these competing RNA structures is a central component of the mechanism guaranteeing that only one exon 6 variant is included in each Dscam mRNA.

Alternative Splicing↗
PubMed1997

In vivo determination of RNA structure-function relationships: analysis of the 790 loop in ribosomal RNA.

The 790 loop is a conserved hairpin located between positions 786 and 796 of Escherichia coli 16 S rRNA that is required for ribosome function. Using a novel genetic approach, all positions in the loop were simultaneously mutated and functional mutant sequences were selected in vivo. This "instant evolution" experiment revealed that approximately 190 of the 262,144 possible mutant sequences were functional. Analysis of functional mutant sequences allowed discrimination between nucleotides directly involved in protein synthesis and those involved primarily in loop structure. Among the functional mutant sequences, positions 789 and 791 were invariant and extensive covariation was observed among the nucleotides at the base of the loop at positions 787, 788, 794 and 795. NMR and thermodynamic analyses of model 790 hairpins in vitro revealed weak pairing interactions between positions 787 and 795 and between positions 788 and 794 consistent with the in vivo mutational analysis. Functional analysis of site-directed mutants containing all possible nucleotide combinations at positions 787 and 795 in vivo showed that stable base-pairs at these positions prevent subunit association.

Chloramphenicol↗
PubMed2006

DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation.

The DExD/H box family of proteins includes a large number of proteins that play important roles in RNA metabolism. Members of this family have been shown to act as RNA helicases or unwindases, using the energy from ATP hydrolysis to unwind RNA structures or dissociate RNA-protein complexes in cellular processes that require modulation of RNA structures. However, it is clear that several members of this family are multifunctional and, in addition to acting as RNA helicases in processes such as pre-mRNA processing, play important roles in transcriptional regulation. In this review I shall concentrate on RNA helicase A (Dhx9), DP103 (Ddx20), p68 (Ddx5) and p72 (Ddx17), proteins for which there is a strong body of evidence showing that they play important roles in transcription, often as coactivators or corepressors through their interaction with key components of the transcriptional machinery, such as CREB-binding protein, p300, RNA polymerase II and histone deacetylases.

Autoantigens↗
PubMed2005

RNA secondary structure prediction by centroids in a Boltzmann weighted ensemble.

Prediction of RNA secondary structure by free energy minimization has been the standard for over two decades. Here we describe a novel method that forsakes this paradigm for predictions based on Boltzmann-weighted structure ensemble. We introduce the notion of a centroid structure as a representative for a set of structures and describe a procedure for its identification. In comparison with the minimum free energy (MFE) structure using diverse types of structural RNAs, the centroid of the ensemble makes 30.0% fewer prediction errors as measured by the positive predictive value (PPV) with marginally improved sensitivity. The Boltzmann ensemble can be separated into a small number (3.2 on average) of clusters. Among the centroids of these clusters, the "best cluster centroid" as determined by comparison to the known structure simultaneously improves PPV by 46.5% and sensitivity by 21.7%. For 58% of the studied sequences for which the MFE structure is outside the cluster containing the best centroid, the improvements by the best centroid are 62.5% for PPV and 31.4% for sensitivity. These results suggest that the energy well containing the MFE structure under the current incomplete energy model is often different from the one for the unavailable complete model that presumably contains the unique native structure. Centroids are available on the Sfold server at http://sfold.wadsworth.org.

Base Pairing↗
PubMed2003

On the algebraic representation of RNA secondary structures with G x U pairs.

Magarshak et al. represented an RNA molecule as a complex vector x and an RNA secondary structure gamma as a complex matrix Sgamma in such a way that the molecule represented by x was compatible with the secondary structure gamma if and only if Sgamma o x=x. They only considered Watson-Crick base pairs and their representation cannot be extended to allow for G x U pairs. In this paper we study a generalization of Magarshak's representation that allows for these pairs, and in particular we provide a family of algebraic structures where that generalization can be carried out. We also show that this representation can be used to compare secondary structures, through transfer matrices which transform the representation of one secondary structure into the representation of the other.

Base Pairing↗
PubMed2003

Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR.

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. In this study, we find that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. We also present evidence that TOR regulates association of Rpd3-Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site-specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Delta and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, our results suggest a chromatin-mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability.

Antifungal Agents↗
PubMed1992

Recognition of tertiary structure in tRNAs by Rh(phen)2phi3+, a new reagent for RNA structure-function mapping.

With photoactivation Rh(phen)2phi3+ promotes strand cleavage at sites of tertiary interaction in tRNA. The rhodium complex, which binds double-helical DNA by intercalation in the major groove, yields no cleavage in double-helical regions of the RNA or in unstructured single-stranded regions. Instead, Rh(phen)2phi3+ appears to target regions which are structured so that the major groove is open and accessible for stacking with the complex, as occurs where bases are triply bonded. So as to examine the specificity of this novel reagent and to evaluate its use in probing structural changes in RNAs, cleavage studies have been conducted on two structurally characterized tRNAs, tRNA(Phe) and tRNA(Asp) from yeast, the unmodified yeast tRNA(Phe) transcript, and a chemically modified tRNA(Phe), as well as on a series of tRNA(Phe) mutants. On tRNA(Phe) strong cleavage is observed at residues G22, G45, U47, psi 55, and U59; weaker cleavage is observed at A44, m7G46, and C48. On tRNA(Asp) cleavage is found at residues A21 through G26, psi 32, and U48, with minor cleavage apparent at A44, G45, A46, psi 55, U59, and U60. There is a striking similarity in cleavage observed on these tRNAs, and the sites of cleavage mark regions of tertiary folding. Cleavage on the unmodified tRNA(Phe) transcript resembles closely that found on native yeast tRNA(Phe), but additional sites, primarily in the anticodon loop and stem, are evident. The results indicate that globally the structures containing or lacking the modified bases appear to be the same; the differences in cleavage observed may reflect a loosening or alteration in the structure due to the absence of the modified bases. Cleavage results on mutants of tRNA(Phe) illustrate Rh(phen)2phi3+ as a sensitive probe in characterizing tRNA tertiary structure. Results are consistent with other assays for structural or functional changes. Uniquely, Rh(phen)2phi3+ appears to target directly sites of tertiary interaction. Cleavage results on mutants which involve base changes within the triply bounded region of the molecule indicate that it is the structure of the triply bonded array rather than the individual nucleotides which are being targeted. Chemical modification to promote selective depurination of the third base (m7G46) involved in the triple in the folded, native tRNA leads to the reduction of cleavage by the metal complex; this result shows directly the importance of the stacked triple base structure for recognition by the metal complex.(ABSTRACT TRUNCATED AT 400 WORDS)

2,2'-Dipyridyl↗
PubMed2006

In silico predicted robustness of viroids RNA secondary structures. I. The effect of single mutations.

Viroids are plant subviral pathogens whose genomes are constituted by a single-stranded and covalently closed small RNA molecule that does not encode for any protein. Despite this genomic simplicity, they are able of inducing devastating symptoms in susceptible plants. Most of the 29 described viroid species fold into a rodlike or quasi-rodlike structure, whereas a few of them fold as branched structures. The shape of these RNA structures is perhaps one of the most characteristic properties of viroids and sometimes is considered their only phenotype. Here we use RNA thermodynamic secondary structure prediction algorithms to compare the mutational robustness of all viroid species. After characterizing the statistical properties of the distribution of mutational effects on structure stability and the wideness of neutral neighborhood for each viroid species, we show an evolutionary trend toward increased structural robustness during viroid radiation, giving support to the adaptive value of robustness. Differences in robustness among the 2 viroid families can be explained by the larger fragility of branched structures compared with the rodlike ones. We also show that genomic redundancy can contribute to the robustness of these simple RNA genomes.

Algorithms↗
PubMed1997

Effect of RNA secondary structure on the kinetics of DNA synthesis catalyzed by HIV-1 reverse transcriptase.

The effect of RNA secondary structure on the kinetics of DNA synthesis catalyzed by HIV-1 RT was determined using a 66 nucleotide RNA template containing a stable 12 base pair hairpin structure. Prior to reaching the hairpin structure, the primer elongation by RT was fast and the kinetics of polymerization was not affected by the presence of the secondary structure. Once within the regions of template secondary structure, polymerization was much slower and RT paused at five distinct sites [Suo, Z., & Johnson, K. A. (1997) Biochemistry (manuscript submitted for publication)]. Kinetic analysis of single nucleotide incorporation at the pause sites showed polymerization occurred by both a fast phase (54-76 s-1) and a slow phase (0.07-0.4 s-1) during a single binding event. The biphasic kinetics suggests that the DNA substrates are initially bound in both productive and nonproductive states at the polymerase site of RT. The nonproductively bound DNA is slowly converted into a productive state without dissociation from the enzyme. At the pause sites, the enzyme amplitudes of the fast phase are small (4.0-15%) while the amplitudes of the slow phase are large (11-40%). In contrast, only the reaction at the fast phase was observed at the nonpause sites and the enzyme amplitudes were large (63-66%) although the nucleotide incorporation rates (62-78 s-1) are similar to the fast phase rates at the pause sites. These indicate that DNA substrates were bound predominantly nonproductively at pause sites and productively at nonpause sites. However, the overall binding affinity of DNA substrates was measured by the nitrocellulose-DEAE double filter binding assay, binding affinity at both pause sites and nonpause sites was similar (9-38 nM). This indicates that substrates are bound tightly at the large binding cleft of HIV-1, although they may not be productively bound at the polymerase active site. These results and those reported elsewhere [Suo, Z., & Johnson, K. A. (1997) Biochemistry (manuscript submitted for publication)] are consistent with a model in which, at pause sites, HIV-1 RT remains bound to DNA substrates waiting for the melting of the next stem base pair of template secondary structure. Upon melting of the stem base pair, polymerization to fill the open template site is fast and largely irreversible, allowing RT to read through the stable hairpin structures.

Base Sequence↗
PubMed2006

A periodic pattern of mRNA secondary structure created by the genetic code.

Single-stranded mRNA molecules form secondary structures through complementary self-interactions. Several hypotheses have been proposed on the relationship between the nucleotide sequence, encoded amino acid sequence and mRNA secondary structure. We performed the first transcriptome-wide in silico analysis of the human and mouse mRNA foldings and found a pronounced periodic pattern of nucleotide involvement in mRNA secondary structure. We show that this pattern is created by the structure of the genetic code, and the dinucleotide relative abundances are important for the maintenance of mRNA secondary structure. Although synonymous codon usage contributes to this pattern, it is intrinsic to the structure of the genetic code and manifests itself even in the absence of synonymous codon usage bias at the 4-fold degenerate sites. While all codon sites are important for the maintenance of mRNA secondary structure, degeneracy of the code allows regulation of stability and periodicity of mRNA secondary structure. We demonstrate that the third degenerate codon sites contribute most strongly to mRNA stability. These results convincingly support the hypothesis that redundancies in the genetic code allow transcripts to satisfy requirements for both protein structure and RNA structure. Our data show that selection may be operating on synonymous codons to maintain a more stable and ordered mRNA secondary structure, which is likely to be important for transcript stability and translation. We also demonstrate that functional domains of the mRNA [5'-untranslated region (5'-UTR), CDS and 3'-UTR] preferentially fold onto themselves, while the start codon and stop codon regions are characterized by relaxed secondary structures, which may facilitate initiation and termination of translation.

3' Untranslated Regions↗
PubMed1994

Structure-sensitive RNA footprinting of yeast nuclear ribonuclease P.

Several enzymatic and chemical reagents were used to probe the secondary structure of Saccharomyces cerevisiae nuclear RNase P RNA in the presence and absence of its protein components. Double-stranded regions were detected with RNase V1 and single-stranded regions with RNase ONE (Escherichia coli RNase I). Nucleotides not paired at Watson-Crick positions were monitored with dimethyl sulfate, kethoxal, and 1-cyclohexyl-3-[2-(N-methylmorpholinio)ethyl]carbodiimide p-toluenesulfonate. The results supported most aspects of the previously proposed, phylogenetically-derived RNA secondary structure, although minor refinements allowed incorporation of both the biochemical and phylogenetic data. Digestion of the RNase P protein(s) with proteinase K gave enhanced reactivities to structure probes at selected positions, indicating regions of the RNA made inaccessible by the presence of the protein subunit(s). The regions of RNA protected in the yeast nuclear holoenzyme were considerably more extensive than that seen in the Escherichia coli holoenzyme, consistent with the observation that the protein moiety generally comprises a larger percentage of the RNase P holoenzyme in eukaryotes than in eubacteria.

Base Composition↗
PubMed1994

Database on the structure of small ribosomal subunit RNA.

The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library.

Animals↗
PubMed2003

Improving the accuracy of NMR structures of RNA by means of conformational database potentials of mean force as assessed by complete dipolar coupling cross-validation.

The description of the nonbonded contact terms used in simulated annealing refinement can have a major impact on nucleic acid structures generated from NMR data. Using complete dipolar coupling cross-validation, we demonstrate that substantial improvements in coordinate accuracy of NMR structures of RNA can be obtained by making use of two conformational database potentials of mean force: a nucleic acid torsion angle database potential consisting of various multidimensional torsion angle correlations; and an RNA specific base-base positioning potential that provides a simple geometric, statistically based, description of sequential and nonsequential base-base interactions. The former is based on 416 nucleic acid crystal structures solved at a resolution of </=2 A and an R-factor </=25%; the latter is based on 131 RNA crystal structures solved at a resolution of </=3 A and an R-factor of </=25%, and includes both the large and small subunits of the ribosome. The application of these two database potentials is illustrated for the structure refinement of an RNA aptamer/theophylline complex for which extensive NOE and residual dipolar coupling data have been measured in solution.

Databases, Nucleic Acid↗
PubMed1997

The role of the cap structure in RNA processing and nuclear export.

The cap structure that is characteristic of all polymerase-II-transcribed RNAs has been shown to play an important role in many aspects of RNA metabolism including RNA processing, RNA nuclear transport, and translation initiation. The effects of the cap structure on these different processes is mediated by proteins that recognise and bind to it, and are therefore generically called cap-binding proteins. For example, the cap-binding protein eIF4E, in a complex with other proteins, mediates the effect of the cap on the initiation of translation. EIF-4E is predominantly localised in the cytoplasm. In the last five years, it has been demonstrated that a second cap-binding protein complex, which is mainly localised in the nucleus, mediates the stimulatory effects of the cap in nuclear processes such as pre-mRNA splicing, RNA 3'-end formation, and RNA nuclear export. The purpose of this review is to summarise our current knowledge on the role of the cap structure and of the cap-binding protein complex in nuclear RNA metabolism and present evidence that at least some processes may be coupled in vivo.

Animals↗
PubMed2004

RNA tertiary structure and cooperative assembly of a large ribonucleoprotein complex.

The mechanisms that govern the ordered assembly of multiprotein ribonucleoprotein complexes are not well understood. The in vitro reconstitution of the small subunit of the bacterial ribosome provides a tractable system for the detailed study of ordered assembly. We present a quantitative thermodynamic description of the hierarchical binding of ribosomal proteins to 16S rRNA during assembly of the platform of the 30S ribosomal subunit. The binding of S8, S11, S15, and the S6:S18 heterodimer to the central domain of 16S rRNA has been measured both individually and in combination using isothermal titration calorimetry and gel mobility shift assays. Both enthalpy and free energy measurements demonstrate the cooperative binding of S15 and the S6:S18 heterodimer, but no cooperativity is observed for either S8 or S11. The results define a thermodynamic framework that describes cooperative platform assembly.

Bacteria↗
PubMed2006

Stress responsive DEAD-box helicases: a new pathway to engineer plant stress tolerance.

Abiotic stresses including various environmental factors adversely affect plant growth and limit agricultural production worldwide. Minimizing these losses is a major area of concern for all countries. Therefore, it is desirable to develop multi-stress tolerant varieties. Salinity, drought, and cold are among the major environmental stresses that greatly influence the growth, development, survival, and yield of plants. UV-B radiation of sunlight, which damages the cellular genomes, is another growth-retarding factor. Several genes are induced under the influence of various abiotic stresses. Among these are DNA repair genes, which are induced in response to the DNA damage. Since the stresses affect the cellular gene expression machinery, it is possible that molecules involved in nucleic acid metabolism including helicases are likely to be affected. The light-driven shifts in redox-potential can also initiate the helicase gene expression. Helicases are ubiquitous enzymes that catalyse the unwinding of energetically stable duplex DNA (DNA helicases) or duplex RNA secondary structures (RNA helicases). Most helicases are members of DEAD-box protein superfamily and play essential roles in basic cellular processes such as DNA replication, repair, recombination, transcription, ribosome biogenesis and translation initiation. Therefore, helicases might be playing an important role in regulating plant growth and development under stress conditions by regulating some stress-induced pathways. There are now few reports on the up-regulation of DEAD-box helicases in response to abiotic stresses. Recently, salinity-stress tolerant tobacco plants have already been raised by overexpressing a helicase gene, which suggests a new pathway to engineer plant stress tolerance [N. Sanan-Mishra, X.H. Pham, S.K. Sopory, N. Tuteja, Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield. Proc. Natl. Acad. Sci. USA 102 (2005) 509-514]. Presently the exact mechanism of helicase-mediated stress tolerance is not understood. In this review we have described all the reported stress-induced helicases and also discussed the possible mechanisms by which they can provide stress tolerance.

DNA Helicases↗