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PubMed1997

Rational and combinatorial strategies for designing oligonucleotides targeted to RNA structures.

Many RNA structures play a key role in the regulation of gene expression. We designed synthetic oligonucleotides able to interact with folded RNA regions (see Toulmé et al., Biochimie (1996) 78, 663-673, for a review). We have demonstrated that a decanucleotide can form a triple helix with the stem of the hairpin responsible for ribosomal frame-shifting of the gag-pro message of HTLV-I, leading to the inhibition of translation. We have isolated, through an in vitro selection procedure, from a library composed of oligonucleotides with a random part of 30 nucleotides, sequences able to bind to the TAR RNA element of HIV-1 with a dissociation constant of 20-50 nM. The association between the two partners involve non-canonical interactions. This extends the range of potential targets for antisense sequences to functional RNA structures.

Drug Design↗

PubMed2000

A structured RNA motif is involved in correct placement of the tRNA(3)(Lys) primer onto the human immunodeficiency virus genome.

Human immunodeficiency virus type 1 (HIV-1) reverse transcription is primed by the cellular tRNA(3)(Lys) molecule that binds with its 3'-terminal 18 nucleotides to the fully complementary primer-binding site (PBS) on the viral RNA genome. Besides this complementarity, annealing of the primer may be stimulated by additional base-pairing interactions between other parts of the tRNA molecule and viral sequences flanking the PBS. According to the RNA secondary structure model of the HIV-1 leader region, part of the PBS sequence is involved in base pairing to form a small stem-loop structure, termed the U5-PBS hairpin. This hairpin may be involved in the process of reverse transcription. To study the role of the U5-PBS hairpin in the viral replication cycle, we introduced mutations in the U5 region that affect the stability of this structured RNA motif. Stabilization and destabilization of the hairpin significantly inhibited virus replication. Upon prolonged culturing of the virus mutant with the stabilized hairpin, revertant viruses were obtained with additional mutations that restore the thermodynamic stability of the U5-PBS hairpin. The thermodynamic stability of the U5-PBS hairpin apparently has to stay within narrow limits for efficient HIV-1 replication. Transient transfection experiments demonstrated that transcription of the proviral genomes, translation of the viral mRNAs, and assembly of the virions with a normal RNA content is not affected by the mutations within the U5-PBS hairpin. We show that stabilization of the hairpin reduced the amount of tRNA primer that is annealed to the PBS. Destabilization of the hairpin did not affect tRNA annealing, but the viral RNA-tRNA complex was less stable. These results suggest that the U5-PBS hairpin is involved in correct placement of the tRNA primer on the viral genome. The analysis of virus mutants and revertants and the RNA structure probing experiments presented in this study are consistent with the existence of the U5-PBS hairpin as predicted in the RNA secondary structure model.

5' Untranslated Regions↗

PubMed2005

Evidence of reciprocal tertiary interactions between conserved motifs involved in organizing RNA structure essential for internal initiation of translation.

Internal ribosome entry site (IRES) elements consist of highly structured RNA regions that determine internal initiation of translation. We have previously shown that the foot-and-mouth disease virus (FMDV) IRES contains a GNRA tetraloop spanning residues G178UAA181. Here we show that tertiary RNA interactions dependent on the GNRA motif determine the structural organization of the central domain. By using mutational analysis in combination with RNA probing, we have identified distant reciprocal interactions between the GNRA motif and the invariant region G240CACG244, termed motif A. Mutations in motif A caused a decrease in IRES activity as severe as the GUAG substitution in the GNRA motif. Substitutions in either GNRA or motif A sequences induced a common reorganization around the conserved R199AAA202 stem-loop, suggesting that the latter contributes to stabilize the GNRA-motif A interaction. This finding was also consistent with a significant increase in the efficiency of RNA-RNA interactions determined in gel shift assays using as probe the hairpin that contains the GNRA motif compared to a transcript encompassing the entire apical region of the central domain. Thus, we propose that the central domain of the FMDV IRES contains a structural conformation essential for IRES activity stabilized by a tertiary contact involving residues in the GNRA tetraloop and motif A conserved sequences.

Base Sequence↗

PubMed2006

RNAMST: efficient and flexible approach for identifying RNA structural homologs.

RNA molecules fold into characteristic secondary structures for their diverse functional activities such as post-translational regulation of gene expression. Searching homologs of a pre-defined RNA structural motif, which may be a known functional element or a putative RNA structural motif, can provide useful information for deciphering RNA regulatory mechanisms. Since searching for the RNA structural homologs among the numerous RNA sequences is extremely time-consuming, this work develops a data preprocessing strategy to enhance the search efficiency and presents RNAMST, which is an efficient and flexible web server for rapidly identifying homologs of a pre-defined RNA structural motif among numerous RNA sequences. Intuitive user interface are provided on the web server to facilitate the predictive analysis. By comparing the proposed web server to other tools developed previously, RNAMST performs remarkably more efficiently and provides more effective and flexible functions. RNAMST is now available on the web at http://bioinfo.csie.ncu.edu.tw/~rnamst/.

Internet↗

PubMed2002

Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation.

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60-100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90-156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.

5' Untranslated Regions↗

PubMed1994

Mutations in an essential U2 small nuclear RNA structure cause cold-sensitive U2 small nuclear ribonucleoprotein function by favoring competing alternative U2 RNA structures.

Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vitro. Cold-sensitive U2 small nuclear RNA adopts an alternative conformation that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternative U2 RNA structure causes the defects, we tested second-site mutations in U2 predicted to disrupt the alternative conformation. We find that such mutations efficiently suppress the cold-sensitive phenotypes and partially restore correct U2 RNA folding. A genetic search for additional suppressors of cold sensitivity revealed two unexpected mutations in the base of an adjacent stem-loop. Direct probing of RNA structure in vivo indicates that the suppressors of cold sensitivity act to improve the stability of the essential stem relative to competing alternative structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RNAs and RNA-binding proteins could be a result of changes in the stability of a functional RNA conformation relative to a competing structure. The presence of an evolutionarily conserved U2 sequence positioned to form an alternative structure argues that this region of U2 is dynamic during the assembly or function of the U2 small nuclear ribonucleoprotein.

Base Sequence↗

PubMed1984

5S RNA structure and interaction with transcription factor A. 1. Ribonuclease probe of the structure of 5S RNA from Xenopus laevis oocytes.

The structure of Xenopus laevis oocyte (Xlo) 5S ribosomal RNA has been probed with single-strand-specific ribonucleases T1, T2, and A with double-strand-specific ribonuclease V1 from cobra venom. The digestion of 5'- or 3'-labeled renatured 5S RNA samples followed by gel purification of the digested samples allowed the determination of primary cleavage sites. Results of these ribonuclease digestions provide support for the generalized 5S RNA secondary structural model derived from comparative sequence analysis. However, three putative single-stranded regions of the molecule exhibited unexpected V1 cuts, found at C36, U73, U76, and U102. These V1 cuts reflect additional secondary structural features of the RNA including A.G base pairs and support the extended base pairing in the stem containing helices IV and V which was proposed by Stahl et al. [Stahl, D. A., Luehrsen, K. R., Woese, C. R., & Pace, N. R. (1981) Nucleic Acids Res. 9, 6129-6137]. A conserved structure for helix V having a common unpaired uracil residue at Xlo position 84 is proposed for all eukaryotic 5S RNAs. Our results are compared with nuclease probes of other 5S RNAs.

Animals↗

PubMed1989

Site-selective cleavage of structured RNA by a staphylococcal nuclease-DNA hybrid.

A hybrid enzyme consisting of an oligodeoxyribonucleotide fused to a unique site on staphylococcal nuclease site-selectively cleaves a number of natural RNAs including Escherichia coli M1 RNA (377 bases), 16S rRNA (1542 bases), and yeast tRNA(Phe). The oligonucleotide directs the nuclease activity of the enzyme to the nucleotides directly adjacent to the complementary target sequence on the substrate RNA. In the case of M1 RNA, hydrolysis occurs primarily at one phosphodiester bond, converting 50% of the starting material to product. Furthermore, the reaction products can be enzymatically manipulated: tRNA(Phe) was cleaved in the anticodon region and was religated to form the full-length tRNA in high yield. Because the specificity of these hybrid enzymes can be easily altered, they should prove to be useful tools for probing RNA structure and function.

Base Sequence↗

PubMed2000

The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems.

The G x U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodynamic stability to Watson-Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G x C or A x U base pairs. The G x U wobble base pair also has unique chemical, structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson-Crick base pairs or other mispairs. These features mark sites containing G x U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biological processes.

Base Pairing↗

PubMed1984

RNA structure analysis using methidiumpropyl-EDTA.Fe(II): a base-pair-specific RNA structure probe.

Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] in the presence of dithiothreitol, is shown to cleave phenylalanine-accepting tRNA (tRNAPhe) in a structure-specific fashion. Molar ratios of MPE.Fe(II) to tRNAPhe of less than 1 preferentially cleave phosphodiester bonds known to occur in double-stranded regions of the tRNAPhe molecule. Microdensitometric analysis of autoradiograms of MPE.Fe(II) cleavage products following gel electrophoresis reveals a correspondence between preferred sites of MPE.Fe(II) cleavage and sites in tRNAPhe most sensitive to cobra venom ribonuclease, a double-strand-specific endoribonuclease. Conversely, sites of cleavage by the single-strand-specific S1 nuclease correspond to those nucleotides that are least susceptible to MPE.Fe(II) hydrolysis. Sensitive helical regions in tRNAPhe include the dihydrouracil and the "T psi C" stems, which cannot be detected by cobra venom ribonuclease because of steric constraints. Phosphodiester bonds within the T psi C and dihydrouracil loop regions, which are not detected by S1 nuclease under rigorously controlled digestion conditions, are revealed by inference from their relative insensitivity to MPE.Fe(II). These results demonstrate the utility of MPE.Fe(II) as a general small molecular weight probe of RNA structure, having a greater accessibility to base-paired regions than do the more bulky enzymic probes.

Base Sequence↗

PubMed2005

RNA structure and RNA-protein interactions in purified yeast U6 snRNPs.

The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem II with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.

Base Sequence↗

PubMed2005

Thermodynamic stability of RNA structures formed by CNG trinucleotide repeats. Implication for prediction of RNA structure.

Trinucleotide repeat expansion diseases (TREDs) are correlated with elongation of CNG DNA and RNA repeats to pathological level. This paper shows, for the first time, complete data concerning thermodynamic stabilities of RNA with CNG trinucleotide repeats. Our studies include the stability of oligoribonucleotides composed of two to seven of CAG, CCG, CGG, and CUG repeats. The thermodynamic parameters of helix propagation correlated with the presence of multiple N-N mismatches within CNG RNA duplexes were also determined. Moreover, the total stability of CNG RNA hairpins, as well as the contribution of trinucleotide repeats placed only in the stem or loop regions, was evaluated. The improved thermodynamic parameters allow to predict much more accurately the thermodynamic stabilities and structures of CNG RNAs.

Models, Molecular↗

PubMed2005

Multiple RNA structure alignment.

Ribonucleic Acid (RNA) structures can be viewed as a special kind of strings where characters in a string can bond with each other. The question of aligning two RNA structures has been studied for a while, and there are several successful algorithms that are based upon different models. In this paper, by adopting the model introduced in Wang and Zhang,(19) we propose two algorithms to attack the question of aligning multiple RNA structures. Our methods are to reduce the multiple RNA structure alignment problem to the problem of aligning two RNA structure alignments. Meanwhile, we will show that the framework of sequence center star alignment algorithm can be applied to the problem of multiple RNA structure alignment, and if the triangle inequality is met in the scoring matrix, the approximation ratio of the algorithm remains to be 2-2(over)n, where n is the total number of structures.

Algorithms↗

PubMed1983

Recognition of the ribosomal RNA structures by purified nucleolar RNA methyltransferase.

Previously we have isolated the specific RNA methyltransferase from the nucleoli of Ehrlich ascites tumor cells. The purified enzyme was found to be specific for methylation of C5 position of cytosine residue in ribosomal RNA in vitro (Obara, 1982b). In the present study, we have investigated the recognition mechanisms of RNA structure by this enzyme from the points of view of both primary and secondary structures. Analysis of in vitro methylation product by ribonuclease T1 digestion indicated the methylation-site(s) was limited to a certain number of nonanucleotide. The next experiments with either Sl nuclease or actinomycin D and ethidium bromide suggested that the enzyme modified only cytidine residue in or located close to the double stranded part of RNA. On the other hand, the characterization of analogues of cytidine residue in the RNA at molecular level showed that the methylation of rRNA was inhibited by either cytidine, CDP or CTP, but little inhibition was observed in the presence of cytosine, 5-methylcytidine and CMP.

Animals↗

PubMed2006

Local gapped subforest alignment and its application in finding RNA structural motifs.

RNA molecules whose secondary structures contain similar substructures often have similar functions. Therefore, an important task in the study of RNA is to develop methods for discovering substructures in RNA secondary structures that occur frequently (also referred to as motifs). In this paper, we consider the problem of computing an optimal local alignment of two given labeled ordered forests F1 and F2. This problem asks for a substructure of F1 and a substructure of F2 that exhibit a high similarity. Since an RNA molecule's secondary structure can be represented as a labeled ordered forest, the problem we study has a direct application to finding potential motifs. We generalize the previously studied concept of a closed subforest to a gapped subforest and present the first algorithm for computing the optimal local gapped subforest alignment of F1 and F2. We also show that our technique can improve the time and space complexity of the previously most efficient algorithm for optimal local closed subforest alignment. Furthermore, we prove that a special case of our local gapped subforest alignment problem is equivalent to a problem known in the literature as the local sequence-structure alignment problem (lssa) and modify our main algorithm to obtain a much faster algorithm for lssa than the one previously proposed. An implementation of our algorithm is available at www.comp.nus.edu.sg/~bioinfo/LGSFAligner/. Its running time is significantly faster than the original lssa program.

Nucleic Acid Conformation↗

PubMed2004

The identification of novel RNA structural motifs using COMPADRES: an automated approach to structural discovery.

Recurring RNA structural motifs are important sites of tertiary interaction and as such, are integral to RNA macromolecular structure. Although numerous RNA motifs have been classified and characterized, the identification of new motifs is of great interest. In this study, we discovered four new conformationally recurring motifs: the pi-turn, the Omega-turn, the alpha-loop and the C2'-endo mediated flipped adenosine motif. Not only do they have complex and interesting structures, but they participate in contacts of high biological significance. In a first for the RNA field, new motifs were discovered by a fully automated algorithm. This algorithm, COMPADRES, utilized a reduced representation of the RNA backbone and was highly successful at discerning unique structural relationships. This study also shows that recurring RNA substructures are not necessarily accompanied by consistent primary or secondary structure.

Adenosine↗

PubMed1997

Displaying the information contents of structural RNA alignments: the structure logos.

MOTIVATION: We extend the standard 'Sequence Logo' method of Schneider and Stevens (Nucleic Acids Res., 18, 6097-6100, 1990) to incorporate prior frequencies on the bases, allow for gaps in the alignments, and indicate the mutual information of base-paired regions in RNA. RESULTS: Given an alignment of RNA sequences with the base pairings indicated, the program will calculate the information at each position, including the mutual information of the base pairs, and display the results in a 'Structure Logo'. Alignments without base pairing can also be displayed in a 'Sequence Logo', but still allowing gaps and incorporating prior frequencies if desired. AVAILABILITY: The code is available from, and an Internet server can be used to run the program at, http://www.cbs.dtu.dk/gorodkin/appl/slogo. html.

Algorithms↗

PubMed1997

Complex formation of the spinach chloroplast psbA mRNA 5' untranslated region with proteins is dependent on the RNA structure.

RNA-protein interactions are part of many regulatory pathways in gene expression. In chloroplasts of higher plants and of green algae, gene regulation by posttranscriptional processes such as differential regulation of mRNA stability and control of translation plays a major role during chloroplast development and light-dependent protein expression. Regulation here is mediated by interactions of RNA-binding proteins with the respective mRNAs. In this work, structural requirements for protein-RNA complex formation between the 5' untranslated region of the spinach psbA mRNA (encoding the D1 protein of photosystem II) and stromal proteins are analyzed. For this, a combination of temperature gradient gel electrophoresis and gel shift analysis is employed to study several variants of the psbA 5' untranslated region. Supported by theoretical interpretation of the data and analysis of the structures by chemical probing, we show that a certain structure of the RNA is necessary for protein complex formation. Already very subtle structural changes within the RNA interfere with binding and thereby with the biological activity of the mRNA.

Base Sequence↗