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At least 559 records · Page 31Linked to original sources
PubMed1997

A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.

Animals↗
PubMed2025

Structure of coxsackievirus cloverleaf RNA and 3Cpro dimer establishes the RNA-binding mechanism of enterovirus protease 3Cpro.

In positive-strand RNA viruses, the genome serves as a template for both protein translation and negative-strand RNA synthesis. Enteroviruses use the cloverleaf RNA structure at the 5' end of the genome to balance these two processes. Cloverleaf acts as a promoter for RNA synthesis and forms a complex with viral 3CD protein, the precursor to 3Cpro protease, and 3Dpol polymerase. The interaction between cloverleaf and 3CD is mediated by the 3Cpro domain, yet how 3Cpro promotes specific RNA-binding is not clear. We report the structure of coxsackievirus cloverleaf RNA-3Cpro complex, wherein two 3Cpro molecules interact with cloverleaf stem-loop D. 3Cpro dimer mainly recognizes the shape of the dsRNA helix through symmetric interactions, suggesting that 3Cpro is a previously undiscovered type of RNA binding protein. We show that 3CD protein also dimerizes on cloverleaf RNA and binds the RNA with higher affinity than 3Cpro. The structure provides insight into the RNA-binding mechanism of 3Cpro or 3CD with other cis-acting replication elements.

RNA, Viral↗
PubMed1979

Quantitative conservation of chromatin-bound RNA polymerases I and II in mitosis. Implications for chromosome structure.

RNA synthesis almost ceases in mitosis. It is ambiguous whether this temporal, negative control of RNA synthesis is solely because of the nature of chromosomes per se, (i.e., their condensed state), or to a physical loss of RNA polymerases along with other nuclear proteins which have been shown to pass into the cytoplasm in mitosis, or to their combined feature. Aside from such regulatory considerations, a question has also been raised as to whether RNA polymerases are constituents of metaphase chromosomes. To clarify these aspects of RNA polymerase-chromatin interaction in mitosis, the enzymes in chromosomes were quantitated and their levels compared to those in interphase nuclei and cells at various phases of the cell cycle. The results show that the amounts of form I, form II, and probably form III enzymes bound to a genome-equivalent of chromatin stay constant during the cell cycle. Thus, the mechanism for the negative control of RNA synthesis in mitosis appears to exist in the chromosomes per se, but not to be directly related to the RNA polymerase levels. This quantitative conservation of chromatin-bound RNA polymerases implies that they may persist as structural components of the chromosomes in mitosis.

Animals↗
PubMedDate not supplied

Metrics on RNA secondary structures.

Many different programs have been developed for the prediction of the secondary structure of an RNA sequence. Some of these programs generate an ensemble of structures, all of which have free energy close to that of the optimal structure, making it important to be able to quantify how similar these different structures are. To deal with this problem, we define a new class of metrics, the mountain metrics, on the set of RNA secondary structures of a fixed length. We compare properties of these metrics with other well known metrics on RNA secondary structures. We also study some global and local properties of these metrics.

Biometry↗
PubMed1998

Inhibition of polyadenylation by stable RNA secondary structure.

The presence of a polyadenylation signal in the repeat (R) region of the HIV-1 genome, which is located at both the 5' and 3' ends of the viral transcripts, requires differential regulation of polyadenylation. The HIV-1 poly(A) site can fold in a stable stem-loop structure that is well-conserved among different human and simian immunodeficiency viruses. In this study, we tested the effect of this hairpin on polyadenylation by introducing mutations that either stabilize or destabilize the RNA structure. The HIV-1 sequences were inserted into the pSV2CAT reporter plasmid upstream of the SV40 early poly(A) site. These constructs were transfected into COS cells and transcripts were analyzed for the usage of the HIV-1 versus SV40 poly(A) site. The wild-type HIV-1 poly(A) site was used efficiently in this context and destabilization of the poly(A) hairpin did not affect the polyadenylation efficiency. In contrast, further stabilization of the hairpin severely inhibited HIV-1 polyadenylation. Additional mutations that repair the thermodynamic stability of this mutant hairpin restored the polyadenylation activity. These results indicate that the mechanism of polyadenylation can be repressed by stable RNA structure encompassing the poly(A) signal. Experiments performed at reduced temperatures also suggest an inverse correlation between the stability of the RNA structure and the efficiency of polyadenylation.

Animals↗
PubMed1996

Visualization of RNA secondary structures using highly parallel computers.

Results of RNA secondary structure prediction algorithm are usually given as a set of hydrogen bonds between bases. However, we cannot know the precise structure of an RNA molecule by only knowing which bases form hydrogen bonds. One way to understand the structure of an RNA molecule is to visualize it using a planar graph so that we can easily know the geometric relations among the substructures such as stacking regions and loops. To do this, we consider bases to be particles on a plane and introduce a repulsive force and an attractive force among these particles and determine their positions according to these forces. A naive algorithm requires O(N2) time but we can reduce it to O(NlogN) with an approximation algorithm which is often used in the area of N-body simulation. Our program is written in parallel object-oriented language 'Schematic' which is recently developed. Efficiency of our implementation on a parallel computer and results of visualization of secondary structures are presented using cadang-cadang coconut viroid as an example.

Algorithms↗
PubMed2005

ARTS: alignment of RNA tertiary structures.

MOTIVATION: A fast growing number of non-coding RNAs have recently been discovered to play essential roles in many cellular processes. Similar to proteins, understanding the functions of these active RNAs requires methods for analyzing their tertiary structures. However, in contrast to the wide range of structure-based approaches available for proteins, there is still a lack of methods for studying RNA structures. RESULTS: We present a new computational method named ARTS (alignment of RNA tertiary structures). The method compares two nucleic acid structures (RNAs or DNAs) and detects a-priori unknown common substructures. These substructures can be either large global folds containing hundreds and even thousands of nucleotides or small local tertiary motifs with at least two successive base pairs. To the best of our knowledge, this is the first method of this type. The method is highly-efficient and was used to conduct an all-against-all comparison of all the RNA structures currently available in the Protein Data Bank. AVAILABILITY: The program, a web-server and supplementary information are available on http://bioinfo3d.cs.tau.ac.il/ARTS

Algorithms↗
PubMed1986

A dynamic programming algorithm for finding alternative RNA secondary structures.

Dynamic programming algorithms that predict RNA secondary structure by minimizing the free energy have had one important limitation. They were able to predict only one optimal structure. Given the uncertainties of the thermodynamic data and the effects of proteins and other environmental factors on structure, the optimal structure predicted by these methods may not have biological significance. We present a dynamic programming algorithm that can determine optimal and suboptimal secondary structures for an RNA. The power and utility of the method is demonstrated in the folding of the intervening sequence of the rRNA of Tetrahymena. By first identifying the major secondary structures corresponding to the lowest free energy minima, a secondary structure of possible biological significance is derived.

Animals↗
PubMed2001

A thermodynamic framework for Mg2+ binding to RNA.

We present a model describing how Mg(2+) binds and stabilizes specific RNA structures. In this model, RNA stabilization arises from two energetically distinct modes of Mg(2+) binding: diffuse- and site-binding. Diffusely bound Mg(2+) are electrostatically attracted to the strong anionic field around the RNA and are accurately described by the Poisson-Boltzmann equation as an ensemble distributed according to the electrostatic potentials around the nucleic acid. Site-bound Mg(2+) are strongly attracted to specifically arranged electronegative ligands that desolvate the ion and the RNA binding site. Thus, site-binding is a competition between the strong coulombic attraction and the large cost of desolvating the ion and its binding pocket. By using this framework, we analyze three systems where a single site-bound Mg(2+) may be important for stability: the P5 helix and the P5b stem loop from the P4-P6 domain of the Tetrahymena thermophila group I intron and a 58-nt fragment of the Escherichia coli 23S ribosomal RNA. Diffusely bound Mg(2+) play a dominant role in stabilizing these RNA structures. These ions stabilize the folded structures, in part, by accumulating in regions of high negative electrostatic potential. These regions of Mg(2+) localization correspond to ions that are observed in the x-ray crystallographic and NMR structures of the RNA. In contrast, the contribution of site-binding to RNA stability is often quite small because of the large desolvation penalty. However, in special cases, site-binding of partially dehydrated Mg(2+) to locations with extraordinarily high electrostatic potential can also help stabilize folded RNA structures.

Magnesium↗
PubMed2004

Detection of a novel sense-antisense RNA-hybrid structure by RACE experiments on endogenous troponin I antisense RNA.

Conformational changes in the troponin/tropomyosin complex significantly alter the mechanical properties of cardiac muscle. Phosphorylation of cardiac troponin I, part of the troponin/tropomyosin complex, reduces calcium affinity, which leads to increased relaxation of cardiac muscle. Because cardiac troponin I plays a central role in tuning the heart to different work demands, detailed knowledge of troponin I protein regulation is required. Our group previously detected naturally occurring antisense RNA for troponin I in human and rat hearts, and here, attempt to unravel the structure of rat cardiac troponin I antisense RNA. We performed rapid amplification of cDNA ends (RACE) experiments and discovered antisense sequences identical to a copy of the sense mRNA, which led us to conclude that the antisense RNA must be transcribed from troponin I mRNA in the cytoplasm. Moreover, we isolated RNA structures comprising sense and antisense sequences in one continuous molecule. As we found no homolog structures described in the literature, we called this "hybrid RNA." Because a duplex formation was demonstrated previously we concluded that hybrid RNA is a consequence of a tight interaction between sense and antisense troponin I RNA in vivo, which we discuss in the article.

Animals↗
PubMed2001

The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5' cap structure by exhibiting RNA 5'-triphosphatase activity.

Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5'-triphosphatase activity that specifically removes the gamma phosphate from the 5' end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m(7)GpppG cap structure was formed at the 5' end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5'-triphosphatase activity.

Acid Anhydride Hydrolases↗
PubMed2004

A new algorithm for RNA secondary structure design.

The function of many RNAs depends crucially on their structure. Therefore, the design of RNA molecules with specific structural properties has many potential applications, e.g. in the context of investigating the function of biological RNAs, of creating new ribozymes, or of designing artificial RNA nanostructures. Here, we present a new algorithm for solving the following RNA secondary structure design problem: given a secondary structure, find an RNA sequence (if any) that is predicted to fold to that structure. Unlike the (pseudoknot-free) secondary structure prediction problem, this problem appears to be hard computationally. Our new algorithm, "RNA Secondary Structure Designer (RNA-SSD)", is based on stochastic local search, a prominent general approach for solving hard combinatorial problems. A thorough empirical evaluation on computationally predicted structures of biological sequences and artificially generated RNA structures as well as on empirically modelled structures from the biological literature shows that RNA-SSD substantially out-performs the best known algorithm for this problem, RNAinverse from the Vienna RNA Package. In particular, the new algorithm is able to solve structures, consistently, for which RNAinverse is unable to find solutions. The RNA-SSD software is publically available under the name of RNA Designer at the RNASoft website (www.rnasoft.ca).

Algorithms↗
PubMed1999

Structural requirements of the higher order RNA kissing element in the enteroviral 3'UTR.

The origin of replication ( oriR ) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure which is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterised the kissing interaction. Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.

3' Untranslated Regions↗
PubMed1998

A polyhedral approach to RNA sequence structure alignment.

Ribonucleic acid (RNA) is a polymer composed of four bases denoted A, C, G, and U. It generally is a single-stranded molecule where the bases form hydrogen bonds within the same molecule leading to structure formation. In comparing different homologous RNA molecules it is important to consider both the base sequence and the structure of the molecules. Traditional alignment algorithms can only account for the sequence of bases, but not for the base pairings. Considering the structure leads to significant computational problems because of the dependencies introduced by the base pairings. In this paper we address the problem of optimally aligning a given RNA sequence of unknown structure to one of known sequence and structure. We phrase the problem as an integer linear program and then solve it using methods from polyhedral combinatorics. In our computational experiments we could solve large problem instances--23S ribosomal RNA with more than 1400 bases--a size intractable for former algorithms.

Algorithms↗
PubMed1997

Solution structure of RNA duplexes containing alternating CG base pairs: NMR study of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 under low salt conditions.

Structures of r(CGCGCG)2 and 2'-O-Me(CGCGCG)2 have been determined by NMR spectroscopy under low salt conditions. All protons and phosphorus nuclei resonances have been assigned. Signals of H5'/5" have been assigned stereospecifically. All 3JH,H and 3JP,H coupling constants have been measured. The structures were determined and refined using an iterative relaxation matrix procedure (IRMA) and the restrained MD simulation. Both duplexes form half-turn, right-handed helices with several conformational features which deviate significantly from a canonical A-RNA structure. Duplexes are characterised as having C3'-endo sugar pucker, very low base-pair rise and high helical twist and inclination angles. Helices are overwound with <10 bp per turn. There is limited inter-strand guanine stacking for CG steps. Within CG steps of both duplexes, the planes of the inter-strand cytosines are not parallel while guanines are almost parallel. For the GC steps this pattern is reversed. The 2'-O-methyl groups are spatially close to the 5'-hydrogens of neighbouring residues from the 3'-side and are directed towards the minor groove of 2'-O-Me(CGCGCG)2 forming a hydrophobic layer. Solution structures of both duplexes are similar; the effect of 2'-O-methylation on the parent RNA structure is small. This suggests that intrinsic properties imposed by alternating CG base pairs govern the overall conformation of both duplexes.

Base Composition↗
PubMed2006

Interpreting oligonucleotide microarray data to determine RNA secondary structure: application to the 3' end of Bombyx mori R2 RNA.

A method to deduce RNA secondary structure on the basis of data from microarrays of 2'-O-methyl RNA 9-mers immobilized in agarose film on glass slides is tested with a 249 nucleotide RNA from the 3' end of the R2 retrotransposon from Bombyx mori. Various algorithms incorporating binding data and free-energy minimization calculations were compared for interpreting the data to provide possible secondary structures. Two different methods give structures with 100 and 87% of the base pairs determined by sequence comparison. In contrast, structures predicted by free-energy minimization alone by Mfold and RNAstructure contain 52 and 72% of the known base pairs, respectively. This combination of high throughput microarray techniques with algorithms using free-energy calculations has potential to allow for fast determination of RNA secondary structure. It should also facilitate the design of antisense and siRNA oligonucleotides.

3' Untranslated Regions↗
PubMed2005

Specific roles of 5' RNA secondary structures in stabilizing transcripts in chloroplasts.

RNA secondary structures, e.g. stem-loops that are often found at the 5' and 3' ends of mRNAs, are in many cases known to be crucial for transcript stability but their role in prolonging the lifetime of transcripts remains elusive. In this study we show for an essential RNA-stabilizing stem-loop at the 5' end of rbcL gene transcripts in Chlamydomonas that it neither prevents ribonucleases from binding to the RNA nor impedes their movement along the RNA strand. The stem-loop has a formative function in that it mediates folding of a short sequence around its base into a specific RNA conformation, consisting of a helical and single-stranded region, i.e. the real structure required for longevity of rbcL transcripts in chloroplasts. Disturbing this structure renders transcripts completely unstable, even if the sequence of this element is not altered. The requirement of a specific 5' sequence and structure for RNA longevity suggests an interaction of this element with a trans-acting factor that protects transcripts from rapid degradation in chloroplasts.

5' Untranslated Regions↗