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At least 19 recordsLinked to original sources
PubMed2000

In vivo, high-resolution analysis of yeast and mammalian RNA-protein interactions, RNA structure, RNA splicing and ribozyme cleavage by use of terminal transferase-dependent PCR.

We have investigated the analysis of RNA by use of terminal transferase-dependent PCR (TDPCR), a procedure previously used for the analysis of DNA and chromatin [J. Komura and A.D.Riggs, Nucleic Acids Res.,26, 1807-1811 (1998)]. When preceded by reverse transcription (RT), TDPCR provides an extremely sensitive, versatile, quantitative and nucleotide-level assay for detecting RNA lesions or structures that block primer extension during the RT step. The procedure is: (i) RT using a gene-specific oligonucleotide; (ii) ribo-tailing of the single-stranded cDNA product by use of terminal deoxy-nucleotidyl transferase; (iii) ligation of a DNA linker to the tailed cDNA by use of T4 DNA ligase; and (iv) PCR using a nested, gene-specific primer and a linker-specific primer. This procedure combines the versatility of a primer extension assay with nucleotide-level resolution, the specificity of nested primers and the sensitivity of PCR. Band patterns obtained are reproducible and quantifiable. We successfully used the technique for the study of yeast RNA structure, splicing intermediates and ribozyme cleavage. Also, in vivo footprint experiments, using mammalian cells and RNase T1, revealed the binding of iron-responsive element binding protein to iron responsive elements in the mRNAs of transferrin receptor and ferritin H-chain.

Base Sequence↗
PubMed2001

Exploring the folding landscape of a structured RNA.

Structured RNAs achieve their active states by traversing complex, multidimensional energetic landscapes. Here we probe the folding landscape of the Tetrahymena ribozyme by using a powerful approach: the folding of single ribozyme molecules is followed beginning from distinct regions of the folding landscape. The experiments, combined with small-angle x-ray scattering results, show that the landscape contains discrete folding pathways. These pathways are separated by large free-energy barriers that prevent interconversion between them, indicating that the pathways lie in deep channels in the folding landscape. Chemical protection and mutagenesis experiments are then used to elucidate the structural features that determine which folding pathway is followed. Strikingly, a specific long-range tertiary contact can either help folding or hinder folding, depending on when it is formed during the process. Together these results provide an unprecedented view of the topology of an RNA folding landscape and the RNA structural features that underlie this multidimensional landscape.

Animals↗
PubMed1994

RNA helicases: modulators of RNA structure.

RNA molecules play an essential role in many cellular processes, often as components of ribonucleoprotein complexes. Like proteins, RNA molecules adopt sequence-specific secondary and tertiary structures that are essential for function; alteration of these structures therefore provides a means of regulating RNA function. The discovery of DEAD box proteins, a large family of proteins that share several highly conserved motifs and have known or putative ATP-dependent RNA helicase activity, has provoked growing interest in the concept that regulation of RNA function may occur through local unwinding of complex RNA structures.

Journal Article↗
PubMed1998

Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release.

Nascent RNA structures may regulate RNA chain elongation either directly through interaction with RNA polymerase or indirectly by disrupting nascent RNA contacts with polymerase or DNA. To distinguish these mechanisms we tested whether the effects of the his leader pause RNA hairpin could be mimicked by pairing of antisense DNA or RNA oligonucleotides to the nascent transcript. The his pause hairpin inhibits nucleotide addition when it forms 11 nucleotides from the transcript 3' end. It also can terminate transcription when base changes extend its stem to </=8 nucleotides from the 3' end. All oligonucleotides that disrupted the pause hairpin reduced the dwell time of RNA polymerase at the pause site dramatically, even when they mimicked the 11-nucleotide 3'-proximal RNA spacing or created a suitably positioned RNA loop. Oligonucleotides that paired </=8 nucleotides from the pause RNA 3' end could trigger transcript release, but only when added to an already paused complex. These results argue that direct interaction of a nascent RNA hairpin with RNA polymerase delays escape from a pause, but that indirect effects of a hairpin may trigger transcript release from a paused complex. Resistance of the paused complex to pyrophosphorolysis and its reversal by antisense oligonucleotides further suggest that interaction of the pause hairpin with RNA polymerase disengages the RNA 3' end from the active site.

DNA-Directed RNA Polymerases↗
PubMed2002

RNA editing in hepatitis delta virus genotype III requires a branched double-hairpin RNA structure.

RNA editing at the amber/W site plays a central role in the replication scheme of hepatitis delta virus (HDV), allowing the virus to produce two functionally distinct forms of the sole viral protein, hepatitis delta antigen (HDAg), from the same open reading frame. Editing is carried out by a cellular activity known as ADAR (adenosine deaminase), which acts on RNA substrates that are at least partially double stranded. In HDV genotype I, editing requires a highly conserved base-paired structure that occurs within the context of the unbranched rod structure characteristic of HDV RNA. This base-paired structure is disrupted in the unbranched rod of HDV genotype III, which is the most distantly related of the three known HDV genotypes and is associated with the most severe disease. Here I show that RNA editing in HDV genotype III requires a branched double-hairpin structure that deviates substantially from the unbranched rod structure, involving the rearrangement of nearly 80 bp. The structure includes a UNCG RNA tetraloop, a highly stable structural motif frequently involved in the folding of large RNAs such as rRNA. The double-hairpin structure is required for editing, and hence for virion formation, but not for HDV RNA replication, which requires the unbranched rod structure. HDV genotype III thus relies on a dynamic conformational switch between the two different RNA structures: the unbranched rod characteristic of HDV RNA and a branched double-hairpin structure that is required for RNA editing. The different mechanisms of editing in genotypes I and III underscore their functional differences and may be related to pathogenic differences as well.

Base Sequence↗
PubMed2004

A guide to ions and RNA structure.

RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. "Diffuse ions" accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A "chelated" ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg(2+) on RNA stability where experimental data are available. An important conclusion is that diffuse ions are a major factor in the stabilization of RNA tertiary structures.

Animals↗
PubMed2003

RNA labeling and immobilization for nano-displacement measurement: probing three-dimensional RNA structure.

RNA molecules play essential roles in many biological processes, including the storage and transfer of information in the cell. These events are mediated via RNA-protein interactions or by catalytic RNA molecules. It is now recognized that unique RNA folds are associated with biological functions. Therefore, to study the intrinsic structural changes and dynamics which regulate the various functions of RNA, it is necessary to probe its three-dimensional structure in solution. In this respect, using single-molecule methodologies may allow study of native RNA molecules independent of their size and in real time. However, this may require the immobilization of RNA on a surface. Here, we report a novel approach to immobilize RNA on a glass. The procedures involve both chemical and enzymatic modifications of long RNA molecules. In addition, we demonstrate the application of an optical tweezers apparatus to measure the length and, hence, the dynamics of immobilized intact ribosomal RNA molecules as a function of different solution conditions.

Adsorption↗
PubMed2003

TectoRNA and 'kissing-loop' RNA: atomic force microscopy of self-assembling RNA structures.

RNA molecules have been much less studied by atomic force microscopy (AFM) than have DNA molecules. In this paper, AFM imaging is presented for two different RNA molecules able to self-assemble into complex supramolecular architectures. The first one is a molecular dimer of a 230-nt RNA fragment coming from the RNA genome of a murine leukaemia virus. The monomeric RNA fragment, which appears by AFM as an elongated structure with a mean aspect ratio of 1.4, assembles into a dimer of elongated structures through the formation of a 'kissing-loop' RNA interaction. The second one is a large supramolecular fibre formed of artificial self-assembling RNA molecular units called tectoRNA. The fibre lengths by AFM suggest that there are 50-70 tectoRNA units per fibre. Some methods and limitations are presented for measuring molecular volumes from AFM images.

Animals↗
PubMed2006

Thousands of corresponding human and mouse genomic regions unalignable in primary sequence contain common RNA structure.

Human and mouse genome sequences contain roughly 100,000 regions that are unalignable in primary sequence and neighbor corresponding alignable regions between both organisms. These pairs are generally assumed to be nonconserved, although the level of structural conservation between these has never been investigated. Owing to the limitations in computational methods, comparative genomics has been lacking the ability to compare such nonconserved sequence regions for conserved structural RNA elements. We have investigated the presence of structural RNA elements by conducting a local structural alignment, using FOLDALIGN, on a subset of these 100,000 corresponding regions and estimate that 1800 contain common RNA structures. Comparing our results with the recent mapping of transcribed fragments (transfrags) in human, we find that high-scoring candidates are twice as likely to be found in regions overlapped by transfrags than regions that are not overlapped by transfrags. To verify the coexpression between predicted candidates in human and mouse, we conducted expression studies by RT-PCR and Northern blotting on mouse candidates, which overlap with transfrags on human chromosome 20. RT-PCR results confirmed expression of 32 out of 36 candidates, whereas Northern blots confirmed four out of 12 candidates. Furthermore, many RT-PCR results indicate differential expression in different tissues. Hence, our findings suggest that there are corresponding regions between human and mouse, which contain expressed non-coding RNA sequences not alignable in primary sequence.

Animals↗
PubMed2006

The importance of RNA structure in RNA editing and a potential proofreading mechanism for correct guide RNA:pre-mRNA binary complex formation.

RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridine bases are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is cleaved specifically, uridine bases are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, non-coding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of their cognate mRNA and anneal to form a binary RNA complex. An exception from this process is the cytochrome oxidase subunit II (COII) mRNA, which encodes its gRNA within its 3' untranslated region. This gRNA lacks the ability to function in trans. Using an in vitro editing assay, we find that improving thermodynamic stability to the anchor region through increased Watson-Crick base-pairing is sufficient to impart trans editing activity. We further show that a point mutation outside the known functional regions of a gRNA induces both a conformational rearrangement of the gRNA and causes a decrease in the rate of editing. Taken together, these results lead us to propose a model for a potential proofreading step in the formation of a gRNA:pre-edited mRNA binary complex. The mechanism relies on the thermodynamic stability supplied to the RNA complex through Watson-Crick base-pairing. Through mutations in the gRNA, we demonstrate the importance of gRNA structure to the RNA editing reaction.

3' Untranslated Regions↗
PubMed2006

Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication.

The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3Dpol), the precursor 3CD, and an RNA template containing the cre/bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.

Amino Acid Sequence↗
PubMed2004

Diversity of base-pair conformations and their occurrence in rRNA structure and RNA structural motifs.

In addition to the canonical base-pairs comprising the standard Watson-Crick (C:G and U:A) and wobble U:G conformations, an analysis of the base-pair types and conformations in the rRNAs in the high-resolution crystal structures of the Thermus thermophilus 30S and Haloarcula marismortui 50S ribosomal subunits has identified a wide variety of non-canonical base-pair types and conformations. However, the existing nomenclatures do not describe all of the observed non-canonical conformations or describe them with some ambiguity. Thus, a standardized system is required to classify all of these non-canonical conformations appropriately. Here, we propose a new, simple and systematic nomenclature that unambiguously classifies base-pair conformations occurring in base-pairs, base-triples and base-quadruples that are associated with secondary and tertiary interactions. This system is based on the topological arrangement of the two bases and glycosidic bonds in a given base-pair. Base-pairs in the internal positions of regular secondary structure helices usually form with canonical base-pair groups (C:G, U:A, and U:G) and canonical conformations (C:G WC, U:A WC, and U:G Wb). In contrast, non-helical base-pairs outside of regular structure helices usually have non-canonical base-pair groups and conformations. In addition, many non-helical base-pairs are involved in RNA motifs that form a defined set of non-canonical conformations. Thus, each rare non-canonical conformation may be functionally and structurally important. Finally, the topology-based isostericity of base-pair conformations can rationalize base-pair exchanges in the evolution of RNA molecules.

Base Pairing↗
PubMed2004

Multiple RNA structure alignment.

RNA structures can be viewed as a kind of special strings with some characters bonded 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 [18], we propose two algorithms to attack the question of aligning multiple RNA structures. We reduce the multiple RNA structure alignment problem to the problem of aligning two RNA structure alignments.

Algorithms↗
PubMed2001

Modulation of RNA function by oligonucleotides recognizing RNA structure.

Numerous RNA structures are responsible for regulatory processes either because they constitute a signal, like the hairpins or pseudoknots involved in ribosomal frameshifting, or because they are binding sites for proteins such as the trans-activating responsive RNA element of the human immunodeficiency virus whose binding to the viral protein Tat and cellular proteins allows full-length transcription of the retroviral genome. Selective ligands able to bind with high affinity to such RNA motifs may serve as tools for dissecting the molecular mechanisms in which they are involved. Such ligands might also constitute prototypes of therapeutic agents when RNA structures play a role in the expression of dysfunctional genes or in the multiplication of pathogens. Different classes of ligands (aminoglycosides, interacalating agents, peptides) are of interest to this aim. However, oligonucleotides deserve particular consideration. They have been extensively used in the frame of the antisense strategy. The apparent simplicity of this rational approach is, at first sight, very attractive. Indeed, numerous successful studies have been published describing the efficient inhibition of translation, splicing, or reverse transcription in cell-free systems, in cultured cells, or in vivo by oligomers complementary to an RNA region. However, RNA structures restrict the access of the target site to the antisense sequence: The competition between the intramolecular association of RNA regions weakens or even abolishes the antisense effect. Various possibilities have been developed to circumvent this limitation. This includes both rational and combinatorial strategies. High-affinity oligomers were designed to invade the RNA structure. Alternatively, triplex-forming oligonucleotides (TFO) and aptamers may recognize the folded RNA motif. Whereas the use of TFOs is rather limited owing to the strong sequence constraints for triple-helix formation, in vitro selection offers a way to explore vast oligoribo or oligodeoxyribo libraries to identify strong, selective oligonucleotide binders. The candidates (aptamers) selected against the TAR RNA element of HIV-1, which form stable loop-loop (kissing) complexes with the target, provide interesting examples of oligonucleotides recognizing a functional RNA structure through an important contribution of tertiary interactions.

Animals↗
PubMed2003

Stable RNA structures can repress RNA synthesis in vitro by the brome mosaic virus replicase.

A 15-nucleotide (nt) unstructured RNA with an initiation site but lacking a promoter could direct the initiation of RNA synthesis by the brome mosaic virus (BMV) replicase in vitro. However, BMV RNA with a functional initiation site but a mutated promoter could not initiate RNA synthesis either in vitro or in vivo. To explain these two observations, we hypothesize that RNA structures that cannot function as promoters could prevent RNA synthesis by the BMV RNA replicase. We documented that four different nonpromoter stem-loops can inhibit RNA synthesis from an initiation-competent RNA sequence in vitro. Destabilizing these structures increased RNA synthesis. However, RNA synthesis was restored in full only when a BMV RNA promoter element was added in cis. Competition assays to examine replicase-RNA interactions showed that the structured RNAs have a lower affinity for the replicase than do RNAs lacking stable structures or containing a promoter element. The results characterize another potential mechanism whereby the BMV replicase can specifically recognize BMV RNAs.

Base Sequence↗
PubMed1993

Chemical nucleases: their use in studying RNA structure and RNA-protein interactions.

Metal complexes that cleave nucleic acids provide a new means to study RNA structure and RNA-protein interactions. Methods that use these chemical nucleases help compensate for the limitations of other techniques used to determine structure. Because the ligands that coordinate the metal generally control the cleavage selectivity of these complexes, it has become possible to design nucleolytic reagents that target specific higher-order structures. In combination with site-directed mutagenesis these conformation-specific probes can be used to delineate long-range interactions. Alternatively, complexes that cut irrespective of sequence and secondary structure have been used in protection (foot-printing) experiments to locate protein binding sites. Because each position of the nucleic acid is susceptible to cleavage, the protection pattern yields a highly resolved definition of the contact site between the protein and RNA. In other applications, metal complexes have been conjugated to functional moieties such as oligonucleotides, peptides, or substrate analogs to direct their binding to a distinct site on a specific RNA molecule. This latter strategy holds significant therapeutic promise for the destruction of pathogenic RNAs.

Binding Sites↗
PubMed1996

The structure of an RNA dodecamer shows how tandem U-U base pairs increase the range of stable RNA structures and the diversity of recognition sites.

BACKGROUND: Non-canonical base pairs are fundamental building blocks of RNA structures. They can adopt geometries quite different from those of canonical base pairs and are common in RNA molecules that do not transfer sequence information. Tandem U-U base pairs occur frequently, and can stabilize duplex formation despite the fact that a single U-U base pair is destabilizing. RESULTS: We determined the crystal structure of the RNA dodecamer GGCGCUUGCGUC at 2.4 A resolution. The molecule forms a duplex containing tandem U-U base pairs, which introduce an overall bend of 11-12 degrees in the duplex resulting from conformational changes at each interface between the tandem U-U base pairs and a flanking duplex sequence. The formation of the U-U base pairs cause small changes in several backbone torsion angles; base stacking is preserved and two hydrogen bonds are formed per base pair, explaining the stability of the structure. CONCLUSIONS: Tandem U-U base pairs can produce stable structures not accessible to normal A-form RNA, which may allow the formation of specific interfaces for RNA-RNA or RNA-protein recognition. These base-pairs show an unusual pattern of hydrogen-bond donors and acceptors in the major and minor grooves, which could also act as a recognition site.

Base Composition↗
PubMed2004

Nonionic side chains modulate the affinity and specificity of binding between functionalized polyamines and structured RNA.

This Communication introduces side-chain-bearing polyamines as molecules for selective recognition of folded RNA structures. The complex folded structures associated with RNA create binding pockets for proteins, and also binding sites for small molecules. Developing organic molecules that can bind RNA with high affinity and specificity is a challenge that must be overcome for RNA to be considered a viable drug target. In this work, six polyamines with different side chains were synthesized to test for effects on binding affinity and specificity to TAR RNA and RRE RNA of HIV. Binding interactions between polyamines and RNAs were examined using two footprinting assays, based on terbium-induced cleavage and magnesium-catalyzed cleavage at higher pH. The binding constants and the binding specificity were highly dependent on the side chains of the polyamines, demonstrating that this class of molecules is a very promising starting point for development of highly selective RNA-binding ligands.

Base Sequence↗