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Results for “RNA-RNA interactions”

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At least 91 records · Page 5Linked to original sources

PubMed1998

Nonradioactive in situ hybridization for detection of hydrophobin mRNA in the phytopathogenic fungus Claviceps purpurea during infection of rye.

Hydrophobins are unique fungal extracellular proteins that produce amphipathic films at interfaces, mediate contact to hydrophobic surfaces and are known to be important in phytopathogenicity. In the pathogenic ascomycete Claviceps purpurea, causing ergot disease in grasses and cereals and ergotism in livestock, a gene encoding an extraordinary type of hydrophobin has been detected, which appeared to be induced during alkaloid synthesis in axenic culture of an ergot-alkaloid producing strain of Claviceps (V. Garre and P. Tudzynski, pers. communication; Arntz and Tudzynski, 1997, Curr. Genet. 31, 357-360). To elucidate presence and function of this hydrophobin during infection of rye, the nonradioactive in situ hybridization technique was successfully adapted to the fungal organism and optimized in the pathogenic interaction system. Semithin cryosections proved to be suitable for microscopical gene expression analysis using immune-mediated alkaline-phosphatase staining for detection of digoxigenin-labeled cRNA probes. Specific hybridization of the prepared antisense riboprobe to hydrophobin mRNA was confirmed in nonradioactive Northern blots. While permeabilization by proteinase K had only a minor effect, the inclusion of detergent into the hybridization solutions enhanced specific RNA-RNA hybridization under maximum stringency. Hydrophobin mRNA was found in fungal cells, growing in axenic culture. In the disease cycle, hydrophobin transcripts were localized in abundance during vegetative fructification in conidiophores that actively produced conidia. No signals were observed in sclerotial hyphae during formation of the alkaloid-containing ergots, although they fluoresced intensely during total RNA detection using acridine orange. Notably, in situ hybridization experiments resulted in specific signals during early infection and colonization phases in the external mycelia and in hyphae penetrating the host epidermal layer. The presumed role of the hydrophobin gene product in ergot pathogenicity is discussed with respect to the described spatio-temporal distribution of the hydrophobin transcripts.

Claviceps↗

PubMedDate not supplied

Circularization of retroviral genomic RNA and the control of RNA translation, packaging and reverse transcription.

Translation, packaging and reverse transcription of the genomic RNA of retroviruses appear to be regulated by short and long range RNA-RNA interactions which take place within the 5'-600 nt and between the 5' and 3' untranslated sequences. The 5' (R and U5) and 3' (Dr and U3) domains of the genomic RNA together with the nucleic acid binding protein (NBP) would control the balance between the open state of the viral genomic RNA, correlated with an efficient RNA translation and the closed state, with the circular viral RNA efficiently packaged into virions. Retroviral NBP might well drive the packaging of the viral RNA as well as improve reverse transcription of the circular virion RNA.

Amino Acid Sequence↗

PubMed1997

Antisense-mediated regulation of Annexin VII gene expression during the transition from growth to differentiation in Dictyostelium discoideum.

Annexin VII is believed to be required for proper Ca(2+)-homeostasis in Dictyostelium discoideum cells. As was previously reported, the expression of Annexin VII gene increased during the transition of D. discoideum Ax-2 cells from growth to differentiation. We have casually cloned an interesting gene, Quit3, by the differential plaque hybridization. Quit3 had no coding region, and was expressed more predominantly in the growth phase than in the differentiation phase. Unexpectedly, this gene was found to encode the complementary sequence of Annexin VII. Therefore, it is most likely that the Quit3 mRNA may regulate the Annexin VII synthesis by the natural antisense transcript via an antisense RNA-RNA interaction, thus resulting in striking increase of Annexin VII production in the phase-shift of cells from growth to differentiation. Since Annexin VII is known to be coded for by a single gene in Dictyostelium, the antisense RNA seemed to be encoded in the same genetic locus as the Annexin VII mRNA.

Amino Acid Sequence↗

PubMed1996

Biochemistry and regulation of pre-mRNA splicing.

During the past year, significant advances have been made in the field of pre-mRNA splicing. It is now clear that members of the serine-arginine-rich protein family are key players in exon definition and function at multiple steps in the spliceosome cycle. Novel findings have been made concerning the role of exon sequences, which function as both constitutive and regulated enhancers of splicing, in trans-splicing and as targets for tissue-specific control of splicing patterns. By combining biochemical approaches in human and yeast extracts with genetic analysis, much has been learned about the RNA-RNA and RNA-protein interactions that are necessary to assemble the various complexes that are found along the pathway to the catalytically active spliceosome.

Gene Expression Regulation, Fungal↗

PubMed2002

RNA folding in vivo.

RNA folding in vivo is influenced by the cellular environment, the vectorial nature of transcription and translation, trans-acting factors and ion homeostasis. Specific RNA-binding proteins promote RNA folding by stabilizing the native structure or by guiding folding. In contrast, RNA chaperones, which are believed to interact nonspecifically with RNA, were proposed to resolve misfolded RNA structures and to promote intermolecular RNA-RNA annealing. Small trans-acting noncoding RNAs are thought to modulate mRNA structures, thereby regulating gene expression. So far, there is some evidence that in vitro and invivo RNA folding pathways share basic features. However, it is unclear whether the rules deduced from in vitro folding experiments generally apply to invivo conditions.

3' Flanking Region↗

PubMed1999

Small RNAs in Escherichia coli.

Bacterial cells contain several small RNAs (sRNAs) that are not translated. These stable, abundant RNAs act by multiple mechanisms, such as RNA-RNA basepairing, RNA-protein interactions and intrinsic RNA activity, and regulate diverse cellular functions, including RNA processing, mRNA stability, translation, protein stability and secretion.

Base Sequence↗

PubMed1997

RNA-RNA interactions between oligonucleotide substrates for aminoacylation.

RNA stem-loop microhelices with helix sequences based on tRNA acceptor stems can be charged with specific amino acids. Experiments were designed to test the possibility that microhelices could laterally associate through complementary loop sequences and thereby bring their attached aminoacyl groups close enough together to form a peptide bond. Computer simulations suggested that formation of such complexes would be sensitive to the number of loop nucleotides needed to span the grooves of the quasi-continuous helix of the intermolecular pseudoknot so formed. These predictions were conformed experimentally by observation of complex formation sensitivity to loop size. Complexes with optimized loop sizes had apparent bimolecular dissociation constants of approximately 100 nM with only three complementary base pairs between the respective loops. Single nucleotide substitutions that disrupted the predicted intermolecular loop-loop base-pairing abolished detectable association. Similarly, placing a gap between the short helix formed by loop-loop pairing and the adjacent acceptor stems also diminished complex formation. These experiments establish an experimental basis for microhelix association for peptide synthesis.

Acetylation↗

PubMed1998

Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions.

In the past few years thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded RNA molecules and to identify contacts within nucleic acids and/or between nucleic acids and proteins, in complex nucleoprotein assemblies. These thio residues such as 4-thiouracil found in E. coli tRNA and its non-natural congeners 4-thiothymine, 6-thioguanine and 6-mercaptopurine absorb light at wavelengths longer than 320 nm and, thus, can be selectively photoactivated. Synthetic or enzymatic procedures have been established, allowing the random or site-specific incorporation of thionucleotide(s) within a RNA (DNA) chain which, in most cases, retains unaltered structural and biological properties. Owing to the high photoreactivity of their triplet state (intersystem yield close to unity), 4-thiouracil and 4-thiothymine derivatives exhibit a high photocrosslinking ability towards pyrimidines (particularly thymine) but also purines. From the nature of the photoproducts obtained in base or nucleotide mixtures and in dinucleotides, the main photochemical pathway was identified as a (2 + 2) photoaddition of the excited C-S bond onto the 5, 6 double bond of pyrimidines yielding thietane intermediates whose structure could be characterized. Depending on the mutual orientation of these bonds in the thietanes, their subsequent dark rearrangement yielded, respectively, either the 5-4 or 6-4 bipyrimidine photoadduct. A similar mechanism appears to be involved in the formation of the unique photoadduct formed between 4-thiothymidine and adenosine. The higher reactivity of thymine derived acceptors can be explained by an additional pathway which involves hydrogen abstraction from the thymine methyl group, followed by radical recombination, leading to methylene linked bipyrimidines. The high photocrosslinking potential of thionucleosides inserted in nucleic acid chains has been used to probe RNA-RNA contacts within the ribosome permitting, in particular, the elucidation of the path of mRNA throughout the small ribosomal subunit. Functional interactions between the mRNA spliced sites and U RNAs could be detected within the spliceosome. Analysis of the photocrosslinks obtained within small endonucleolytic ribozymes in solution led to a tertiary folded pseudo-knot structure for the HDV ribozyme and allowed the construction of a Y form of a hammerhead ribozyme, which revealed to be in close agreement with the structure observed in crystals. Thionucleosides incorporated in nucleic acids crosslink efficiently amino-acid residues of proteins in contact with them. Despite the fact that little is known about the nature of the photoadducts formed, this approach has been extensively used to identify protein components interacting at a defined nucleic acid site and applied to various systems (replisome, spliceosome, transcription complexes and ribosomes).

Animals↗

PubMed1996

New loop-loop tertiary interactions in self-splicing introns of subgroup IC and ID: a complete 3D model of the Tetrahymena thermophila ribozyme.

BACKGROUND: Group I introns self-splice via two consecutive trans-esterification reactions in the presence of guanosine cofactor and magnesium ions. Comparative sequence analysis has established that a catalytic core of about 120 nucleotides is conserved in all known group I introns. This core is generally not sufficient for activity, however, and most self-splicing group I introns require non-conserved peripheral elements to stabilize the complete three-dimensional (3D) structure. The physico-chemical properties of group I introns make them excellent systems for unraveling the structural basis of the RNA-RNA interactions responsible for promoting the self-assembly of complex RNAs. RESULTS: We present phylogenetic and experimental evidence for the existence of three additional tertiary base pairings between hairpin loops within peripheral components of subgroup IC1 and ID introns. Each of these new long range interactions, called P13, P14 and P16, involves a terminal loop located in domain 2. Although domains 2 of IC and ID introns share very strong sequence similarity, their terminal loops interact with domains 5 and 9 (subgroup IC1) and domain 6 (subgroup ID). Based on these tertiary contacts, comparative sequence analysis, and published experimental results such as Fe(II)-EDTA protection patterns, we propose 3D models for two entire group I introns, the subgroup IC1 intron in the large ribosomal precursor RNA of Tetrahymena thermophila and the SdCob.1 subgroup ID intron found in the cytochrome b gene of Saccharomyces douglasii. CONCLUSIONS: Three-dimensional models of group I introns belonging to four different subgroups are now available. They all emphasize the modular and hierarchical organization of the architecture of group I introns and the widespread use of base-pairings between terminal hairpin loops for stabilizing the folded and active structures of large and complex RNA molecules.

Animals↗

PubMed1996

Crystal structures of an A-form duplex with single-adenosine bulges and a conformational basis for site-specific RNA self-cleavage.

BACKGROUND: Bulged nucleotides are common secondary structural motifs in RNA molecules and are often involved in RNA-RNA and RNA-protein interactions. RNA is selectively cleaved at bulge sites (when compared to other sites within stems) in the presence of divalent metal cations. The effects of bulge nucleotides on duplex stability and topology have been extensively investigated, but no detailed X-ray structures of bulge-containing RNA fragments have been available. RESULTS: We have crystallized a self-complementary RNA-DNA chimeric 11-nucleotide sequence containing single-adenosine bulges under two different conditions, giving two distinct crystal forms. In both lattices the adenosines are looped out, leaving the stacking interactions in the duplex virtually unaffected. The bulges cause the duplex to kink in both cases. In one of the structures, the conformation of the bulged nucleotide places its modeled 2'-oxygen in line with the adjacent phosphate on the 3' side, where it is poised for nucleophilic attack. CONCLUSIONS: Single adenosine bulges cause a marked opening of the normally narrow RNA major groove in both crystal structures, rendering the bases more accessible to interacting molecules compared with an intact stem. The geometries around the looped-out adenosines are different in the two crystal forms, indicating that bulges can confer considerable local plasticity on the usually rigid RNA double helix. The results provide a conformational basis for the preferential, metal-assisted self-cleavage of RNA at bulged sites.

Crystallography, X-Ray↗

PubMed2002

Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction.

The bacterial Hfq protein modulates the stability or the translation of mRNAs and has recently been shown to interact with small regulatory RNAs in E. coli. Here we show that Hfq belongs to the large family of Sm and Sm-like proteins: it contains a conserved sequence motif, known as the Sm1 motif, forms a doughnut-shaped structure, and has RNA binding specificity very similar to the Sm proteins. Moreover, we provide evidence that Hfq strongly cooperates in intermolecular base pairing between the antisense regulator Spot 42 RNA and its target RNA. We speculate that Sm proteins in general cooperate in bimolecular RNA-RNA interaction and that protein-mediated complex formation permits small RNAs to interact with a broad range of target RNAs.

Amino Acid Sequence↗

PubMed2002

The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs.

The Escherichia coli host factor I, Hfq, binds to many small regulatory RNAs and is required for OxyS RNA repression of fhlA and rpoS mRNA translation. Here we report that Hfq is a bacterial homolog of the Sm and Sm-like proteins integral to RNA processing and mRNA degradation complexes in eukaryotic cells. Hfq exhibits the hallmark features of Sm and Sm-like proteins: the Sm1 sequence motif, a multisubunit ring structure (in this case a homomeric hexamer), and preferential binding to polyU. We also show that Hfq increases the OxyS RNA interaction with its target messages and propose that the enhancement of RNA-RNA pairing may be a general function of Hfq, Sm, and Sm-like proteins.

Amino Acid Sequence↗

PubMed2002

Composition and functional characterization of the yeast spliceosomal penta-snRNP.

Pre-mRNA introns are spliced in a macromolecular machine, the spliceosome. For each round of splicing, the spliceosome assembles de novo in a series of ATP-dependent steps involving numerous changes in RNA-RNA and RNA-protein interactions. As currently understood, spliceosome assembly proceeds by addition of discrete U1, U2, and U4/U6*U5 snRNPs to a pre-mRNA substrate to form functional splicing complexes. We characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors. The particle is functional in extracts and, when supplied with soluble factors, is capable of splicing pre-mRNA. We propose that the spliceosomal snRNPs associate prior to binding of a pre-mRNA substrate rather than with pre-mRNA via stepwise addition of discrete snRNPs.

Protein Biosynthesis↗

PubMed2000

Splicing enhancement in the yeast rp51b intron.

Splicing enhancement in higher eukaryotes has been linked to SR proteins, to U1 snRNP, and to communication between splice sites across introns or exons mediated by protein-protein interactions. It has been previously shown that, in yeast, communication mediated by RNA-RNA interactions between the two ends of introns is a basis for splicing enhancement. We designed experiments of randomization-selection to isolate splicing enhancers that would work independently from RNA secondary structures. Surprisingly, one of the two families of sequences selected was essentially composed of 5' splice site variants. We show that this sequence enhances splicing independently of secondary structure, is exportable to heterologous contexts, and works in multiple copies with additive effects. The data argue in favor of an early role for splicing enhancement, possibly coincident with commitment complex formation. Genetic compensation experiments with U1 snRNA mutants suggest that U1 snRNP binding to noncanonical locations is required for splicing enhancement.

5' Untranslated Regions↗

PubMed2001

Yeast U1 snRNP-pre-mRNA complex formation without U1snRNA-pre-mRNA base pairing.

Base pairing between the 5' end of U1 snRNA and the conserved 5' splice site of pre-mRNA is important for commitment complex formation in vitro. However, the biochemical mechanisms by which pre-mRNA is initially recognized by the splicing machinery is not well understood. To evaluate the role of this base pairing interaction, we truncated U1 snRNA to eliminate the RNA-RNA interaction and surprisingly found that U1 snRNP can still form a nearly normal RNA-protein complex and maintain sequence specificity. We propose that some feature of U1 snRNP, perhaps one or more protein factors, is more important than the base pairing for initial 5' splice site recognition. In addition, at least five sets of interactions contribute to complex formation or stability. Only one of these is base pairing between the 5' splice site and the 5' end of U1 snRNA, without which the U1 snRNP-pre-mRNA complex is less stable and has a somewhat altered conformation.

5' Untranslated Regions↗

PubMed2001

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture.

The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 2'OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (psi) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of psi at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of psi also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex deltaG degrees 37. These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.

Hot Temperature↗

PubMed2001

Geometric nomenclature and classification of RNA base pairs.

Non-Watson-Crick base pairs mediate specific interactions responsible for RNA-RNA self-assembly and RNA-protein recognition. An unambiguous and descriptive nomenclature with well-defined and nonoverlapping parameters is needed to communicate concisely structural information about RNA base pairs. The definitions should reflect underlying molecular structures and interactions and, thus, facilitate automated annotation, classification, and comparison of new RNA structures. We propose a classification based on the observation that the planar edge-to-edge, hydrogen-bonding interactions between RNA bases involve one of three distinct edges: the Watson-Crick edge, the Hoogsteen edge, and the Sugar edge (which includes the 2'-OH and which has also been referred to as the Shallow-groove edge). Bases can interact in either of two orientations with respect to the glycosidic bonds, cis or trans relative to the hydrogen bonds. This gives rise to 12 basic geometric types with at least two H bonds connecting the bases. For each geometric type, the relative orientations of the strands can be easily deduced. High-resolution examples of 11 of the 12 geometries are presently available. Bifurcated pairs, in which a single exocyclic carbonyl or amino group of one base directly contacts the edge of a second base, and water-inserted pairs, in which single functional groups on each base interact directly, are intermediate between two of the standard geometries. The nomenclature facilitates the recognition of isosteric relationships among base pairs within each geometry, and thus facilitates the recognition of recurrent three-dimensional motifs from comparison of homologous sequences. Graphical conventions are proposed for displaying non-Watson-Crick interactions on a secondary structure diagram. The utility of the classification in homology modeling of RNA tertiary motifs is illustrated.

Base Pairing↗

PubMed2002

Frequent occurrence of the T-loop RNA folding motif in ribosomal RNAs.

Analysis of atomic resolution structures of the rRNAs within the context of the 50S and the 30S ribosomal subunits have revealed the presence of nine examples of a recurrent structural motif, first observed in the TpsiC loop of tRNAs. The key component of this T-loop motif is a UA trans Watson-Crick/Hoogsteen base pair stacked on a Watson-Crick pair on one side. This motif is stabilized by several noncanonical hydrogen bonds, facilitating RNA-RNA as well as RNA-protein interactions. In particular, the sugar edge of the purine on the 3' side of the pivotal uridine in the UA pair frequently forms a noncanonical base pair with a distant residue. The bulged-out bases, usually seen as part of the motif, also use their Watson-Crick edges to interact with nearby residues via base-specific hydrogen bonds. In certain occurrences, a backbone reversal is stabilized by specific hydrogen bonds as is observed in the U-turn motifs and the adenosine residue of the key UA pair interacts with a third base via its Watson-Crick edge, essentially generating a base triple.

Base Pairing↗