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Results for “RNA-RNA interactions”

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PubMed1998

The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro.

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA-RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5-200kD and U5-100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5-200kD protein but lack U5-100kD, suggesting that the U5-200kD protein could mediate U4/U6 duplex unwinding. Finally, U5-200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5-200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Adenosine Triphosphate↗

PubMed1999

Site-directed hydroxyl radical probing of 30S ribosomal subunits by using Fe(II) tethered to an interruption in the 16S rRNA chain.

Two in vitro transcripts, one corresponding to the 5' and central domains (residues 1-920) of 16S rRNA and the other corresponding to its 3' domain (residues 922-1542), assemble efficiently in trans with 30S ribosomal proteins to form a compact ribonucleoprotein particle that cosediments with natural 30S subunits. Isolated particles are similar in appearance to natural 30S subunits with electron microscopy and contain a full complement of the small subunit ribosomal proteins. The particles have a reduced ability to bind tRNA (attributable to the location of the discontinuity in a conserved region of the rRNA) near features that have been implicated in tRNA binding. Association of these two halves of 16S rRNA in trans must be stabilized by either previously unidentified RNA-RNA contacts or interactions mediated by ribosomal proteins because there are no known direct interactions between them. The trans construct was used to probe the three-dimensional RNA neighborhood around position 922 of 16S rRNA by generating hydroxyl radicals from Fe(II) tethered to the 5' end of the 3' transcript. Hydroxyl radical-induced cuts in the 16S rRNA chain were localized by primer extension to nucleotides 923-929 and 1192-1198, providing evidence for the mutual proximity of the 920 and 1192 regions.

Centrifugation, Density Gradient↗

PubMed1997

The position dependence of translational regulation via RNA-RNA and RNA-protein interactions in the 5'-untranslated region of eukaryotic mRNA is a function of the thermodynamic competence of 40 S ribosomes in translational initiation.

Cap proximity is a requirement to enable secondary structures and RNA-binding proteins to repress translational initiation via the 5'-untranslated region (5'-UTR) of mammalian mRNAs. We show that in Saccharomyces cerevisiae, unlike mammalian cells, the in vitro translational repressive effect of the mammalian iron regulatory protein 1 (IRP1) is independent of the site of its target in the 5'-UTR, the iron-responsive element (IRE). In vitro studies demonstrate that the binding affinity of IRP1 is also unaffected by the position of the IRE. Using IRE loop mutants, we observe an almost complete loss of IRP1-dependent repression in yeast concomitant with a 150-fold reduction in binding affinity for the IRE target. This mirrors the natural quantitative range of iron-induced adjustment of IRE/IRP1 affinity in mammalian cells. By enhancing the stability of the IRE stem-loop, we also show that its intrinsic folding energy acts together with the binding energy of IRP1 to give an additive capacity to restrict translational initiation. An IRE.IRP1 complex in a cap-distal position in yeast blocks scanning 40 S ribosomes on the 5'-UTR. It follows that the position effect of mammalian site-specific translational repression is dictated by the competence of the mammalian preinitiation complex to destabilize inhibitory structures at different steps of the initiation process.

Base Sequence↗

PubMed2001

The kissing hairpin sequence promotes recombination within the HIV-I 5' leader region.

The role of RNA-RNA template interactions in facilitating recombination during reverse transcription of minus strand DNA has been examined. The tested hypothesis is that template switching by reverse transcriptase is promoted at sites where homologous regions of two RNAs are brought in close proximity via stable intertemplate interactions. Frequency and distribution of template switching between homologous donor and acceptor RNAs were examined within the human immunodeficiency virus type I (HIV-I) 5'-untranslated region (UTR) containing the dimer initiation sequence (DIS). Results were compared with control nondimerizing templates from the pol region. The dimerizing UTR templates displayed a 4-fold higher transfer efficiency than the control. A striking 53% of transfers in the UTR mapped near the DIS, of which two-thirds occurred immediately 5' to this sequence. In the UTR template, deletion of the DIS hairpin disrupted template dimerization and caused a 4-fold drop in transfer efficiency. Insertion of the DIS within the pol template increased both dimerization and transfer efficiency. Transfer distributions revealed that in both sets of templates, DIS-induced dimerization increased the efficiency of transfers across the whole template, with the transfers peaking around the dimerization site. Overall, these results suggest that template dimerization facilitated by the unique geometry of the DIS-promoted kissing interactions effectively promotes recombination within the HIV-I 5'-UTR.

5' Untranslated Regions↗

PubMed2001

Regulatory activity of distal and core RNA elements in Tombusvirus subgenomic mRNA2 transcription.

Positive-strand RNA viruses that encode multiple cistrons often mediate expression of 3'-encoded open reading frames via RNA-templated transcription of subgenomic (sg) mRNAs. Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus that transcribes two such sg mRNAs during infections. We have previously identified a distal element (DE), located approximately 1100 nucleotides upstream from the initiation site of sg mRNA2 transcription, part of which must base pair with a portion of a core element (CE), located just 5' to the initiation site, for efficient transcription to occur (Zhang, G., Slowinski, V., and White, K. A. (1999) RNA 5, 550-561). Here we have analyzed further this long distance RNA-RNA interaction and have investigated the regulatory roles of other subelements within the DE and CE. Our results indicate that (i) the functional base-pairing interaction between these elements occurs in the positive strand and that the interaction likely acts to properly position other subelements, (ii) two previously undefined subelements within the DE and CE are important and essential, respectively, for efficient sg mRNA2 accumulation, and (iii) the production of (-)-strand sg mRNA2 can be uncoupled from the synthesis of its (+)-strand complement. These data provide important insight into the mechanism of sg mRNA2 transcription.

RNA, Messenger↗

PubMed2001

In vitro evidence for a long range pseudoknot in the 5'-untranslated and matrix coding regions of HIV-1 genomic RNA.

The 5'-untranslated leader region of human immunodeficiency virus type 1 (HIV-1) RNA contains multiple signals that control distinct steps of the viral replication cycle such as transcription, reverse transcription, genomic RNA dimerization, splicing, and packaging. It is likely that fine tuned coordinated regulation of these functions is achieved through specific RNA-protein and RNA-RNA interactions. In a search for cis-acting elements important for the tertiary structure of the 5'-untranslated region of HIV-1 genomic RNA, we identified, by ladder selection experiments, a short stretch of nucleotides directly downstream of the poly(A) signal that interacts with a nucleotide sequence located in the matrix region. Confirmation of the sequence of the interacting sites was obtained by partial or complete inhibition of this interaction by antisense oligonucleotides and by nucleotide substitutions. In the wild type RNA, this long range interaction was intramolecular, since no intermolecular RNA association was detected by gel electrophoresis with an RNA mutated in the dimerization initiation site and containing both sequences involved in the tertiary interaction. Moreover, the functional importance of this interaction is supported by its conservation in all HIV-1 isolates as well as in HIV-2 and simian immunodeficiency virus. Our results raise the possibility that this long range RNA-RNA interaction might be involved in the full-length genomic RNA selection during packaging, repression of the 5' polyadenylation signal, and/or splicing regulation.

5' Untranslated Regions↗

PubMed2002

Crystal structures of the Pyrococcus abyssi Sm core and its complex with RNA. Common features of RNA binding in archaea and eukarya.

The Sm proteins are conserved in all three domains of life and are always associated with U-rich RNA sequences. Their proposed function is to mediate RNA-RNA interactions. We present here the crystal structures of Pyrococcus abyssi Sm protein (PA-Sm1) and its complex with a uridine heptamer. The overall structure of the protein complex, a heptameric ring with a central cavity, is similar to that proposed for the eukaryotic Sm core complex and found for other archaeal Sm proteins. RNA molecules bind to the protein at two different sites. They interact specifically inside the ring with three highly conserved residues, defining the uridine-binding pocket. In addition, nucleotides also interact on the surface formed by the N-terminal alpha-helix as well as a conserved aromatic residue in beta-strand 2 of the PA-Sm1 protein. The mutation of this conserved aromatic residue shows the importance of this second site for the discrimination between RNA sequences. Given the high structural homology between archaeal and eukaryotic Sm proteins, the PA-Sm1.RNA complex provides a model for how the small nuclear RNA contacts the Sm proteins in the Sm core. In addition, it suggests how Sm proteins might exert their function as modulators of RNA-RNA interactions.

Amino Acid Sequence↗

PubMed2004

5'-3' RNA-RNA interaction facilitates cap- and poly(A) tail-independent translation of tomato bushy stunt virus mrna: a potential common mechanism for tombusviridae.

Tomato bushy stunt virus (TBSV) is the prototypical member of the genus Tombusvirus in the family Tombusviridae. The (+)-strand RNA genome of TBSV lacks both a 5' cap and a 3' poly(A) tail and instead contains a 3'-terminal RNA sequence that acts as a cap-independent translational enhancer (3' CITE). In this study, we have determined the RNA secondary structure of the translation-specific central segment of the 3' CITE, termed region 3.5 (R3.5). MFOLD structural modeling combined with solution structure mapping and comparative sequence analysis indicate that R3.5 adopts a branched structure that contains three major helices. Deletion and substitution studies revealed that two of these extended stem-loop (SL) structures are essential for 3' CITE activity in vivo. In particular, the terminal loop of one of these SLs, SL-B, was found to be critical for translation. Compensatory mutational analysis showed that SL-B functions by base pairing with another SL, SL3, in the 5' untranslated region of the TBSV genome. Thus, efficient translation of TBSV mRNA in vivo requires a 5'-3' RNA-RNA interaction that effectively circularizes the message. Similar types of interactions are also predicted to occur in TBSV subgenomic mRNAs between their 5' untranslated regions and the 3' CITE, and both genomic and subgenomic 5'-3' interactions are well conserved in all members of the genus Tombusvirus. In addition, a survey of other genera in Tombusviridae revealed the potential for similar 5'-3' RNA-RNA-based interactions in their viral mRNAs, suggesting that this mechanism extends throughout this large virus family.

Base Sequence↗

PubMed2005

YxiN is a modular protein combining a DEx(D/H) core and a specific RNA-binding domain.

DEx(D/H) proteins, typically described as RNA helicases, participate in rearrangement of RNA-RNA and possibly RNA-protein complexes in the cell. Aside from the conserved DEx(D/H) core, members of this protein family often contain N- and C-terminal extensions that are responsible for additional functions. The Bacillus subtilis DEx(D/H)-box protein YxiN and its Escherichia coli ortholog DbpA contain an approximately 80 amino acid C-terminal extension that has been proposed to specifically interact with a region of 23 S ribosomal RNA including hairpin 92. In this study, the DEx(D/H)-box core and the C-terminal domain of YxiN were expressed and characterized as separate proteins. The isolated DEx(D/H)-box core, YxCat, had weak, nonspecific RNA binding activity and showed RNA-stimulated ATPase activity with a Km(ATP) that resembled several non-specific DEx(D/H) proteins. The isolated C-terminal domain, YxRBD, bound RNA with the high affinity and specificity seen with full-length YxiN. Thus, YxiN is a modular protein combining the activities of the YxCat and YxRBD domains. Footprinting of YxiN and YxRBD on a 172-nucleotide fragment of 23 S rRNA was used to identify the sites of interaction of the C-terminal and helicase domains with the RNA.

Adenosine Triphosphatases↗

PubMed2006

Modifications in Thermus thermophilus 23 S ribosomal RNA are centered in regions of RNA-RNA contact.

Ribosomal RNA from all organisms contains post-transcriptionally modified nucleotides whose function is far from clear. To gain insight into the molecular interactions of modified nucleotides, we investigated the modification status of Thermus thermophilus 5 S and 23 S ribosomal RNA by mass spectrometry and chemical derivatization/primer extension. A total of eleven modified nucleotides was found in 23 S rRNA, of which eight were singly methylated nucleotides and three were pseudouridines. These modified nucleotides were mapped into the published three-dimensional ribosome structure. Seven of the modified nucleotides located to domain IV, and four modified nucleotides located to domain V of the 23 S rRNA. All posttranscriptionally modified nucleotides map in the center of the ribosome, and none of them are in contact with ribosomal proteins. All except one of the modified nucleotides were found in secondary structure elements of the 23 S ribosomal RNA that contact either 16 S ribosomal RNA or transfer RNA, with five of these nucleotides physically involved in intermolecular RNA-RNA bridges. These findings strongly suggest that the post-transcriptional modifications play a role in modulating intermolecular RNA-RNA contacts, which is the first suggestion on a specific function of endogenous ribosomal RNA modifications.

Binding Sites↗

PubMed2006

Down-regulation of porins by a small RNA bypasses the essentiality of the regulated intramembrane proteolysis protease RseP in Escherichia coli.

Adaptation to extracytoplasmic stress in Escherichia coli depends on the activation of sigmaE, normally sequestered by the membrane protein RseA. SigmaE is released in response to stress through the successive RseA cleavage by DegS and the RIP protease RseP. SigmaE and proteases that free it from RseA are essential. We isolated a multicopy suppressor that alleviated RseP and DegS requirement. The suppressor encodes a novel small RNA, RseX. Its activity required the RNA-binding protein Hfq. We used the property that small RNAs are often involved in RNA-RNA interactions to capture RseX putative partners; ompA and ompC mRNA, which encode two major outer membrane proteins, were identified. RseX activity was shown to confer an Hfq-dependent coordinate OmpA and OmpC down-regulation. Because RseP is shown to be no longer essential in a strain lacking OmpA and OmpC, we conclude that RseP, which is required for normal sigmaE activation, prevents toxicity due to the presence of two specific outer membrane proteins that are down-regulated by RseX.

Bacterial Outer Membrane Proteins↗

PubMed2006

Mutations in the intersubunit bridge regions of 23 S rRNA.

The large and small subunits of the ribosome are joined by a series of bridges that are conserved among mitochondrial, bacterial, and eukaryal ribosomes. In addition to joining the subunits together at the initiation of protein synthesis, a variety of other roles have been proposed for these bridges. These roles include transmission of signals between the functional centers of the two subunits, modulation of tRNA-ribosome and factor-ribosome interactions, and mediation of the relative movement of large and small ribosomal subunits during translocation. The majority of the bridges involve RNA-RNA interactions, and to gain insight into their function, we constructed mutations in the 23 S rRNA regions involved in forming 7 of the 12 intersubunit bridges in the Escherichia coli ribosome. The majority of the mutants were viable in strains expressing mutant rRNA exclusively but had distinct growth phenotypes, particularly at 30 degrees C, and the mutant ribosomes promoted a variety of miscoding errors. Analysis of subunit association activities both in vitro and in vivo indicated that, with the exception of the bridge B5 mutants, at least one mutation at each bridge site affected 70 S ribosome formation. These results confirm the structural data linking bridges with subunit-subunit interactions and, together with the effects on decoding fidelity, indicate that intersubunit bridges function at multiple stages of protein synthesis.

Base Sequence↗

PubMed2006

RNA-RNA interaction prediction and antisense RNA target search.

Recent studies demonstrating the existence of special noncoding "antisense" RNAs used in post transcriptional gene regulation have received considerable attention. These RNAs are synthesized naturally to control gene expression in C. elegans, Drosophila, and other organisms; they are known to regulate plasmid copy numbers in E. coli as well. Small RNAs have also been artificially constructed to knock out genes of interest in humans and other organisms for the purpose of finding out more about their functions. Although there are a number of algorithms for predicting the secondary structure of a single RNA molecule, no such algorithm exists for reliably predicting the joint secondary structure of two interacting RNA molecules or measuring the stability of such a joint structure. In this paper, we describe the RNA-RNA interaction prediction (RIP) problem between an antisense RNA and its target mRNA and develop efficient algorithms to solve it. Our algorithms minimize the joint free energy between the two RNA molecules under a number of energy models with growing complexity. Because the computational resources needed by our most accurate approach is prohibitive for long RNA molecules, we also describe how to speed up our techniques through a number of heuristic approaches while experimentally maintaining the original accuracy. Equipped with this fast approach, we apply our method to discover targets for any given antisense RNA in the associated genome sequence.

Adenosine Triphosphatases↗

PubMed1998

Oligonucleotide-mediated inhibition of genomic RNA dimerization of HIV-1 strains MAL and LAI: a comparative analysis.

An essential step in the replication cycle of retroviruses is the dimerization of two copies of the genomic RNA. In vitro and in vivo studies have demonstrated that dimerization is mediated at least partially by RNA-RNA interactions. In HIV-1, the cis-element most important for dimerization is the dimerization initiation site (DIS), a stem-loop structure with an autocomplementary loop located between the primer binding site and the splice donor site in the 5' leader region of genomic RNA. We have studied the inhibition of dimerization of RNA corresponding to the first 615 nt of HIV-1 strains MAL and LAI in vitro using RNA and DNA oligonucleotides. The oligonucleotides were identical to or complementary to the DIS of the MAL and LAI strains, which are representative of the two most common DIS motifs found in natural isolates. The loop sequence of the DIS of the MAL isolate is AGGUGCACA, and that of the LAI sequence is AAGCGCGCA (the autocomplementary sequences are GUGCAC and GCGCGC, respectively). Several of the oligonucleotides were very efficient inhibitors of dimerization. However, homologous oligonucleotides displayed vastly different inhibition efficiencies between the two strains despite relatively modest sequence differences. Some of the oligonucleotides bound the viral RNA via a loop-loop interaction alone, whereas others recruited stem nucleotides to form an extended duplex even in the absence of loop complementarity. Furthermore, oligonucleotide inhibition was ineffective at low temperature, suggesting that a conformational change in the DIS is necessary for disruption of the dimeric structure of the DIS or binding of oligonucleotide or both.

Base Sequence↗

PubMed2003

Silent DNA: speaking RNA language?

The sequence of silent DNA in the human genome (intergenic spacers, introns and synonymous codon positions of protein-coding genes) was found here to have the higher thermostability of corresponding RNA/RNA and RNA/DNA duplexes as compared with randomized sequence. This difference increased with elevation of GC content. The revealed effect was not due to correlation of RNA/RNA and RNA/DNA thermostabilities with thermostability of the DNA/DNA duplex, which, on the contrary, was lower than in the randomized sequence and lagged behind the elevation of GC content. The same picture was observed in the genomes of other warm-blooded vertebrates but not in the lower organisms. This finding suggests that RNA-RNA and RNA-DNA interactions could be involved in the putative function of silent DNA.

Animals↗

PubMed2006

Thermodynamics of RNA-RNA binding.

BACKGROUND: Reliable prediction of RNA-RNA binding energies is crucial, e.g. for the understanding on RNAi, microRNA-mRNA binding and antisense interactions. The thermodynamics of such RNA-RNA interactions can be understood as the sum of two energy contributions: (1) the energy necessary to 'open' the binding site and (2) the energy gained from hybridization. METHODS: We present an extension of the standard partition function approach to RNA secondary structures that computes the probabilities Pu[i, j] that a sequence interval [i, j] is unpaired. RESULTS: Comparison with experimental data shows that Pu[i, j] can be applied as a significant determinant of local target site accessibility for RNA interference (RNAi). Furthermore, these quantities can be used to rigorously determine binding free energies of short oligomers to large mRNA targets. The resource consumption is comparable with a single partition function computation for the large target molecule. We can show that RNAi efficiency correlates well with the binding energies of siRNAs to their respective mRNA target. AVAILABILITY: RNAup will be distributed as part of the Vienna RNA Package, www.tbi.univie.ac.at/~ivo/RNA/

Algorithms↗

PubMed2001

Non-coding RNAs: the architects of eukaryotic complexity.

Around 98% of all transcriptional output in humans is non-coding RNA. RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, imprinting, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signaling. I suggest that the central dogma is incomplete, and that intronic and other non-coding RNAs have evolved to comprise a second tier of gene expression in eukaryotes, which enables the integration and networking of complex suites of gene activity. Although proteins are the fundamental effectors of cellular function, the basis of eukaryotic complexity and phenotypic variation may lie primarily in a control architecture composed of a highly parallel system of trans-acting RNAs that relay state information required for the coordination and modulation of gene expression, via chromatin remodeling, RNA-DNA, RNA-RNA and RNA-protein interactions. This system has interesting and perhaps informative analogies with small world networks and dataflow computing.

Animals↗

PubMed1997

Rules for RNA recognition of GNRA tetraloops deduced by in vitro selection: comparison with in vivo evolution.

Terminal loops with a GNRA consensus sequence are a prominent feature of large self-assembling RNA molecules. In order to investigate tertiary interactions involving GNRA loops, we have devised an in vitro selection system derived from a group I ribozyme. Two selections, destined to isolate RNA sequences that would recognize two of the most widespread loops (GUGA and GAAA), yielded variants of previously identified receptors for those loops, and also some yet unrecognized, high-affinity binders with novel specificities towards members of the GNRA family. By taking advantage of available crystal structures, we have attempted to rationalize these results in terms of RNA-RNA contacts and to expose some of the structural principles that govern GNRA loop-mediated tertiary interactions; the role of loop nucleotide 2 in ensuring specific recognition by receptors is emphasized. More generally, comparison of the products of in vitro and natural selection is shown to provide insights into the mechanisms underlying the in vivo evolution of self-assembling RNA molecules.

Binding Sites↗