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At least 181 records · Page 10Linked to original sources
PubMed1997

RNA-RNA interaction is required for the formation of specific bicoid mRNA 3' UTR-STAUFEN ribonucleoprotein particles.

The formation of the anterior pattern of the Drosophila embryo is dependent on the localization of the mRNA of the morphogen Bicoid (bcd) to the anterior pole of the egg cell. Staufen protein (STAU) is required in a late step of the localization to anchor the bcd mRNA in the anterior cytoplasm. We have shown previously that endogenous STAU associates specifically with injected bcd mRNA 3'-untranslated region (UTR), resulting in the formation of characteristic RNA-protein particles that are transported along microtubules of the mitotic spindles in a directed manner. The regions recognized by STAU in this in vivo assay are predicted to form three stem-loop structures involving large double-stranded stretches. Here, we show that the STAU interaction requires a double-stranded conformation of the stems within the RNA localization signal. In addition, base pairing between two single-stranded loops plays a major role in particle formation. This loop-loop interaction is intermolecular, not intramolecular; thus dimers or multimers of the RNA localization signal must be associated with STAU in these particles. The bcd mRNA 3' UTR can also dimerize in vitro in the absence of STAU. Thus, in addition to RNA-protein interactions, RNA-RNA interaction might be involved in the formation of ribonucleoprotein particles for transport and localization.

Animals↗
PubMed1998

Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site.

Replication of a low-copy-number IncIalpha plasmid ColIb-P9 depends on expression of the repZ gene encoding the replication initiator protein. repZ expression is negatively controlled by the small antisense Inc RNA, and requires formation of a pseudoknot in the RepZ mRNA consisting of stem-loop I, the Inc RNA target, and a downstream sequence complementary to the loop I. The loop I sequence comprises 5'-rUUGGCG-3', conserved in many prokaryotic antisense systems, and was proposed to be the important site of copy number control. Here we show that the level of repZ expression is rate-limiting for replication and thus copy number, by comparing the levels of repZ expression and copy number from different mutant ColIb-P9 derivatives defective in Inc RNA and pseudoknot formation. Kinetic analyses using in vitro transcribed RNAs indicate that Inc RNA binding and the pseudoknot formation are competitive at the level of initial base paring to loop I. This initial interaction is stimulated by the presence of the loop U residue in the 5'-rUUGGCG-3' motif. These results indicate that the competition between the two RNA-RNA interactions at the specific site is a novel regulatory mechanism for establishing the constant level of repZ expression and thus copy number.

Bacterial Proteins↗
PubMed1999

tRNA prefers to kiss.

Six RNA aptamers that bind to yeast phenylalanine tRNA were identified by in vitro selection from a random-sequence pool. The two most abundantly represented aptamers interact with the tRNA anticodon loop, each through a sequence block with perfect Watson-Crick complementarity to the loop. It was possible to truncate one of these aptamers to a simple hairpin loop that forms a classical 'kissing complex' with the anticodon loop. Three other aptamers have nearly complete complementarity to the anticodon loop. The sixth aptamer has two sequence blocks, one complementary to the tRNA T loop and the other to the D loop; this aptamer binds better to a mutant tRNA that disrupts the normal D-loop/T-loop tertiary interaction than to the wild-type tRNA. Selection of complements to tRNA loops occurred despite an attempt to direct binding to tertiary structural features of tRNA. This serves as a reminder of how special the RNA-RNA interactions are that are not based on complementarity. Nonetheless, these aptamers must present the tRNA complement in some special structural context; the simple single-strand complement of the anticodon loop did not bind tRNA effectively.

Anticodon↗
PubMed2000

Progression of a loop-loop complex to a four-way junction is crucial for the activity of a regulatory antisense RNA.

The antisense RNA, CopA, regulates the replication frequency of plasmid R1 through inhibition of RepA translation by rapid and specific binding to its target RNA (CopT). The stable CopA-CopT complex is characterized by a four-way junction structure and a side-by-side alignment of two long intramolecular helices. The significance of this structure for binding in vitro and control in vivo was tested by mutations in both CopA and CopT. High rates of stable complex formation in vitro and efficient inhibition in vivo required initial loop-loop complexes to be rapidly converted to extended interactions. These interactions involve asymmetric helix progression and melting of the upper stems of both RNAs to promote the formation of two intermolecular helices. Data presented here delineate the boundaries of these helices and emphasize the need for unimpeded helix propagation. This process is directional, i.e. one of the two intermolecular helices (B) must form first to allow formation of the other (B'). A binding pathway, characterized by a hierarchy of intermediates leading to an irreversible and inhibitory RNA-RNA complex, is proposed.

Bacterial Proteins↗
PubMed2000

Yra1p, a conserved nuclear RNA-binding protein, interacts directly with Mex67p and is required for mRNA export.

Mex67p and Mtr2p constitute an essential mRNA export complex that interacts with poly(A)+ RNA and nuclear pore proteins. We have identified Yra1p, an intranuclear protein with in vitro RNA-RNA annealing activity, which directly binds to Mex67p. The complex between Yra1p and Mex67p was reconstituted in vitro and shown by UV-crosslinking to bind directly to RNA. Mutants of YRA1 are impaired in nuclear poly(A)+ RNA export at restrictive growth conditions. ALY, the mouse homologue of Yra1p and a transcriptional coactivator, can bind in vitro to yeast and human Mex67p and partly complements the otherwise non-viable yra1 null mutant. Thus, Yra1p is the first RNA-binding protein characterized, which bridges the shuttling Mex67p/Mtr2p exporter to intranuclear mRNA transport cargoes.

Amino Acid Sequence↗
PubMed2003

A replication silencer element in a plus-strand RNA virus.

Replication represents a key step in the infectious cycles of RNA viruses. Here we describe a regulatory RNA element, termed replication silencer, that can down-regulate complementary RNA synthesis of a positive-strand RNA virus via an RNA-RNA interaction. This interaction occurs between the 5-nucleotide-long, internally positioned replication silencer and the extreme 3'-terminus of the viral RNA comprising part of the minimal minus-strand initiation promoter. Analysis of RNA synthesis in vitro, using model defective interfering (DI) RNA templates of tomato bushy stunt virus and a partially purified, RNA-dependent RNA polymerase preparation from tombusvirus-infected plants, revealed that this interaction inhibits minus-strand synthesis 7-fold. This functional interaction was supported further by: (i) RNA structure probing; (ii) phylogenetic analysis; (iii) inhibition of activity by short complementary DNAs; and (iv) compensatory mutational analysis. The silencer was found to be essential for accumulation of DI RNAs in protoplasts, indicating that it serves an important regulatory role(s) in vivo. Because similar silencer-promoter interactions are also predicted in other virus genera, this type of RNA-based regulatory mechanism may represent a widely utilized strategy for modulating replication.

Base Sequence↗
PubMed1982

A ribonucleoprotein fragment of the 30 S ribosome of E. coli: evidence for long range RNA-RNA interactions.

A stable homogeneous ribonucleoprotein fragment of the 30 S ribosomal subunit of E. coli has been prepared by mild nuclease digestion and heating in a constant ionic environment. The fragment contains about half of the 16 S ribosomal RNa and six proteins: S4, S7, S9, S13, S16 and S19. The RNA moiety contains the reported binding sites of all six proteins. After deproteinization, 80% of the RNA migrated as two major electrophoretic bands, which were isolated and sequenced. Each band contained sequences from the 5' and 3' thirds of the 16 S RNA but none from the central third. That these two noncontiguous RNA domains migrated together electrophoretically in Mg++-containing gels after deproteinization constitutes direct evidence that the 16 S RNA is folded in the intact ribosome so as to bring the two domains close together and that there are RNA-RNA interactions between them in the presence of Mg++.

Base Sequence↗
PubMed1984

Xenopus laevis 28S ribosomal RNA: a secondary structure model and its evolutionary and functional implications.

Based upon the three experimentally derived models of E. coli 23S rRNA (1-3) and the partial model for yeast 26S rRNA (4), which was deduced by homology to E. coli, we derived a secondary structure model for Xenopus laevis 28S rRNA. This is the first complete model presented for eukaryotic 28S rRNA. Compensatory base changes support the general validity of our model and offer help to resolve which of the three E. coli models is correct in regions where they are different from one another. Eukaryotic rDNA is longer than prokaryotic rDNA by virtue of introns, expansion segments and transcribed spacers, all of which are discussed relative to our secondary structure model. Comments are made on the evolutionary origins of these three categories and the processing fates of their transcripts. Functionally important sites on our 28S rRNA secondary structure model are suggested by analogy for ribosomal protein binding, the GTPase center, the peptidyl transferase center, and for rRNA interaction with tRNA and 5S RNA. We discuss how RNA-RNA interactions may play a vital role in translocation.

Animals↗
PubMed1984

U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle.

U4 and U6 RNAs of mammalian cells possess extensive intermolecular sequence complementarity and hence have the potential to base pair. A U4/U6 RNA complex, detectable in nondenaturing polyacrylamide gels, is released when human small nuclear ribonucleoproteins (snRNPs) containing U1, U2, U4, U5, and U6 RNAs are dissociated with proteinase K in the presence of sodium dodecyl sulfate. The released RNA/RNA complex dissociates with increasing temperature, consistent with the existence of specific base-pairing between the two RNAs. Since U6 RNA is selectively released from intact snRNPs under the same conditions required to dissociate the U4/U6 RNA complex, the RNA-RNA interaction may be sufficient to maintain U4 and U6 RNAs in the same snRNP particle. The biological implications of these findings are discussed.

Base Composition↗
PubMed1985

Long range RNA-RNA interactions in the 30 S ribosomal subunit of E. coli.

We have attempted to identify long-range interactions in the tertiary structure of RNA in the E. coli 30 S ribosome. Native subunits were cleaved with ribonuclease and separated into nucleoprotein fragments which were deproteinized and fractionated into multi-oligonucleotide complexes under conditions intended to preserve RNA-RNA interactions. The final products were denatured by urea and heat and their constituent oligonucleotides resolved and sequenced. Many complexes contained complementary sequences known to be bound together in the RNA secondary structure, attesting to the validity of the technique. Other co-migrating oligonucleotides, not joined in the secondary structure, contained mutually complementary sequences in locations that allow base-pairing interaction without disrupting pre-existing secondary structure. In seven instances the complementary relationship was found to have been preserved during phylogenetic diversification.

Base Sequence↗
PubMed1987

Human glutamate tRNA forms stable hybrids in vitro with 28S ribosomal RNA.

A human glutamate tRNA has been shown to form stable hybrids with 28S ribosomal RNA. This tRNA was purified from HeLa cell cytoplasmic RNA by RNA-RNA solution hybridization followed by the isolation of tRNA-28S rRNA complexes by hybridization-selection with ribosomal DNA or by recovery of the 28S peak from formamide-sucrose gradients. The single hybridizing tRNA species was identified as tRNAGluCUC by sequencing: pU-C-C-C-U-G-G-U-G-m2G-U-C-phi-A-G-U-G-G-D-phi-A-G-G-A-U-U- C-G-G-C-G-C-U-C-U-C-A-C-C-G-C-G-G-C-m5C-m5C-G-G-G-Tm-phi-C-G-A- U-U-C-C-C-G-G-U-C-A-G-G-G-A-A-C-C-AOH. Computer analysis located a nucleotide sequence near the middle of human 28S rRNA which is complementary to 15-26 nucleotides between residues 20 and 50 of this tRNA. An interaction between this tRNA and 28S rRNA suggests that tRNAGluCUC may have functions in the cell in addition to translation.

Base Sequence↗
PubMed1990

The structure of a pre-mRNA molecule in solution determined with a site directed cross-linking reagent.

We describe the use of site specific psoralen (SSP) to determine the solution structure of a segment of the human beta globin pre-mRNA. In these experiments, SSP is first delivered as monoadducts to specific nucleotides in the pre-mRNA and subsequently used to form intramolecular RNA-RNA cross-links. The use of this reagent greatly decreases the number of the cross-linked products as compared to generalized psoralen cross-linking. The experiments confirm the locations of previously determined aminomethyltrimethylpsoralen (AMT) cross-links in the human precursor mRNA. In addition, new cross-links consistent with an alternative secondary structure and a small number of cross-links that represent higher order interactions have been determined. Altogether, 42 of 47 cross-links identified in this analysis can be accounted for in a small number of alternative secondary structures and higher order interactions. The site directed cross-linking technique will be useful for the precise determination of RNA secondary and tertiary structures under a variety of experimental conditions.

Base Sequence↗
PubMed1991

A chemical interference study on the interaction of ribosomal protein L11 from Escherichia coli with RNA molecules containing its binding site from 23S rRNA.

The interaction between ribosomal protein L11 from Escherichia coli and in vitro synthesized RNA containing its binding site from 23S rRNA was characterized by identifying nucleotides that interfered with complex formation when chemically modified by diethylpyrocarbonate or hydrazine. Chemically modified RNA was incubated with L11 under conditions appropriate for specific binding of L11 and the resulting protein-RNA complex was separated from unbound RNA on Mg(2+)-containing polyacrylamide gels. The ability to isolate L11 complexes on such gels was affected by the extent of modification by either reagent. Protein-bound and free RNAs were recovered and treated with aniline to identify their content of modified bases. Exclusion of RNA containing chemically altered bases from L11-associated material occurred for 29 modified nucleotides, located throughout the region corresponding to residues 1055-1105 in 23S rRNA. Ten bases within this region did not reproducibly inhibit binding when modified. Multiple bands of RNA were consistently observed on the nondenaturing gels, suggesting that significant intermolecular RNA-RNA interactions had occurred.

Aniline Compounds↗
PubMed1991

A conserved sequence element in ribonuclease III processing signals is not required for accurate in vitro enzymatic cleavage.

Ribonuclease III of Escherichia coli is prominently involved in the endoribonucleolytic processing of cell and viral-encoded RNAs. Towards the goal of defining the RNA sequence and structural elements that establish specific catalytic cleavage of RNase III processing signals, this report demonstrates that a 60 nucleotide RNA (R1.1 RNA) containing the bacteriophage T7 R1.1 RNase III processing signal, can be generated by in vitro enzymatic transcription of a synthetic deoxyoligonucleotide and accurately cleaved in vitro by RNase III. Several R1.1 RNA sequence variants were prepared to contain point mutations in the internal loop which, on the basis of a hypothetical 'dsRNA mimicry' structural model of RNase III processing signals, would be predicted to inhibit cleavage by disrupting essential tertiary RNA-RNA interactions. These R1.1 sequence variants are accurately and efficiently cleaved in vitro by RNase III, indicating that the dsRNA mimicry structure, if it does exist, is not important for substrate reactivity. Also, we tested the functional importance of the strongly conserved CUU/GAA base-pair sequence by constructing R1.1 sequence variants containing base-pair changes within this element. These R1.1 variants are accurately cleaved at rates comparable to wild-type R1.1 RNA, indicating the nonessentiality of this conserved sequence element in establishing in vitro processing reactivity and selectivity.

Base Sequence↗
PubMed1975

Evidence for tertiary structural RNA-RNA interactions within the protein S4 binding site at the 5'-end of 16S ribosomal RNA of Escherichia coli.+.

Evidence is presented for tertiary structural interaction(s) (interactions(s) between two regions of an RNA molecule that are widely separated in the RNA sequence) within the 5'-one third of the 16S ribosomal RNA of Escherichia coli that constitutes the binding site of protein S4. The two main interacting RNA regions were separated by about 120 nucleotides (sections Q to M) of the 16S RNA sequence. A second, smaller gap, of 13 nucleotides, occurred within section C". The two main interacting regions contain about 150 nucleotides (sections H" to Q) and 160 nucleotides (sections M to C"). They are folded back on one another and, especially in the presence of protein S4, are strongly protected against ribonuclease digestion. The intermediate region (sections Q to M), however, is relatively accessible to ribonucleases in the S4-RNP. By partial removal of subfragments from the RNA complex it was possible to localise the two main interacting sites within sections H" - H and sections I" - C". Three main criteria for the specificity of the RNA-RNA interactions were invoked and satisfied. The possibility of other tertiary structural RNA-RNA interactions occurring in other regions of the 16S RNA is discussed. Finally, all the structural information on the S4-RNP is summarised and a tentative model is proposed.

Base Sequence↗
PubMed1993

Secondary structure of the HIV-2 leader RNA comprising the tRNA-primer binding site.

The initiation of reverse transcription of a retroviral RNA genome occurs by a tRNA primer bound near the 5' end of the genomic RNA at a position called the primer-binding site (PBS). To understand the molecular basis for this RNA-RNA interaction, the secondary structure of the leader RNA of the human immunodeficiency virus type 2 (HIV-2) RNA was analyzed. In vitro synthesized HIV-2 RNA was probed with various structure-specific enzymes and chemicals. A computer program was then used to predict the secondary structure consistent with these data. In addition, the nucleotide sequences of different HIV-2 isolates were used to screen for the occurrence of covariation among putative base pairs. The primary sequences have diverged rapidly in some HIV-2 isolates, however, some strikingly conserved secondary structure elements were identified. Most nucleotides in the leader region are involved in base pairing. An exception is the PBS sequence, of which 15 out of 18 nucleotides are exposed in an internal loop. These findings suggest that the overall structure of the HIV-2 genome has evolved to facilitate an optimal interaction with its tRNA primer.

Base Sequence↗
PubMed1993

Translational control by a long range RNA-RNA interaction; a basepair substitution analysis.

One of the two mechanisms that regulate expression of the replicase cistron in the single stranded RNA coliphages is translational coupling. This mechanism prevents ribosomes from binding at the start of the replicase cistron unless the upstream coat cistron is being translated. Genetic analysis had identified a maximal region of 132 nucleotides in the coat gene over which ribosomes should pass to activate the replicase start. Subsequent deletion studies in our laboratory had further narrowed down the regulatory region to 12 nucleotides. Here, we identify a long-distance RNA-RNA interaction of 6 base pairs as the basis of the translational polarity. The 3' side of the complementarity region is located in the coat-replicase intercistronic region, some 20 nucleotides before the start codon of the replicase. The 5' side encodes amino acids 31 and 32 of the coat protein. Mutations that disrupt the long-range interaction abolish the translational coupling. Repair of basepairing by second site base substitutions restores translational coupling.

Base Composition↗
PubMed1994

Interaction between the first and last nucleotides of pre-mRNA introns is a determinant of 3' splice site selection in S. cerevisiae.

The splicing of group II and nuclear pre-mRNAs introns occurs via a similar splicing pathway and some of the RNA-RNA interactions involved in these splicing reactions show structural similarities. Recently, genetic analyses performed in a group II intron and the yeast nuclear actin gene suggested that non Watson-Crick interactions between intron boundaries are important for the second splicing step efficiency in both classes of introns. We here show that, in the yeast nuclear rp51A intron, a G to A mutation at the first position activates cryptic 3' splice sites with the sequences UAC/ or UAA/. Moreover, the natural 3' splice site could be reactivated by a G to C substitution of the last intron nucleotide. These results demonstrate that the interaction between the first and last intron nucleotides is a conserved feature of nuclear pre-mRNA splicing in yeast and is involved in the mechanism of 3' splice site selection.

Base Sequence↗