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Results for “RNA-RNA interactions”

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PubMed2004

Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs.

The bacterial Sm-like protein Hfq facilitates RNA-RNA interactions involved in post-transcriptional regulation of the stress response. Specifically, Hfq helps pair noncoding RNAs (ncRNAs) with complementary regions of target mRNAs. To probe the mechanism of this pairing, we generated a series of Hfq mutants and measured their affinity for RNAs like those with which Hfq must associate in vivo. We tested the mutants' DsrA-dependent activation of rpoS, and their ability to stabilize DsrA ncRNA against degradation in vivo. Our results suggest that Hfq has two independent RNA-binding surfaces. In addition to a well-known site around the core of the Hfq hexamer, we observe interactions with the distal face of Hfq, a new locus with which mRNAs and poly(A) sequences associate. Our model explains how Hfq can simultaneously bind a ncRNA and its mRNA target to facilitate the strand displacement reaction required for Hfq-dependent translational regulation.

Amino Acid Sequence↗

PubMed2026

hnRNPK condensates facilitate enhancer-promoter looping and RNA polymerase II recruitment.

Enhancer RNAs interact with promoter-derived RNAs to dictate enhancer-promoter looping, but the RNA-binding protein that mediates this process has remained unidentified. Here we identify hnRNPK as a general structural regulator that preferentially binds to nascent RNAs transcribed from enhancer and promoter regions, promoting enhancer-promoter looping and transcriptional activation. We further show that hnRNPK forms phase-separated, cavity-containing condensates that encapsulate RNA polymerase II (Pol II) via its RPB3 subunit, facilitating chromatin looping and potentially enabling recruitment of Pol II from enhancers to promoters through protein dimerization. Notably, a mutation associated with Au-Kline syndrome in hnRNPK (c.953+1dupG) alters its condensates from a liquid-like to a gel-like state, leading to developmental defects in knock-in mice. Fibroblasts derived from these mutants display reduced enhancer-promoter looping and decreased Pol II recruitment at promoters of key developmental genes. These findings suggest that hnRNPK is a structural regulator of enhancer-promoter communication and highlight the importance of RNA-RNA interactions mediated by RNA-binding proteins in transcriptional regulation.

RNA Polymerase II↗

PubMed2004

A complex network of RNA-RNA interactions controls subgenomic mRNA transcription in a tombusvirus.

Eukaryotic (+)-strand RNA viruses utilize a wide variety of gene expression strategies to achieve regulated production of their viral proteins. A common mechanism used by many is to transcribe viral subgenomic (sg) mRNAs. Transcription of sg mRNA2 in tombusviruses allows for expression of the p19 suppressor of gene silencing and p22 movement proteins. We have investigated the mechanism of transcription of this sg mRNA in Tomato bushy stunt virus and have determined that this process is facilitated by no less than three different RNA modules that are located throughout the viral genome. These RNA units perform distinct tasks and function via long-distance RNA-RNA interactions. Systematic deconstruction of the RNA network and analysis of related RNA promoter elements allowed us to identify fundamental properties necessary for productive sg mRNA2 transcription. Collectively, our results (i) establish specific roles for the different RNA components of a multipartite RNA-based control system, (ii) support a premature termination mechanism for tombusvirus sg mRNA transcription and (iii) reveal a close mechanistic relationship between sg mRNA transcription, viral RNA replication and RNA recombination.

Base Pairing↗

PubMed2006

Proximity of conserved U6 and U2 snRNA elements to the 5' splice site region in activated spliceosomes.

Major structural changes occur in the spliceosome during its catalytic activation, which immediately precedes the splicing of pre-mRNA. Whereas changes in snRNA conformation are well documented at the level of secondary RNA-RNA interactions, little is known about the tertiary structure of this RNA-RNA network, which comprises the spliceosome's catalytic core. Here, we have used the hydroxyl-radical probe Fe-BABE, tethered to the tenth nucleotide (U(+10)) of the 5' end of a pre-mRNA intron, to map RNA-RNA proximities in spliceosomes. These studies revealed that several conserved snRNA regions are close to U(+10) in activated spliceosomes, namely (i) the U6 snRNA ACAGAG-box region, (ii) portions of the U6 intramolecular stem-loop (U6-ISL) including a nucleotide implicated in the first catalytic step (U74), and (iii) the region of U2 that interacts with the branch point. These data constrain the relative orientation of these structural elements with respect to U(+10) in the activated spliceosome. Upon conversion of the activated spliceosome to complex C, the accessibility of U6-ISL to hydroxyl-radical cleavage is altered, suggesting rearrangements after the first catalytic step.

Base Sequence↗

PubMed2005

The 5-Me of thyminyl (T) interaction with the neighboring nucleobases dictate the relative stability of isosequential DNA-RNA hybrid duplexes.

Relative energetic contributions from the base-pairing [DeltaG(degrees)(bp)] vis-a-vis stacking [DeltaG(degrees)(stacking)] to the total free-energy of stabilization [DeltaG(degrees)(37)] for 14 pairs of isosequential hybrid DNA-RNA duplexes (taken from E. A. Lesnik and S. M. Freier, Biochemistry ,1995, 34, 10807) have been dissected in order to understand the differences in the intrinsic nature of the electrostatic forces that are responsible for the self-assembly of the heteroduplexes compared to homoduplexes. The pKa differences between the monomeric nucleotide 3'-ethylphosphates [(d/rN)pEt] as well as nucleotide 3',5'-bis-ethylphosphates [Etp(d/rN)pEt] in both 2'-deoxy (dN) and ribo (rN) series (N = A/G/C/T/U), as the model donor and acceptor (in which stacking is completely eliminated) mimicking those of the internucleotide monomer building blocks of a duplex, can be qualitatively used (P. Acharya, P. Cheruku, S. Chatterjee, S. Acharya and J. Chattopadhyaya, J. Am. Chem. Soc., 2004, 126, 2862) to understand the strength of base-pairing energies in different DNA-RNA (DR), RNA-DNA (RD), DNA-DNA (DD), and RNA-RNA (RR) duplexes. The study has led us to show the following. (1) As the number of excess %T in DR duplexes compared to the isosequential RD duplexes increase the differences in their thermal stabilization [DeltaT(m)](DR-RD) increase and vice-versa(2) The total relative stabilizations, [DeltaDeltaG(degrees)(37)](DR-RD) among the 14 pairs of isosequential DR and RD duplexes (E. A. Lesnik and S. M. Freier, Biochemistry ,1995, 34, 10807) are wholly dependent on the differences in the number of 5-Me(T) stacking interactions with the nearest-neighbors in the D strands of DR duplexes compared to that of the RD duplexes (3) In the relative stabilization of the DR or RD duplexes differences in the free-energy of stackings [DeltaDeltaG(degrees)(stacking)](DR-RD) play a more significant role than the differences in the free-energy of base-pairing, [DeltaDeltaG(degrees)(bp)](DR-RD). In contradistinction, our experimental data shows that RNA-RNA duplexes are more stable than DNA-DNA duplexes because of larger energy gain from the base-pairing in the former compared to the latter (P. Acharya, P. Cheruku, S. Chatterjee, S. Acharya and J. Chattopadhyaya, J. Am. Chem. Soc., 2004, 126, 2862).

Base Pairing↗

PubMed2005

Lsm proteins and RNA processing.

Sm and Lsm proteins are ubiquitous in eukaryotes and form complexes that interact with RNAs involved in almost every cellular process. My laboratory has studied the Lsm proteins in the yeast Saccharomyces cerevisiae, identifying in the nucleus and cytoplasm distinct complexes that affect pre-mRNA splicing and degradation, small nucleolar RNA, tRNA processing, rRNA processing and mRNA degradation. These activities suggest RNA chaperone-like roles for Lsm proteins, affecting RNA-RNA and/or RNA-protein interactions. This article reviews the properties of the Sm and Lsm proteins and structurally and functionally related proteins in archaea and eubacteria.

Animals↗

PubMed2000

Riboregulation by DsrA RNA: trans-actions for global economy.

DsrA is an 87 nucleotide Escherichia coli RNA with extraordinary regulatory properties. The profound impact of its actions stems from DsrA regulating translation of two global transcription regulators, H-NS and RpoS (sigmas), by sequence-specific RNA-RNA interactions. H-NS is a major nucleoid-structuring and global repressor protein, and RpoS is the stationary phase and stress response sigma factor of RNA polymerase. DsrA changes its conformation to bind to these two different mRNA targets and thereby inhibits H-NS translation, while stimulating that of RpoS in a mechanistically distinct fashion. DsrA apparently binds both the start and the stop codons of hns mRNA and sharply decreases the mRNA half-life. DsrA also binds sequences in the 5'-untranslated leader region of rpoS mRNA, enhancing rpoS mRNA stability and RpoS translation. A cohort of genes, governed by H-NS repression and RpoS activation, are thus regulated. Low temperatures increase the levels of DsrA, with differential effects on H-NS and RpoS. Additionally, the RNA chaperone protein Hfq is involved with DsrA regulation, as well as with other small RNAs that also act on RpoS to co-ordinate stress responses. We address the possible functions of this genetic regulatory mechanism, as well as the advantages of using small RNAs as global regulators to orchestrate gene expression.

Escherichia coli↗

PubMed2006

Base-tetrad swapping results in dimerization of RNA quadruplexes: implications for formation of the i-motif RNA octaplex.

Nucleic acids adopt different multistranded helical architectures to perform various biological functions. Here, we report a crystal structure of an RNA quadruplex containing "base-tetrad swapping" and bulged nucleotide at 2.1-Angstroms resolution. The base-tetrad swapping results in a dimer of quadruplexes with an intercalated octaplex fragment at the 5' end junction. The intercalated base tetrads provide the basic repeat unit for constructing a model of intercalated RNA octaplex. The model we obtained shows fundamentally different characteristics from duplex, triplex, and quadruplex. We also observed two different orientations of bulged uridine residues that are related to the interaction with surroundings. This structural evidence reflects the conformational flexibility of bulged nucleotides in RNA quadruplexes and implies the potential roles of bulged nucleotides as recognition and interaction sites in RNA-protein and RNA-RNA interactions.

Crystallography, X-Ray↗

PubMed2002

tRNA-mediated transcription antitermination in vitro: codon-anticodon pairing independent of the ribosome.

Uncharged tRNA acts as the effector for transcription antitermination of genes in the T box family in Bacillus subtilis and other Gram-positive bacteria. Genetic studies suggested that expression of these genes is induced by stabilization of an antiterminator element in the leader RNA of each target gene by the cognate uncharged tRNA. The specificity of the tRNA response is dependent on a single codon in the leader, which was postulated to pair with the anticodon of the corresponding tRNA. It was not known whether the leader RNA-tRNA interaction requires additional factors. We show here that tRNA-dependent antitermination occurs in vitro in a purified transcription system, in the absence of ribosomes or accessory factors, demonstrating that the RNA-RNA interaction is sufficient to control gene expression by antitermination. The tRNA response exhibits similar specificity in vivo and in vitro, and the antitermination reaction in vitro is independent of NusA and functions with either B. subtilis or Escherichia coli RNA polymerase.

Anticodon↗

PubMed2000

A trans-acting RNA as a control switch in Escherichia coli: DsrA modulates function by forming alternative structures.

DsrA is an 87-nucleotide regulatory RNA of Escherichia coli that acts in trans by RNA-RNA interactions with two different mRNAs, hns and rpoS. DsrA has opposite effects on these transcriptional regulators. H-NS levels decrease, whereas RpoS (final sigma(s)) levels increase. Here we show that DsrA enhances hns mRNA turnover yet stabilizes rpoS mRNA, either directly or via effects on translation. Computational and RNA footprinting approaches led to a refined structure for DsrA, and a model in which DsrA interacts with the hns mRNA start and stop codon regions to form a coaxial stack. Analogous bipartite interactions exist in eukaryotes, albeit with different regulatory consequences. In contrast, DsrA base pairs in discrete fashion with the rpoS RNA translational operator. Thus, different structural configurations for DsrA lead to opposite regulatory consequences for target RNAs.

Bacterial Proteins↗

PubMed2002

Specificity of RNA-RNA helix recognition.

Functional RNAs often form compact structures characterized by closely packed helices. Crystallographic analysis of several large RNAs revealed a prevalent interaction in which unpaired adenosine residues dock into the minor groove of a receptor helix. This A-minor motif, potentially the most important element responsible for global RNA architecture, has also been suggested to contribute to the fidelity of protein synthesis by discriminating against near-cognate tRNAs on the ribosome. The specificity of A-minor interactions is fundamental to RNA tertiary structure formation, as well as to their proposed role in translational accuracy. To investigate A-minor motif specificity, we analyzed mutations in an A-minor interaction within the Tetrahymena group I self-splicing intron. Thermodynamic and x-ray crystallographic results show that the A-minor interaction strongly prefers canonical base pairs over base mismatches in the receptor helix, enabling RNA interhelical packing through specific recognition of Watson-Crick minor groove geometry.

Base Pairing↗

PubMed1982

Effects of DNA base analogs on transcription termination at the tryptophan operon attenuator of EScherichia coli.

We have devised a method to specifically incorporate deoxyribonucleotide base analogs in vitro into either strand of the tryptophan (trp) operon attenuator region, using primed synthesis on bacteriophage M13 derivatives carrying cloned trp attenuator DNA. We have employed these techniques to extend previous studies implicating both RNA-RNA and RNA-DNA interactions in transcription termination in an attempt to determine the nature of the contribution from the template DNA molecule in termination regions. In general, we find that the dramatic effects upon transcription termination seen with base analog incorporation into mRNA do not occur when similar analogs are incorporated into the DNA. Only the analog 2,6-diaminopurine deoxyribonucleotide triphosphate (dDapTP), which strengthens A.T or A.U base pairing, elicits a significant response: in the template DNA strand, the presence of this analog increases read-through at the trp attenuator. The analog 5-bromouracil deoxyribonucleoside triphosphate (BrdUTP), which also strengthens pairing with its complementary base, has no detectable effect on termination when it is placed in either strand of the trp attenuator or the mutant attenuator trp a1419. Surprisingly, though the analog 5-iodocytosine deoxyribonucleoside triphosphate (IdCTP) does not affect termination, it has a great effect on initiation of transcription, depressing trp promoter activity as well as stimulating transcription from other regions. These results support the postulated interaction between terminal uridines in mRNA and the template DNA strand in enhancing termination and suggest that there are no significant additional contributions from the DNA. In addition, the novel use of M13 derivatives for incorporating analogs into the DNA on a preparative scale provides a technique for introducing mutations in a general but controlled fashion as a new means for studying other regulatory regions.

DNA, Bacterial↗

PubMed1983

Association of 16S and 23S ribosomal RNAs to form a bimolecular complex.

Association of the 30S and 50S subunits to generate the 70S ribosomes of Escherichia coli has long been known but the mechanism of this interaction remains obscure. Light-scattering studies indicate that naked 16S and 23S RNAs can also associate under conditions similar to those required for the assembly of ribosomes from the constituent RNAs and proteins. The RNA-RNA association also takes place in the presence of ethanol, which promotes folding of 16S and 23S RNAs into specific compact structures with the morphological features of 30S and 50S ribosomes, respectively. Equimolar amounts of the two RNAs are involved in the association. The formation of a stoichiometric complex was shown by light scattering, sucrose density gradient centrifugation, and composite polyacrylamide/agarose gel electrophoresis. The presence of the two species of RNA in the complex was also shown by gel electrophoresis. The association of naked 16S and 23S RNAs suggests that RNA-RNA interaction may play an important role in the association of 30S and 50S subunits.

Escherichia coli↗

PubMed1991

Reconstitution of a group I intron self-splicing reaction with an activator RNA.

The self-splicing rRNA intron of Tetrahymena thermophila belongs to a subgroup of group I introns that contain a conserved extra stem-loop structure termed P5abc. A Tetrahymena mutant precursor RNA lacking this P5abc is splicing-defective under standard conditions (5 mM MgCl2/200 mM NH4Cl, pH 7.5) in vitro. However, the mutant precursor RNA by itself is capable of performing the self-splicing reaction without P5abc under different conditions (15 mM MgCl2/2 mM spermidine, pH 7.5). We have investigated the functional role of the P5abc in the mechanism of the self-splicing reaction. When an RNA consisting of the P5abc but lacking the rest of the Tetrahymena intron is incubated with the mutant precursor, the self-splicing reaction proceeds highly efficiently under standard conditions (5 mM MgCl2/200 mM NH4Cl, pH 7.5). Two steps of the bimolecular self-splicing reaction can be performed accurately by a shortened precursor RNA containing all essential components required in the self-splicing reaction and an activator RNA consisting of the P5abc. Gel-mobility-shift assays suggest that two molecules associate by a direct RNA-RNA interaction during the splicing reaction. The results imply that there might exist other small RNAs whose role is to activate ribozymes.

Animals↗

PubMed1993

Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA.

Retroviruses package two homologous single-stranded RNA genomes within a gag protein-RNA complex. In mature virion particles, the two RNA strands are thought to associate primarily through direct RNA-RNA interactions, although the structural basis for this stable association is unknown. We now report that a 127-nucleotide (nt) HIV-1NL4-3 RNA fragment (positions 732-858) encompassing the 5' end of the gag gene dimerizes spontaneously under high ionic strength conditions in the absence of any protein cofactor. The HIV-1 RNA dimer is dramatically and specifically stabilized by the monovalent cation potassium. Thermal dissociation of the dimer occurs at 80 degrees C in 100 mM K+ (5 mM Mg2+) but at significantly lower temperatures in the presence of either smaller or larger monovalent cations (100 mM Li+, 40 degrees C; 100 mM Na+, 55 degrees C; 100 mM Cs+, 30 degrees C). Deletion analyses of the 3' end of the 127-nt fragment reveal that an HIV-1 RNA fragment as short as 94 nt (732-825) can dimerize spontaneously, but a further 9-base deletion of the purine-rich sequence, GGGGGAGAA from positions 817 through 825, eliminates dimerization. These experimental results support a model in which HIV-1 RNA dimerizes by forming an interstrand quadruple helix stabilized by guanine (and/or purine)-base tetrads in analogy to the well-known dimerization of telomeric DNA. We speculate that this structure may also mediate the association of genomic HIV-1 RNA in vivo, revealing how RNA itself can achieve the self-recognition required for subsequent genetic recombination.

Base Sequence↗

PubMed1995

In vitro trans-splicing in Saccharomyces cerevisiae.

The interactions established at the 5'-splice site during spliceosome assembly are likely to be important for both precise recognition of the upstream intron boundary and for positioning this site in the active center of the spliceosome. Definition of the RNA-RNA and the RNA-protein interactions at the 5' splice site would be facilitated by the use of a small substrate amenable to modification during chemical synthesis. We describe a trans-splicing reaction performed in Saccharomyces cerevisiae extracts in which the 5' splice site and the 3' splice site are on separate molecules. The RNA contributing the 5' splice site is only 20 nucleotides long and was synthesized chemically. The trans-splicing reaction is accurate and has the same sequence, ATP, and Mg2+ requirements as cis-splicing. We also report how deoxy substitutions around the 5'-splice site affect trans-splicing efficiency.

Actins↗

PubMed1995

Probing of tertiary interactions in RNA: 2'-hydroxyl-base contacts between the RNase P RNA and pre-tRNA.

A general method has been developed to analyze all 2' hydroxyl groups involved in tertiary interactions in RNA in a single experiment. This method involves comparing the activity of populations of circularly permuted RNAs that contain or lack potential hydrogen-bond donors at each position. The 2' hydroxyls of the pre-tRNA substrate identified as potential hydrogen bond donors in intermolecular interactions with the ribozyme from eubacterial RNase P (P RNA) are located in the T stem and T loop, acceptor stem, and 3' CCA regions. To locate the hydrogen-bond acceptors for one of those 2' hydroxyls in the P RNA, a phylogenetically conserved adenosine was mutated to a guanosine. When this mutant P RNA was used, increased cleavage activity of a single circularly permuted substrate within the population was observed. The cleavage efficiency (kcat/Km) of a singly 2'-deoxy-substituted substrate at this position in the T stem was also determined. For the wild-type P RNA, the catalytic efficiency was significantly decreased compared with that of the all-ribo substrate, consistent with the notion that this 2' hydroxyl plays an important role. For the P RNA mutant, no additional effect was found upon 2'-deoxy substitution. We propose that this particular 2' hydroxyl in the pre-tRNA interacts specifically with this adenosine in the P RNA. This method should be useful in examining the role of 2' hydroxyl groups in other RNA-RNA and RNA-protein complexes.

Adenosine↗

PubMed1996

A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

Base Composition↗