RNA-RNA Annealing Research Articles

The Significance of the DUF283 Domain for the Activity of Human Ribonuclease Dicer.

Dicers are multidomain proteins, usually comprising an amino-terminal putative helicase domain, a DUF283 domain (domain of unknown function), a PAZ domain, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Dicer homologs play an important role in the biogenesis of small regulatory RNAs by cleaving single-stranded precursors adopting stem-loop structures (pre-miRNAs) and double-strand RNAs into short RNA duplexes containing functional microRNAs or small interfering RNAs, respectively. Growing evidence shows that apart from the canonical role, Dicer proteins can serve a number of other functions. For example, results of our previous studies showed that human Dicer (hDicer), presumably through its DUF283 domain, can facilitate hybridization between two complementary RNAs, thus, acting as a nucleic acid annealer. Here, to test this assumption, we prepared a hDicer deletion variant lacking the amino acid residues 625-752 corresponding to the DUF283 domain. The respective 128-amino acid fragment of hDicer was earlier demonstrated to accelerate base-pairing between two complementary RNAs in vitro. We show that the ΔDUF(625-752) hDicer variant loses the potential to facilitate RNA-RNA base pairing, which strongly proves our hypothesis about the importance of the DUF283 domain for the RNA-RNA annealing activity of hDicer. Interestingly, the in vitro biochemical characterization of the obtained deletion variant reveals that it displays different RNA cleavage properties depending on the pre-miRNA substrate.

The Interaction between tRNALys3 and the Primer Activation Signal Deciphered by NMR Spectroscopy

The initiation of reverse transcription of the human immunodeficiency virus type 1 (HIV-1) requires the opening of the three-dimensional structure of the primer tRNALys 3 for its annealing to the viral RNA at the primer binding site (PBS). Despite the fact that the result of this rearrangement is thermodynamically more stable, there is a high-energy barrier that requires the chaperoning activity of the viral nucleocapsid protein. In addition to the nucleotide complementarity to the PBS, several regions of tRNALys 3 have been described as interacting with the viral genomic RNA. Among these sequences, a sequence of the viral genome called PAS for “primer activation signal” was proposed to interact with the T-arm of tRNALys 3, this interaction stimulating the initiation of reverse transcription. In this report, we investigate the formation of this additional interaction with NMR spectroscopy, using a simple system composed of the primer tRNALys 3, the 18 nucleotides of the PBS, the PAS (8 nucleotides) encompassed or not in a hairpin structure, and the nucleocapsid protein. Our NMR study provides molecular evidence of the existence of this interaction and highlights the role of the nucleocapsid protein in promoting this additional RNA-RNA annealing. This study presents the first direct observation at a single base-pair resolution of the PAS/anti-PAS association, which has been proposed to be involved in the chronological regulation of the reverse transcription.

Dynamic Competition for the Proximal RNA Binding Site of Hfq Drives Efficient Annealing of Small Noncoding RNA and mRNA

Hfq, a bacterial RNA chaperone, stimulates specific RNA-RNA interactions by inducing RNA unwinding or RNA-RNA annealing. However, it is still unclear how Hfq can selectively achieve two completely opposite functions--annealing, and unwinding--depending on RNA substrates. Also, basic properties like an oligomeric state of an active form is under debate. Here we describe single-molecule fluorescence studies on Hfq-mediated annealing of DsrA, a small noncoding regulatory RNA of E. coli, and its mRNA target rpoS, which encodes the σStranscription factor. Our results reveal that a single hexamer of Hfq stimulates DsrA-rpoS annealing by simultaneously binding both RNAs using same RNA binding site on the proximal surface of Hfq. The competition for the same binding site of the two RNAs makes the RNA-Hfq interaction dynamic, but drives more efficient annealing. When the Hfq-binding sequence exists only in one of the two RNAs, RNA unwinding can occur due to the reduced stability of the RNA caused by the partial unwinding of the RNA upon Hfq binding on the specific binding site.

The Activity of siRNA in Mammalian Cells is Related to the Kinetics of siRNA-target Recognition In Vitro: Mechanistic Implications

The specificity of siRNA-mediated suppression of gene expression involves base-base recognition between siRNA and its single-stranded RNA target. We investigated the kinetics of this process in vitro by using full-length ICAM-1 target RNA and biologically active and inactive ICAM-1-directed siRNA, respectively. To mimic the situation in living cells we used the well-characterised facilitator of RNA-RNA annealing and strand exchange cetyltrimethylammonium bromide, which increases strongly the kinetics of RNA-RNA interactions at conditions that do not affect RNA structure nor the presumed structure-function relationship. For the biologically active siRNA si2B, we find faster binding, i.e. recognition of the target and a slower backward reaction when compared with the biologically inactive siRNA si1. This is reflected by an approximately 400-fold more favorable equilibrium constant of si2B. Kinetic evidence favors an associative mechanism of recognition of the target strand by the double-stranded siRNA. The minimal model for siRNA-target recognition described here is consistent with the high biological activity of si2B only if one assumes a step subsequent to target recognition, which might be degradation of the target RNA when complexed with the antisense strand of siRNA or when considering rapid destruction of the released sense strand of siRNA.

The ubiquitous nature of RNA chaperone proteins

RNA chaperones are ubiquitous and abundant proteins found in all living organisms and viruses, where they interact with various classes of RNA. These highly diverse families of nucleic acid-binding proteins possess activities enabling rapid and faithful RNA-RNA annealing, strand transfer, and exchange and RNA ribozyme-mediated cleavage under physiological conditions. RNA chaperones appear to be critical to functions as important as maintenance of chromosome ends, DNA transcription, preRNA export, splicing and modifications, and mRNA translation and degradation. Here we review some of the properties of RNA chaperones in RNA-RNA interactions that take place during cellular processes and retrovirus replication. Examples of cellular and viral proteins are dicussed vis à vis the relationships between RNA chaperone activities in vitro and functions. In this new "genomic era" we discuss the possible use of small RNA chaperones to improve the synthesis of cDNA libraries for use in large screening reactions using DNA chips.

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.

Length dependence of RNA-RNA annealing

The association of complementary nucleic acids can be described by a second order rate constant k. For extended molecules, including complex nucleic acids, values of k were shown to be proportional to the square root of the chain length L of the shorter nucleic acid strand at temperatures between t m and t m−30 °C. For homopolymers this is true over a wider temperature range. Below temperatures of t m−30 °C, annealing rate constants may sharply decrease due to the formation of intramolecular structures. It seems to be reasonable to assume that the formation of intramolecular structures of nucleic acids reduces the density of nucleation sites for annealing and, thereby, lowers the rates of association. Here, we examined the relationship between RNA chain length and the kinetics of RNA-RNA annealing at physiological ionic strength and temperature. We used a complete sequence space derived from chloramphenicol acetyltransferase (cat) sequences to average over all structures for each given length. For groups of progressively longer antisense RNA species and a 800 nucleotides long complementary RNA, the observed annealing rate constants k obs were measured in vitro. The structure-averaged values for k obs of RNA-RNA annealing were not related to the square root of the chain length. Instead, they were found to be proportional to 10 αL (α=0.0017). Here, a theoretical model is suggested in which the observed length dependence is mainly influenced by ionic interactions between complementary RNA strands. The observed length dependence has substantial implications for the biological behavior of long-chain complementary RNA including the design of antisense RNA. The efficacy of antisense RNA in living cells is known to be related to annealing kinetics in vitro. Thus, on a statistical basis and independent of individual structures, long-chain rather than short-chain antisense RNA should lead to stronger inhibition.

The association of complementary ribonucleic acids can be strongly increased without lowering Arrhenius activation energies or significantly altering structures.

The association rates of complementary nucleic acids can be increased by 2-3 orders of magnitude in vitro by cellular proteins and low molecular weight compounds including cetyltrimethylammonium bromide (CTAB). In this work, we provide experimental evidence that the CTAB-mediated enhancement of RNA-RNA annealing by approximately 3 orders of magnitude is due to a favorable activation entropy (DeltaS) and not due to a decrease of the Arrhenius activation energy (Ea) nor to major structural changes of the RNA. Two alternative models for the CTAB-facilitated RNA-RNA annealing will be discussed. First, CTAB could form a positively charged liquid matrix which could steer complementary RNA molecules and thereby increase their collision frequency and annealing rate. Second, increased annealing rates could be explained by stabilization of a non-base-specific precomplex of both complementary RNA molecules in solution.

Theoretical and experimental selection parameters for HBV-directed antisense RNA are related to increased RNA-RNA annealing.

Annealing kinetics of antisense species against two different target regions of the hepatitis B virus (HBV) were measured by kinetic in vitro selection. Individual association rates were related to energies calculated for local sequence segments and predicted structures of the complete pregenomic target RNA. A relationship between the presence of external loops and joint sequences with fast pairing was observed whereas internal loops did not favor fast RNA-RNA annealing. The findings were used to predict a fast-annealing HBV-directed antisense oligodeoxyribonucleotide that turned out to pair with its target RNA at an association rate constant of k=9.2 x 10(4) M(-1) s(-1), which is substantially faster than the annealing rates of artificial antisense RNA so far included in in vitro selection assays.

Mechanistic insights into p53-promoted RNA-RNA annealing

The tumour suppressor protein p53 promotes the annealing of complementary nucleic acids in vitro. We observed an up to 1600-fold increase of RNA-RNA annealing by recombinant p53 protein which was shown to bind to RNA in a sequence-independent way. Nuclease mapping experiments suggest that p53 binds to intramolecular duplex portions and only marginally changes the overall secondary structure of RNA at conditions of increased annealing. Thus, the mechanism of p53-promoted RNA-RNA annealing does not seem to be dependent on an activity that melts or changes RNA structure. The activation enthalpy of RNA-RNA annealing is decreased in the presence of p53, i.e. the p53 protein could stabilize the transition state whereas the activation entropy is unfavourable. A comparison with thermodynamic data measured for other facilitators strongly suggests that the mechanism of p53-promoted RNA-RNA annealing is distinct from the mechanism by which other facilitators work. The annealing activity of p53 is almost abolished in the presence of magnesium indicating that it can be sharply regulated in vitro and, in principle, could also be regulated in vivo.

Transcriptional specificity of a multisubunit RNA polymerase induced by Bacillus subtilis bacteriophage PBS2

Bacillus subtilis phage PBS2 induced the synthesis of two temporally defined categories of phage-specified transcripts. The transcription of phage "early" genes was induced almost immediately after infection; this RNA synthesis did not require phage protein synthesis. Phage "late" gene transcription, on the other hand, was induced at an intermediate time in the lytic cycle; this RNA synthesis required the production of phage proteins. Both classes of transcription were resistant to the drug rifampin and, therefore, apparently did not require the rifampin-sensitive component of the host RNA polymerase. A rifampin-resistant, DNA-dependent RNA polymerase was purified from bacteria infected with PBS2. The highly purified phage polymerase consisted of five distinct subunits that remained associated during zone centrifugation, isoelectric focusing, and disc gel electrophoresis. The synthesis of each of the five polypeptides was induced at an intermediate time in the phage lytic cycle. As judged by hybridization competition, hybridization to DNA restriction fragments, and RNA-RNA annealing, the phage-induced RNA polymerase preferentially and asymmetrically transcribed PBS2 late genes in vitro. These findings suggest that the PBS2 RNA polymerase controls the expression of phage genes late in the lytic cycle.

Transcription of supercoiled mitochondrial DNA by bacterial RNA polymerase.

Mitochondrial DNA isolated from cysts of Artemia salina (brine shrimp) was found to have a closed‐circular structure 5.1 μm long and a buoyant density of 1.697 g/cm3. Escherichia coli RNA polymerase can transcribe this DNA. RNA synthesis is greatly stimulated by the σ subunit and is influenced by the presence of KCl. After formation of an initial complex between the enzyme and the mitochondrial DNA only a limited synthesis of RNA took place in the presence of rifampicin.The products obtained with holo enzyme and core enzyme in the presence of excess nucleoside triphosphates have a mean molecular weight of 1.5 × 106 and greater than 3 × 106 respectively, as determined by acrylamide‐agarose gel electrophoresis. Addition of the termination factor ϱ to the reaction mixture reduced the molecular weight of the products to 1.0 × 106 at a KCl concentration of 0.05 M but not at 0.1 M KCl.Under limiting UTP concentrations, a few discrete RNA bands of shorter size (about 8 × 105, 105 and 3 × 104 mol. wt) are synthesized. Addition of ϱ factor to the reaction mixture caused the disappearance of the higher‐molecular‐weight band (8 × 105) while the other RNA bands remained.Little self‐annealing was observed upon heating the [3H]RNA synthesized in vitro at 70 °C. On the other hand, when the [3H]RNA was heated in the presence of unlabeled mitochondrial rRNA, about 54% of the labeled product was resistant to degradation by a mixture of T1 and pancreatic RNase. In hybridization‐competition experiments, annealing of the [3H]RNA synthesized in vitro was reduced by 65% upon addition of unlabeled mitochondrial rRNA. RNA‐RNA annealing is thought to be the main cause for this competition reaction since similar results were obtained by simultaneous or step‐wise hybridization procedures. It is concluded that transcription in vitro of closed circular‐mitochondrial DNA from Artemia by E. coli RNA polymerase proceeds mainly on the anti‐rRNA strand.