‘RNA walk’ a novel approach to study RNA–RNA interactions between a small RNA and its target

In this study we describe a novel method to investigate the RNA–RNA interactions between a small RNA and its target that we termed ‘RNA walk’. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT–PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by ‘RNA walk’ and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that ‘RNA walk, is a generic method to map target RNA small RNAs interactions in vivo.     

Bacterial Hen1 is a 3′ terminal RNA ribose 2′-O-methyltransferase component of a bacterial RNA repair cassette

Hen1 is an RNA ribose 2′-O-methyltransferase that modifies the 3′ terminal nucleoside of eukaryal small regulatory RNAs. Here, we report that Hen1 homologs are present in bacterial proteomes from eight different phyla. Bacterial Hen1 is encoded by the proximal ORF of a two-gene operon that also encodes polynucleotide kinase-phosphatase (Pnkp), an RNA repair enzyme. Purified recombinant Clostridium thermocellum Hen1 is a homodimer of a 465-amino acid polypeptide. CthHen1 catalyzes methyl transfer from AdoMet to the 3′ terminal nucleoside of an RNA oligonucleotide, but is unreactive with a synonymous DNA oligonucleotide or an RNA with a single 3′-terminal deoxyribose sugar. CthHen1 is optimally active at alkaline pH and dependent on manganese. Activity is inhibited by AdoHcy and abolished by mutations D291A and D316A in the putative AdoMet-binding pocket. The C-terminal fragment, Hen1-(259–465), comprises an autonomous monomeric methyltransferase domain.     

Recombination and RNA processing: a common strand?

Genetic recombination is a basic cellular process required for altering genome structure. The RecA protein of Escherichia coli has a central role in homologous recombination, and a eukaryotic protein with similar properties has been discovered in the yeast Saccharomyces cerevisiae. Unexpectedly, this RecA-like protein has additional biochemical activities, and its function may not be restricted to recombination.     

Probing RNA–antibiotic interactions: a FTIR study

The crystal structure determination of antibiotic binding sites on the 30S ribosomal subunit and the increasing demand for developing RNA-based drugs has prompted us to study the direct binding of spectinomycin, vancomycin and bleomycin with yeast total RNA using Fourier transform infrared (FTIR) spectroscopy. We report that the OH of spectinomycin and the peptide group of vancomycin can bind to the bases of RNA, which might depend on Mg²⁺ concentration. Bleomycin on the other hand does not show such a drastic effect on yeast total RNA. This study might help in developing innovative strategies utilizing RNA molecules to perform a variety of essential biological functions.     

Tailoring RNA modular units on a common scaffold: A modular ribozyme with a catalytic unit for β-nicotinamide mononucleotide-activated RNA ligation

A novel ribozyme that accelerates the ligation of β-nicotinamide mononucleotide (β-NMN)-activated RNA fragments was isolated and characterized. This artificial ligase ribozyme (YFL ribozyme) was isolated by a “design and selection” strategy, in which a modular catalytic unit was generated on a rationally designed modular scaffold RNA. Biochemical analyses of the YFL ribozyme revealed that it catalyzes RNA ligation in a template-dependent manner, and its activity is highly dependent on its architecture, which consists of a modular scaffold and a catalytic unit. As the design and selection strategy was used for generation of DSL ribozyme, isolation of the YFL ribozyme indicated the versatility of this strategy for generation of functional RNAs with modular architectures. The catalytic unit of the YFL ribozyme accepts not only β-NMN but also inorganic pyrophosphate and adenosine monophosphate as leaving groups for RNA ligation. This versatility of the YFL ribozyme provides novel insight into the possible roles of β-NMN (or NADH) in the RNA world.     

Swallowing dynein: a missing link in RNA localization?

Localization of bicoid messenger RNA to the anterior cortex of the developing oocyte is essential for correct anterior-posterior patterning of the Drosophila embryo. It now seems that the Swallow protein functions as an adaptor, bridging bicoid mRNA to dynein, a molecular motor that would transport the complex anteriorly along microtubules.     

Is a closing "GA pair" a rule for stable loop-loop RNA complexes?

RNA hairpin aptamers specific for the trans-activation-responsive (TAR) RNA element of human immunodeficiency virus type 1 were identified by in vitro selection (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). The high affinity sequences selected at physiological magnesium concentration (3 mm) were shown to form a loop-loop complex with the targeted TAR RNA. The stability of this complex depends on the aptamer loop closing "GA pair" as characterized by preliminary electrophoretic mobility shift assays. Thermal denaturation monitored by UV-absorption spectroscopy and binding kinetics determined by surface plasmon resonance show that the GA pair is crucial for the formation of the TAR-RNA aptamer complex. Both thermal denaturation and surface plasmon resonance experiments show that any other "pairs" leads to complexes whose stability decreases in the order AG > GG > GU > AA > GC > UA > CA, CU. The binding kinetics indicate that stability is controlled by the off-rate rather than by the on-rate. Comparison with the complex formed with the TAR* hairpin, a rationally designed TAR RNA ligand (Chang, K. Y., and Tinoco, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8705-8709), demonstrates that the GA pair is a key determinant which accounts for the 50-fold increased stability of the TAR-aptamer complex (K(d) = 2.0 nm) over the TAR-TAR* one (K(d) = 92. 5 nm) at physiological concentration of magnesium. Replacement of the wild-type GC pair next to the loop of RNA I' by a GA pair stabilizes the RNA I'-RNA II' loop-loop complex derived from the one involved in the control of the ColE1 plasmid replication. Thus, the GA pair might be the preferred one for stable loop-loop interactions.     

Identification of antisense RNA stem–loops that inhibit RNA–protein interactions using a bacterial reporter system

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem–loop RNAs, presumably due to the high thermodynamic stability of the resulting loop–loop and loop–linear interactions. In this study, the identification of RNA stem–loops that inhibit U1A protein binding to the hpII RNA through RNA–RNA interactions was attempted using a bacterial reporter system based on phage λ N-mediated antitermination. As a result, loop sequences possessing 7–8 base complementarity to the 5′ region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem–loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem–loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem–loops that disrupt various types of RNA–protein interactions.     

Nucleotide sequence and evolution of a mammalian α-Tubulin messenger RNA

Bacterial clones containing complementary DNA sequences specific for rat brain α-tubulin messenger RNA were constructed. One plasmid, pILαTl, contains >95% of the sequences found in the mRNA: the entire coding sequence as well as extensive 5′ and 3′ untranslated sequences. Comparison of the rat amino acid sequence with the known chicken α-tubulin sequence (Valenzuela et al., 1981) reveals the extraordinary evolutionary stability of α-tubulin protein. The presence of only two interspecies amino acid differences within analogous 411 amino acid sequences predicts that amino acid substitutions in this protein are fixed with a unit evolutionary period (Wilson et al., 1977) of 550 million years (i.e. the time required for a 1% difference to arise within a specific protein in two diverging evolutionary lineages). An analysis of the silent nucleotide differences, permissible because of the degeneracy of the genetic code, demonstrates that these might not occur in a random fashion. The high guanine-cytosine bias in silent codon positions within the chicken α-tubulin sequence, previously noted by Valenzuela et al. (1981), is not conserved within the rat sequence. This decrease in guanine-cytosine bias is accompanied by a selective loss of CpG dinucleotides in the rat sequence.     

Finding specific RNA motifs: function in a zeptomole world?

We have developed a new method for estimating the abundance of any modular (piecewise) RNA motif within a longer random region. We have used this method to estimate the size of the active motifs available to modern SELEX experiments (picomoles of unique sequences) and to a plausible RNA World (zeptomoles of unique sequences: 1 zmole = 602 sequences). Unexpectedly, activities such as specific isoleucine binding are almost certainly present in zeptomoles of molecules, and even ribozymes such as self-cleavage motifs may appear (depending on assumptions about the minimal structures). The number of specified nucleotides is not the only important determinant of a motif's rarity: The number of modules into which it is divided, and the details of this division, are also crucial. We propose three maxims for easily isolated motifs: the Maxim of Minimization, the Maxim of Multiplicity, and the Maxim of the Median. These maxims together state that selected motifs should be small and composed of as many separate, equally sized modules as possible. For evenly divided motifs with four modules, the largest accessible activity in picomole scale (1-1000 pmole) pools of length 100 is about 34 nucleotides; while for zeptomole scale (1-1000 zmole) pools it is about 20 specific nucleotides (50% probability of occurrence). This latter figure includes some ribozymes and aptamers. Consequently, an RNA metabolism apparently could have begun with only zeptomoles of RNA molecules.