The effect of loop size in antisense and target RNAs on the efficiency of antisense RNA control.

Most natural antisense RNAs display a high degree of secondary structure with stem-loops as their most prominent feature. Mutations affecting the inhibitory activity of these RNAs most often map in or close to loop regions in both the antisense and target RNAs. The primary recognition loops often contain 5-7 unpaired nucleotides. Nucleotide changes in the loops affect the binding rate and, hence, the inhibitory effect on the activity of the target RNA. Here we address the question whether loop sizes affect binding rates between antisense and target RNAs, using the replication control system of plasmid R1 as a model system. By creating a series of loop size mutants we show that loop size alterations have strong effects on the binding rates between the two reactant RNAs in vitro, and that most of the mutations analyzed display corresponding effects on antisense RNA control in vivo. Our data suggest that the three-dimensional structures of antisense and target RNA stem-loops are crucial for determining binding rates. The implications of these results for the design of efficient artificial antisense RNA control systems are discussed.     

HIV-1 TAR as anchoring site for optimized catalytic RNAs

Ribozymes have a great potential for developing specific gene silencing molecules. One of the main limitations to ensure the efficient application of ribozymes is to achieve effective binding to the target. Stem-loop domains support efficient formation of the kissing complex between natural antisense molecules and their target sequence. We have characterized catalytic antisense RNA hybrid molecules composed of a hammerhead ribozyme and a stem-loop antisense domain. A series of artificial RNA substrates containing the TAR-RNA stem-loop and a target for the hammerhead ribozyme were constructed and challenged with a catalytic antisense RNA carrying the TAR complementary stem-loop. The catalytic antisense RNA cleaves each of these substrates significantly more efficiently than the parental hammerhead ribozyme. Deletion of the TAR domain in the substrate abolishes the positive effect. These results suggest that the enhancement is due to the interaction of both complementary stem-loop motifs. A similar improvement was corroborated when targeting the LTR region of HIV-1 with either hammerhead- and hairpin-based catalytic antisense RNAs. Our results indicate that the TAR domain can be used as an anchoring site to facilitate the access of ribozymes to their specific target sequences within TAR-containing RNAs. Finally, we propose the addition of stable stem-loop motifs to the ribozyme domain as a rational way for constructing catalytic antisense RNAs.     

A two unit antisense RNA cassette test system for silencing of target genes.

This communication describes a two unit antisense RNA cassette system for use in gene silencing. Cassettes consist of a recognition unit and an inhibitory unit which are transcribed into a single RNA that carries sequences of non-contiguous complementarity to the chosen target RNA. The recognition unit is designed as a stem-loop for rapid formation of long- lived binding intermediates with target sequences and resembles the major stem-loop of a naturally occurring antisense RNA, CopA. The inhibitory unit consists of either a sequence complementary to a ribosome binding site or of a hairpin ribozyme targeted at a site within the chosen mRNA. The contributions of the individual units to inhibition was assessed using the lacI gene as a target. All possible combinations of recognition and inhibitory units were tested in either orientation. In general, inhibition of lacI expression was relatively low. Fifty per cent inhibition was obtained with the most effective of the constructs, carrying the recognition stem-loop in the antisense orientation and the inhibitory unit with an anti-RBS sequence. Several experiments were performed to assess activities of the RNAs in vitro and in vivo : antisense RNA binding assays, cleavage assays, secondary structure analysis as well as Northern blotting and primer extension analysis of antisense and target RNAs. The problems associated with this antisense RNA approach as well as its potential are discussed with respect to possible optimization strategies.     

Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction.

The bacterial Hfq protein modulates the stability or the translation of mRNAs and has recently been shown to interact with small regulatory RNAs in E. coli. Here we show that Hfq belongs to the large family of Sm and Sm-like proteins: it contains a conserved sequence motif, known as the Sm1 motif, forms a doughnut-shaped structure, and has RNA binding specificity very similar to the Sm proteins. Moreover, we provide evidence that Hfq strongly cooperates in intermolecular base pairing between the antisense regulator Spot 42 RNA and its target RNA. We speculate that Sm proteins in general cooperate in bimolecular RNA-RNA interaction and that protein-mediated complex formation permits small RNAs to interact with a broad range of target RNAs.     

Adenovirus-delivered antisense RNA and shRNA exhibit different silencing efficiencies for the endogenous transforming growth factor-beta (TGF-beta) type II receptor

Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-beta type II receptor (TGFbetaRII). We used a dicistronic reporter, consisting of the coding sequences for TGFbetaRII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGFbetaRII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGFbetaRII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGFbetaRII. In contrast, the shRNAs were able to silence endogenous TGFbetaRII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGFbetaRII/GFP reporter reduced endogenous TGFbetaRII protein expression by 70% in A549 cells and reduced TGFbeta signaling by >80% in HeLa cells.     

Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus

By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense-box RNAs, where the latter only exhibit partial complementarity to RNA targets. The most prominent group of antisense RNAs is transcribed in the opposite orientation to the transposase genes, encoded by insertion elements (transposons). Thus, these antisense RNAs may regulate transposition of insertion elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted to target the 3'-untranslated regions of certain mRNAs. Furthermore, one of the ncRNAs that does not show antisense elements is transcribed from a repeat unit of a cluster of small regularly spaced repeats in S. solfataricus which is potentially involved in replicon partitioning. In conclusion, this is the first report of stably expressed antisense RNAs in an archaeal species and it raises the prospect that antisense-based mechanisms are also used widely in Archaea to regulate gene expression.     

Anchoring hairpin ribozymes to long target RNAs by loop-loop RNA interactions

Efficient ribozyme-mediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. Stable stem-loop domains are key elements for efficiency of natural antisense RNAs. This work tests the possibility of using such naturally existing structural motifs for anchoring hairpin ribozymes when targeting long RNAs. Assays were performed with four catalytic antisense RNAs, based on the hairpin ribozyme (HP), that carried a stable stem-loop motif at their 3' end. Extensions consisted of one of the following motifs: the stem-loop II of the natural antisense RNA-CopA, its natural target in CopT, the TAR-RNA motif, or its complementary sequence alphaTAR. Interestingly, the presence of any of these antisense motifs resulted in an enhancement of catalytic performance against the ribozyme's 14-nucleotide-long target RNA (Swt). A series of artificial, long RNA substrates containing the Swt sequence and the natural TAR-RNA stem-loop were constructed and challenged with a catalytic antisense RNA carrying the TAR-complementary stem-loop. This cleaves each of these substrates significantly more efficiently than HP. The deletion of the TAR domain in the substrate, or its substitution by its complementary counterpart alphaTAR, abolishes the positive effect. These results suggest that the enhancement is owed to the interaction of both complementary stem-loop domains. Moreover, they demonstrate that the TAR domain can be used as an anchoring site to facilitate the access of hairpin ribozymes to their specific target sequences within TAR-containing RNAs.     

Effects of different target sites on antisense RNA-mediated regulation of gene expression

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3'' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability. [BMB Reports 2014; 47(11): 619-624]     

Regulation of gene expression by trans-encoded antisense RNAs

Members of a class of antisense RNAs are encoded by genes that are located at loci other than those of their target genes. Three examples of antisense RNA genes are discussed here. micF is found in Escherichia coli and other bacteria and functions to control outer membrane protein F levels in response to environmental stimuli. dicF is also found in E. coli and is involved in the regulation of cell division, lin-4 is found in the nematode Caenorhabditis elegans and functions during larval development. Nucleotide sequences of at least two of these genes appear to be phylogenetically conserved. The trans-encoded antisense RNAs are small RNAs which display only partial complementarity to their target RNAs. Models for RNA/RNA interactions have been proposed. It is possible that currently known unlinked antisense RNA genes are part of a larger class of heretofore undiscovered regulatory RNA genes. Possible ways of detecting other unlinked antisense RNA genes are discussed.     

Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation

This communication describes improvement strategies used on a previously described two-unit antisense RNA cassette system. This cassette system encodes RNA with noncontiguous regions of complementarity to a bacterial target RNA, lacI mRNA. One of the units of complementarity was contained within an RNA stem-loop resembling that of the very efficient, naturally occurring antisense RNA CopA. As relatively low inhibitory activity was obtained previously, we tested variants in which several stem-loops were combined within one RNA, each of them directed against a different stretch of target RNA. One to four stem-loop RNAs were tested and found to be relatively ineffective, likely because of low metabolic stability. To increase the intracellular stability of these and other antisense RNAs, a stabilizer element (stem-loop derived from gene 32 mRNA of phage T4) was inserted at their 5'-ends. The results indicate that addition of this element indeed increased antisense RNA efficiency in vivo. As expected, this effect was primarily due to a longer antisense RNA half-life, as shown by RNA abundance (Northern analysis) and decay rates (rifampicin runout experiments). In summary, the results reported indicate that rational design of antisense RNA is feasible, but that the degree of inhibition (approximately 75% maximum inhibition) accomplished here could still be improved.     

Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference

In RNA-mediated interference (RNAi), externally provided mixtures of sense and antisense RNA trigger concerted degradation of homologous cellular RNAs. We show that RNAi requires duplex formation between the two trigger strands, that the duplex must include a region of identity between trigger and target RNAs, and that duplexes as short as 26 bp can trigger RNAi. Consistent with in vitro observations, a fraction of input dsRNA is converted in vivo to short segments of approximately 25 nt. Interference assays with modified dsRNAs indicate precise chemical requirements for both bases and backbone of the RNA trigger. Strikingly, certain modifications are well tolerated on the sense, but not the antisense, strand, indicating that the two trigger strands have distinct roles in the interference process.     

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.     

Using binding profiles to predict binding sites of target RNAs

Prediction of RNA-RNA interaction is a key to elucidating possible functions of small non-coding RNAs, and a number of computational methods have been proposed to analyze interacting RNA secondary structures. In this article, we focus on predicting binding sites of target RNAs that are expected to interact with regulatory antisense RNAs in a general form of interaction. For this purpose, we propose bistaRNA, a novel method for predicting multiple binding sites of target RNAs. bistaRNA employs binding profiles that represent scores for hybridized structures, leading to reducing the computational cost for interaction prediction. bistaRNA considers an ensemble of equilibrium interacting structures and seeks to maximize expected accuracy using dynamic programming. Experimental results on real interaction data validate good accuracy and fast computation time of bistaRNA as compared with several competitive methods. Moreover, we aim to find new targets given specific antisense RNAs, which provides interesting insights into antisense RNA regulation. bistaRNA is implemented in C++. The program and Supplementary Material are available at http://rna.naist.jp/program/bistarna/.     

Four-way junctions in antisense RNA-mRNA complexes involved in plasmid replication control: a common theme?

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. In plasmid R1, we have recently shown that the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by the formation of two intermolecular helices, resulting in a four-way junction structure and a side-by-side helical alignment. Based on lead-induced cleavage and ribonuclease (RNase) V(1) probing combined with molecular modeling, a strikingly similar topology is supported for the complex formed between the antisense RNA (Inc) and mRNA (RepZ) of plasmid Col1b-P9. In particular, the position of the four-way junction and the location of divalent ion-binding site(s) indicate that the structural features of these two complexes are essentially the same in spite of sequence differences. Comparisons of several target and antisense RNAs in other plasmids further indicate that similar binding pathways are used to form the inhibitory antisense-target RNA complexes. Thus, in all these systems, the structural features of both antisense and target RNAs determine the topologically possible and kinetically favored pathway that is essential for efficient in vivo control.     

Antisense RNA injections in fertilized frog eggs reveal an RNA duplex unwinding activity

Previous experiments have shown that mRNA translation in frog oocytes can be inhibited by the injection of a complementary antisense RNA. Here we explore the use of antisense RNAs to study the functions of localized maternal mRNAs during postfertilization development. While developmental abnormalities were observed in injected fertilized eggs, these abnormalities could not be attributed to the antisense RNA since they were induced at a similar frequency in control embryos. Biochemical tests show that the injected antisense RNA does not form stable hybrids in vivo with its complementary endogenous mRNA. In addition, a novel activity that unwinds RNA:RNA duplexes was found. This activity exists at high levels in eggs and early embryos and is absent or very much diminished in oocytes and late blastula embryos. These results suggest that antisense RNAs may be of limited use in studying the functions of maternal RNAs in Xenopus.     

Antisense RNA targeting of primase interferes with bacteriophage replication in Streptococcus thermophilus

The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages. Expression of antisense RNAs complementary to the putative primase gene (pri3.1) from S. thermophilus phage kappa 3 provided significant protection from kappa 3 and two other Sfi21-type phages. Expression of pri3.10-AS, an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of kappa 3 to 3 x 10(-3) and reduced its burst size by 20%. Mutant phages capable of overcoming antisense inhibition were not recovered. Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene. Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 x 10(-3). The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5' region of the gene. The impacts of primase-targeted antisense RNAs on phage development were examined. The expression of pri3.10-AS resulted in reductions in target RNA abundance and the number of phage genomes synthesized. Targeting a key genome replication function with antisense RNA provided effective phage protection in S. thermophilus.