Effects of single-base bulges on intercalator binding to small RNA and DNA hairpins and a ribosomal RNA fragment

The way in which a single-base bulge might affect the structure of an RNA helix has been examined by preparing a series of six RNA hairpins, all with seven base pairs and a four-nucleotide loop. Five of the hairpins have single-base bulges at different positions. The intercalating cleavage reagent (methidiumpropyl)-EDTA-Fe(II) [MPE-Fe(II)] binds preferentially at a CpG sequence in the helix lacking a bulge and in four of the five hairpins with bulges. Hairpins with a bulge one or two bases to the 3' side of the CpG sequence bind ethidium 4-5-fold more strongly than the others. V1 RNase, which is sensitive to RNA backbone conformation in helices, detects a conformational change in all of the helices when ethidium binds; the most dramatic changes, involving the entire hairpin stem, are in one of the two hairpins with enhanced ethidium affinity. Only a slight conformational change is detected in the hairpin lacking a bulge. A bulge adjacent to a CpG sequence in a 100-nucleotide ribosomal RNA fragment enhances MPE-Fe(II) binding by an order of magnitude. These results extend our previous observations of bulges at a single position in an RNA hairpin [White, S. A., & Draper, D.E. (1987) Nucleic Acids Res. 15, 4049] and show that (1) a structural change in an RNA helix may be propagated for several base pairs, (2) bulges tend to increase the number of conformations available to a helix, and (3) the effects observed in small RNA hairpins are relevant to larger RNAs with more extensive structure. A bulge in a DNA hairpin identical in sequence with the RNA hairpins does not enhance MPE-Fe(II) binding affinity, relative to a control DNA hairpin. The effects of bulges on ethidium intercalation are evidently modulated by helix structure.     

Sequence,chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.     

Secondary structure and dynamics of the r(CGG) repeat in the mRNA of the fragile X mental retardation 1 (FMR1) gene.

The CGG triplet repeat found within the 5'UTR of the FMR1 gene is involved in the pathogenesis of both fragile X syndrome and fragile X-associated tremor/ataxia syndrome (FXTAS). The repeat has been shown to form both hairpins and tetraplexes in DNA; however, the secondary structure of CGG-repeat RNA has not been well defined. To this end, we have performed NMR spectroscopy on in vitro transcribed CGG-repeat RNAs and see clear evidence of intramolecular hairpins, with no evidence of tetraplex structures. Both C*G and G*G base pairs form in the hairpin stem, though in a dynamic equilibrium of conformations. In addition, we investigated the effect of an AGG repeat interruption on hairpin stability; such interruptions are often interspersed within the CGG repeat element and are thought to modulate secondary structure of the RNA. While the AGG repeat lowers the Tm of the hairpin at low Mg2+ concentrations, this difference disappears at physiological Mg2+ levels.     

Identification of a functional core in the RNA component of RNase MRP of budding yeasts

RNase MRP is an endonuclease participating in ribosomal RNA processing. It consists of one RNA and at least nine protein subunits. Using oligonucleotide-directed mutagenesis, we analyzed the functional role of five of the hairpins in the secondary structure of the RNA subunit of Saccharomyces cerevisiae RNase MRP. Deletion of an entire hairpin was either lethal or resulted in very poor growth. However, peripheral portions constituting up to 70% of a hairpin could be deleted without effects on cell growth rate or processing of rRNA. To determine whether these hairpins perform redundant functions, we analyzed mutants combining four or five benign hairpin deletions. Simultaneous removal of four of these hairpin segments had no detectable effect. Removing five created a temperature- and cold-sensitive enzyme, but these deficiencies could be partially overcome by a mutation in one of the RNase MRP protein subunits, or by increasing the copy number of several of the protein subunit genes. These observations suggest that the peripheral elements of the RNA hairpins contain no structures or sequences required for substrate recognition, catalysis or binding of protein subunits. Thus, the functionally essential elements of the RNase MRP RNA appear to be concentrated in the core of the subunit.     

Forced evolution of a regulatory RNA helix in the HIV-1 genome.

The 5'and 3'end of the HIV-1 RNA genome forms a repeat (R) element that encodes a double stem-loop structure (the TAR and polyA hairpins). Phylogenetic analysis of the polyA hairpin in different human and simian immunodeficiency viruses suggests that the thermodynamic stability of the helix is fine-tuned. We demonstrated previously that mutant HIV-1 genomes with a stabilized or destabilized hairpin are severely replication-impaired. In this study, we found that the mutant with a destabilized polyA hairpin structure is conditionally defective. Whereas reduced replication is measured in infections at the regular temperature (37 degrees C), this mutant is more fit than the wild-type virus at reduced temperature (33 degrees C). This observation of a temperature-dependent replication defect underscores that the stability of this RNA structure is critical for function. An extensive analysis of revertant viruses was performed to further improve the understanding of the critical sequence and structural features of the element under scrutiny. The virus mutants with a stabilized or destabilized hairpin were used as a starting point in multiple, independent selections for revertant viruses with compensatory mutations. Both mutants reverted to hairpins with wild-type stability along various pathways by acquisition of compensatory mutations. We identified 19 different revertant HIV-1 forms with improved replication characteristics, providing a first look at some of the peaks in the total sequence landscape that are compatible with virus replication. These experiments also highlight some general principles of RNA structure building.     

Induction of gene silencing by hairpin RNA expression in Tetrahymena thermophila reveals a second small RNA pathway

Unlike in other eukaryotes, in which it causes gene silencing, RNA interference (RNAi) has been linked to programmed DNA deletion in the ciliate Tetrahymena thermophila. Here we have developed an efficient method to inducibly express double-stranded RNA hairpins and demonstrated that they cause gene silencing through targeted mRNA degradation in all phases of the life cycle, including growth, starvation, and mating. This technique offers a new tool for gene silencing in this model organism. Induction of RNA hairpins causes dramatic upregulation of Dicer and Argonaute family genes, revealing a system capable of rapidly responding to double-stranded RNA. These hairpins are processed into 23- to 24-nucleotide (nt) small RNAs, which are distinctly different from the 28- to 30-nt small RNAs known to be associated with DNA deletion. Thus, two different small RNA pathways appear to be responsible for gene silencing and DNA deletion. Surprisingly, expression of the RNA hairpin also causes targeted DNA deletion during conjugation, although at low efficiencies, which suggests a possible crossover of these two molecular paths.     

Modulators of prostate cancer cell proliferation and viability identified by short-hairpin RNA library screening

There is significant need to identify novel prostate cancer drug targets because current hormone therapies eventually fail, leading to a drug-resistant and fatal disease termed castration-resistant prostate cancer. To functionally identify genes that, when silenced, decrease prostate cancer cell proliferation or induce cell death in combination with antiandrogens, we employed an RNA interference-based short hairpin RNA barcode screen in LNCaP human prostate cancer cells. We identified and validated four candidate genes (AKT1, PSMC1, STRADA, and TTK) that impaired growth when silenced in androgen receptor positive prostate cancer cells and enhanced the antiproliferative effects of antiandrogens. Inhibition of AKT with a pharmacologic inhibitor also induced apoptosis when combined with antiandrogens, consistent with recent evidence for PI3K and AR pathway crosstalk in prostate cancer cells. Recovery of hairpins targeting a known prostate cancer pathway validates the utility of shRNA library screening in prostate cancer as a broad strategy to identify new candidate drug targets.     

Translin associated protein X is essential for cellular proliferation

DNA vectors that express short hairpin RNAs (shRNAs) provide a new tool for reverse genetic analysis for selective long-term reduction of gene expression in mammalian cells. Using shRNA constructs with a cytomegalovirus promoter and an actin intron between the hairpins for stabilization, we reduce expression of an exogenously expressed gene, GFP and the endogenous protein, Translin-associated factor X (TRAX), in stably transfected Hela cell lines. The reduction of TRAX in Hela cells causes reduced cell proliferation. This decrease is specific as there is no equivalent reduction of the TRAX interacting protein, Testis brain RNA-binding protein, or any significant increase in a number of interferon-related target genes.     

The fragile X syndrome repeats form RNA hairpins that do not activate the interferon-inducible protein kinase, PKR, but are cut by Dicer

We show here that under physiologically reasonable conditions, CGG repeats in RNA readily form hairpins. In contrast to its DNA counterpart that forms a complex mixture of hairpins and tetraplexes, r(CGG)₂₂ forms a single stable hairpin with no evidence for any other folded structure even at low pH. RNA with the sequence (CGG)₉AGG (CGG)₁₂AGG(CGG)₉₇, found in a fragile X syndrome pre-mutation allele, forms a number of different hairpins. The most prominent hairpin forms in the 3′ part of the repeat and involves the 97 uninterrupted CGG repeats. In contrast to the CUG-RNA hairpins formed by myotonic dystrophy type 1 repeats, we found no evidence that CGG-RNA hairpins activate PKR, the interferon-inducible protein kinase that is activated by a wide range of double-stranded RNAs. However, we do show that the CGG-RNA is digested, albeit inefficiently, by the human Dicer enzyme, a step central to the RNA interference effect on gene expression. These data provide clues to the basis of the toxic effect of CGG-RNA that is thought to occur in fragile X pre-mutation carriers. In addition, RNA hairpins may also account for the stalling of the 40S ribosomal subunit that is thought to contribute to the translation deficit in fragile X pre-mutation and full mutation alleles.     

Characterization of the mechanical unfolding of RNA pseudoknots

The pseudoknot is an important RNA structural element that provides an excellent model system for studying the contributions of tertiary interactions to RNA stability and to folding kinetics. RNA pseudoknots are also of interest because of their key role in the control of ribosomal frameshifting by viral RNAs. Their mechanical properties are directly relevant to their unfolding by ribosomes during translation. We have used optical tweezers to study the kinetics and thermodynamics of mechanical unfolding and refolding of single RNA molecules. Here we describe the unfolding of the frameshifting pseudoknot from infectious bronchitis virus (IBV), three constituent hairpins, and three mutants of the IBV pseudoknot. All four pseudoknots cause −1 programmed ribosomal frameshifting. We have measured the free energies and rates of mechanical unfolding and refolding of the four frameshifting pseudoknots. Our results show that the IBV pseudoknot requires a higher force than its corresponding hairpins to unfold. Furthermore, its rate of unfolding changes little with increasing force, in contrast with the rate of hairpin unfolding. The presence of Mg²⁺ significantly increases the kinetic barriers to unfolding the IBV pseudoknot, but has only a minor effect on the hairpin unfolding. The greater mechanical stability of pseudoknots compared to hairpins, and their kinetic insensitivity to force supports the hypothesis that −1 frameshifting depends on the difficulty of unfolding the mRNA.     

A systematic analysis of the effect of target RNA structure on RNA interference

RNAi efficiency is influenced by local RNA structure of the target sequence. We studied this structure-based resistance in detail by targeting a perfect RNA hairpin and subsequently destabilized its tight structure by mutation, thereby gradually exposing the target sequence. Although the tightest RNA hairpins were completely resistant to RNAi, we observed an inverse correlation between the overall target hairpin stability and RNAi efficiency within a specific thermodynamic stability (ΔG) range. Increased RNAi efficiency was shown to be caused by improved binding of the siRNA to the destabilized target RNA hairpins. The mutational effects vary for different target regions. We find an accessible target 3′ end to be most important for RNAi-mediated inhibition. However, these 3′ end effects cannot be reproduced in siRNA-target RNA-binding studies in vitro, indicating the important role of RISC components in the in vivo RNAi reaction. The results provide a more detailed insight into the impact of target RNA structure on RNAi and we discuss several possible implications. With respect to lentiviral-mediated delivery of shRNA expression cassettes, we present a ΔG window to destabilize the shRNA insert for vector improvement, while avoiding RNAi-mediated self-targeting during lentiviral vector production.     

Thermodynamic,structural and fluorescent characteristics of DNA hairpins containing functionalized pyrrolo-2′-deoxycytidines

Herein, we present comprehensive physicochemical and structural analysis of various DNA hairpins modified with pyrrolo-2′-deoxycytidine (Py-dC) derivatives. The introduction of modified Py-dC in most cases causes minor decrease of hairpin thermodynamic stability. The energetically unfavorable effect is more pronounced when modified residue is present within hairpin loop. Our studies indicate that thermodynamic effects induced by all Py-dC derivatives are net results of increased stacking interactions caused by larger surface of pyrrolo-2′-deoxycytidine aromatic ring and unfavorable effect implied by the presence of additional side chains. The CD spectra of all modified hairpins are similar to unmodified hairpin indicating that the presence of Py-dC derivatives does not disrupt the secondary structure of DNA. Interestingly, the presence of various side chains can increase fluorescent discrimination of paired and unpaired regions of DNA. The fluorescence observed for hairpins modified within loop is significantly quenched when Py-dC derivative is present in the stem region.     

Evidence for a four-strand exchange catalyzed by the RecA protein

Strand exchange between two duplexes is usually initiated as a three-strand event that requires the presence of a single-stranded overhang or gap in one of the two molecules. Here we show that the RecA protein can catalyze a four-strand exchange. Specifically, it can recombine short hairpin substrates with homologous stems provided that one of the hairpins possesses a chimeric DNA/RNA backbone. This four-strand exchange reaction goes to completion in the presence of ATPgammaS and releases a stable heteroduplex upon removal of the RecA protein. Under identical conditions, strand exchange between two DNA hairpins is incomplete and generates a nascent heteroduplex that rapidly dissociates when the RecA protein is denatured. Since presynaptic filament formation does not appear to melt either type of hairpin, we propose that exchange occurs between homologously aligned duplexes that are extended and unwound within a RecA filament. The first reaction provides a mechanism for gene targeting by chimeric double-hairpin oligonucleotides while the second reaction explains the ability of the RecA protein to transiently align double-stranded DNA molecules.     

Preference for ribose over deoxyribose in loop-closing base pairs of extra stable nucleic acid hairpins

We have investigated the effect of switching ribose to deoxyribose at the closing base-pair of an extra-stable RNA hairpin. Specifically, we studied the sequence 5'-GGAC(UUCG)GUCC, a dodecanucleotide that folds into a well-characterized, "extra stable" RNA hairpin structure. Recently, we showed that hairpins containing a 2',5'-linked (UUCG) loop instead of the native 3',5'-linked loop also exhibit extra-stability (Hannoush and Damha, J. Am. Chem. Soc., 2001, 123, 12368-12374). In this article, we show that the ribose units located at the loop-closing positions (i.e., rC4 and rG9) contribute significantly to the stabilization of RNA hairpins, particularly those containing the 3',5'-UUCG loop. Interestingly, the requirement of rC4 and rG9 is more relaxed for DNA hairpins containing the 2',5'-UUCC loop and, in fact, they may be replaced altogether (ribose--> deoxyribose) without affecting stability. The results broaden our understanding of the behavior of highly stable (UUCG) hairpin loops and how they respond to structural perturbation of the loop-closing base pairs.