RNA Dimerization Research Articles

SHAPE analysis of the 5′ end of the Mason-Pfizer monkey virus (MPMV) genomic RNA reveals structural elements required for genome dimerization

Earlier genetic and structural prediction analyses revealed that the packaging determinants of Mason Pfizer monkey virus (MPMV) include two discontinuous core regions at the 5' end of its genomic RNA. RNA secondary structure predictions suggested that these packaging determinants fold into several stem-loops (SLs). To experimentally validate this structural model, we employed selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), which examines the flexibility of the RNA backbone at each nucleotide position. Our SHAPE data validated several predicted structural motifs, including U5/Gag long-range interactions (LRIs), a stretch of single-stranded purine (ssPurine)-rich region, and a distinctive G-C-rich palindromic (pal) SL. Minimum free-energy structure predictions, phylogenetic, and in silico modeling analyses of different MPMV strains revealed that the U5 and gag sequences involved in the LRIs differ minimally within strains and maintain a very high degree of complementarity. Since the pal SL forms a helix loop containing a canonical "GC" dyad, it may act as a RNA dimerization initiation site (DIS), enabling the virus to package two copies of its genome. Analyses of wild-type and pal mutant RNAs revealed that disruption of pal sequence strongly affected RNA dimerization. However, when in vitro transcribed trans-complementary pal mutants were incubated together showed RNA dimerization was restored authenticating that the pal loop (5'-CGGCCG-3') functions as DIS.

Structure-Guided Discovery of a Novel Aminoglycoside Conjugate Targeting HIV-1 RNA Viral Genome

The dimerization initiation site (DIS) of the HIV-1 genomic RNA is a conserved stem-loop that promotes viral genome dimerization by forming a loop-loop complex. The DIS constitutes a potentially interesting target because it is crucial for several key steps of the viral replication. In this work we describe the synthesis of a rationally designed aminoglycoside conjugate that binds the HIV-1 DIS viral RNA with high specificity, as shown by an extensive in vitro binding characterization. We propose a three-dimensional model of the drug-RNA interaction that perfectly fits with binding data. Our results show the feasibility of targeting the HIV DIS viral RNA dimer and open the way to the rationale design of a new class of antiviral drugs. In addition, due to similarities between the HIV-1 DIS RNA and the bacterial aminoacyl decoding site (A site) RNA, we show that this novel aminoglycoside conjugate also binds the bacterial A site with a similar affinity as natural aminoglycoside antibiotics.

High-resolution reversible folding of hyperstable RNA tetraloops using molecular dynamics simulations

We report the de novo folding of three hyperstable RNA tetraloops to 1-3 Å rmsd from their experimentally determined structures using molecular dynamics simulations initialized in the unfolded state. RNA tetraloops with loop sequences UUCG, GCAA, or CUUG are hyperstable because of the formation of noncanonical loop-stabilizing interactions, and they are all faithfully reproduced to angstrom-level accuracy in replica exchange molecular dynamics simulations, including explicit solvent and ion molecules. This accuracy is accomplished using unique RNA parameters, in which biases that favor rigid, highly stacked conformations are corrected to accurately capture the inherent flexibility of ssRNA loops, accurate base stacking energetics, and purine syn-anti interconversions. In a departure from traditional quantum chemistrycentric approaches to force field optimization, our parameters are calibrated directly from thermodynamic and kinetic measurements of intra- and internucleotide structural transitions. The ability to recapitulate the signature noncanonical interactions of the three most abundant hyperstable stem loop motifs represents a significant milestone to the accurate prediction of RNA tertiary structure using unbiased all-atom molecular dynamics simulations.

RNA dimer synthesis using montmorillonite as a catalyst: The role of surface layer charge

The oligomerisation of activated nucleotides to form RNA is catalyzed by montmorillonite. However, the mechanism of this process and its use as a model for similar prebiotic chemistry is still under investigation. To date, amongst the more than two hundred clay minerals investigated as catalysts, only a few are excellent. We turned our attention to the less efficient catalysts to see how they performed in the synthesis of nucleotide dimers in which a nucleoside and an activated nucleotide produced only linear products of the form MpN. We have found that representative clay minerals, Otay and Chambers specifically, although poor at catalyzing the direct oligomerization of activated nucleotides, are able to promote these dimer syntheses. Sixteen reactions and thirty two products have been studied and found to vary in their outcome. The yields of 2′–5′ and 3′–5′-products were always less than those obtained with the excellent catalyst Volclay®. The results of this research provide a basis for further understanding of the physical processes in the mechanism of this catalysis and suggest that more clay minerals than hitherto expected may have the ability to catalyze RNA synthesis at the dimer level. These results have an important bearing upon the RNA world scenario for the origin of life.

A novel method to examine individual RNA structures within a mixed population reveals insights into the HIV-1 RNA dimerization process

Different models have been proposed for the HIV-1 RNA monomer and dimer structures, often by studying mutants that disrupt the palindromic nature of the dimerisation initiation site (DIS) to stabilise the monomer. We have developed a new technique known as “ingel SHAPE (selective 2’OH acylation analysed by primer extension)”. Individual RNA structures within a mixed structural population are isolated by electrophoresis under native conditions, and secondary structural probing takes place within the gel. High-throughput analysis generates a secondary structural map of each RNA, making it possible to examine the structures of different conformers under native conditions, without the need to stabilise each structure by mutagenesis or by using non-physiological buffers. We have validated the technique using TAR RNA, and show it to be accurate and highly reproducible. We have then used it to show that under native conditions the unspliced HIV-1 RNA monomer and dimer have different acylation sensitivities across the packaging signal region, but that the DIS is unreactive in both monomer and dimer. These data imply that a structural switch takes place; further examination showed that our results fit most closely to a model where, in the monomer, the dimerisation initiation site interacts with the U5 region, and that the native dimer structure corresponds closely with previously proposed structures of the monomeric RNA. This new technique allows us to visualise the nucleotides involved in the RNA structural switch, and those that remain static.

In vivo SELEX of single-stranded domains in the HIV-1 leader RNA

The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two segments we document the selection of the (nearly) wild-type sequence, thus demonstrating the importance of these primary nucleotide sequences and the power of the in vivo SELEX approach. However, for the third segment a large variety of sequences is compatible with efficient HIV-1 replication. Interestingly, the A-rich sequence of this segment is highly conserved among HIV-1 isolates, which likely reflects the evolutionary tendency of HIV-1 to adopt A-rich sequences.

Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging

The 5' untranslated region (5' UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNA(Lys3) annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5'-UTR RNA. In this study, we show that annealing of tRNA(Lys3) or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5' UTR enhances its dimerization in vitro. Structural analysis of the 5'-UTR RNA using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5' UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNA(Lys3) to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNA(Lys3) to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions.

Identification of the herbaceous host range of Apple scar skin viroid and analysis of its progeny variants

Apple scar skin viroid (ASSVd) is the type member of the genus Apscaviroid of the family Pospiviroidae and is known to cause scarring and dappling of apple fruits. The herbaceous host range of ASSVd was identified using an infectious dimeric clone. Among 13 plant species inoculated with the viroid, nine were found to be systemic hosts and three served as indicator hosts. The viroid could be transferred to these hosts through agroinoculation, rubbing of dimeric RNA transcripts, dimeric DNA plasmids and through sap inoculation. Single‐strand confirmation polymorphism (SSCP) analysis of the progeny obtained after inoculation of the infectious ASSVd clone showed the presence of four sequence variants (V1, V2, V3 and V4) which was also confirmed by sequencing. Two sequence variants (V1 and V3) were identified as dominant, representing 47·72 and 36·36% of the total progeny, respectively. The V4 sequence variant was similar to the wild type and occurred at a frequency of only 4·5% of the total progeny.

Mechanism of HIV-1 RNA Dimerization in the Central Region of the Genome and Significance for Viral Evolution

The genome of HIV-1 consists of two identical or nearly identical RNA molecules. The RNA genomes are held in the same, parallel orientation by interactions at the dimer initiation site (DIS). Previous studies showed that in addition to interactions at DIS, sequences located 100 nucleotides downstream from the 5' splice site can dimerize in vitro through an intermolecular G-quartet structure. Here we report that the highly conserved G-rich sequence in the middle portion of the HIV-1 genome near the central polypurine tract (cPPT) dimerizes spontaneously under high ionic strength in the absence of protein. The antisense RNA does not dimerize, strongly indicating that RNA dimerization does not exclusively involve A:U and G:C base pairing. The cation-dependent reverse transcriptase pausing profile, CD spectra profile, and cation-dependent association and thermal dissociation characteristics indicate G-quartet structures. Different forms of G-quartets are formed including monomers and, significantly, intermolecular dimers. Our results indicate that RNA genome dimerization and parallel alignment initiated through interactions at DIS may be greatly expanded and stabilized by formation of an intermolecular G-quartet at a distant site near the cPPT. It is likely that formation of G-quartet structure near the cPPT in vivo keeps the RNA genomes in proximity over a long range, promoting genetic recombination in numerous hot spots.

Three-Dimensional RNA Structure of the Major HIV-1 Packaging Signal Region

SummaryHIV-1 genomic RNA has a noncoding 5′ region containing sequential conserved structural motifs that control many parts of the life cycle. Very limited data exist on their three-dimensional (3D) conformation and, hence, how they work structurally. To assemble a working model, we experimentally reassessed secondary structure elements of a 240-nt region and used single-molecule distances, derived from fluorescence resonance energy transfer, between defined locations in these elements as restraints to drive folding of the secondary structure into a 3D model with an estimated resolution below 10 Å. The folded 3D model satisfying the data is consensual with short nuclear-magnetic-resonance-solved regions and reveals previously unpredicted motifs, offering insight into earlier functional assays. It is a 3D representation of this entire region, with implications for RNA dimerization and protein binding during regulatory steps. The structural information of this highly conserved region of the virus has the potential to reveal promising therapeutic targets.

Dimeric RNA recognition regulates HIV-1 genome packaging.

How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses.

Influence of RNA structural elements on Ty1 retrotransposition

The long-terminal repeat (LTR)-retrotransposon Ty1 is a mobile genetic element that replicates through an RNA intermediate. Retroelement genomic transcripts contain internal structures fundamental to gene expression and propagation. In addition, long non-coding antisense RNAs overlap the 5′-terminal region of the genomic RNA and confer post-translational copy number control. Although LTR- retrotransposons are functionally related to retroviruses, little is known about the structural determinants required for genomic RNA packaging or reverse transcription. This commentary summarizes two recent papers that provide the first snapshot of genomic RNA structures from the retrotransposon Ty1 involved in transposition. We combined structural approaches with functional and genetic assays to determine if antisense RNAs anneal with the genomic RNA. Analysis of various steps in the Ty1 life cycle showed that a novel RNA pseudoknot contributes to retrotransposon function. Comparing different RNA states provides additional information about regions potentially involved in Ty1 RNA dimerization or packaging.

197 Modeling the maturation pathway of the HIV-1 5’-UTR RNA dimer

HIV-1 genomic RNA is packaged as a dimer into the virions. The initial metastable RNA dimer is believed to be formed by virtue of “kissing interactions” between two copies of the palindromic apical loops of stem-loop SL1 of the 5’-untranslated region (5’-UTR) of the genomic RNA. Viral nucleocapsid protein NCp7 promotes maturation of the RNA dimer into more stable form, which involves extended or linear form of SL1 dimer (reviewed in Paillart et al., 2004; Moore & Hu, 2009; Lu et al., 2011). In vitro experiments have shown that this conversion occurs at stoichiometric amounts of NCp7 without breaking interactions between the two copies of the SL1 apical loops (Mujeeb et al., 2007). We have proposed a hypothetical pathway and calculated models of the intermediate structures for the SL1 stem-loop dimer maturation that does not require simultaneous dissociation of all base pairs in SL1 stems; this pathway involves formation of an RNA analog of the Holliday junction intermediate between the two stems of the SL1 dimer and a following branch migration towards the palindromic duplex (Ulyanov et al., 2011). Here, we extend these models to the dimer of the 1–344 fragment of HIV-1 RNA, which includes all of the 5’-UTR and the gag start AUG codon region, and show that the branch-migration mechanism of the dimer maturation is also feasible for the full 5’-UTR RNA. All RNA models have been calculated with the miniCarlo program (Zhurkin et al., 1991).

A Novel Bent Intermediate in the Dimerization of HIV-1 DIS RNA

Dimerization Initiation Sequence (DIS) plays a crucial role in genome dimerization through formation of a “kissing complex” intermediate between two homologous DIS sequences. DIS is a conserved hairpin loop motif on the 5' UTR of the HIV-1 genome. This bimolecular kissing complex ultimately leads to the formation of an extended RNA duplex. Studying the mechanism and kinetics of this RNA interaction is the key to ex-ploiting DIS as a possible HIV drug target.Here, we report a novel study that makes an important contribution to understanding the dimerization mechanism of HIV-1 RNA in vitro. This work employed single-molecule fluorescence resonance energy transfer (smFRET) to monitor the dimerization of minimal HIV-1 RNA sequence containing DIS. Most significantly, we observe a previously uncharacterized folding intermediate that plays a critical role in the dimerization mechanism. Our data reveal that dimerization involves three distinct steps in dynamic equilibrium and the equilibrium between the steps are regulated by Mg2+ ions. Two of the steps are identified as previously proposed structures: the kissing complex and the extended duplex. In addition to these, our data reveal a previously unobserved folding intermediate, consistent with a bent kissing complex conformation, similar to the TAR-TAT complex. Mutations of the highly conserved purines flanking the DIS loop destabilize this intermediate which indicates that these purines may play an important role in the HIV-1 RNA dimerization in vivo. The mechanistic insights gained from these experiments would represent significant progress in understanding the HIV-1 dimerization mechanism.

10 The montmorillonite-catalyzed synthesis of RNA dimer

A synthesis has been developed, providing nucleotide dimers comprising natural or unnatural nucleoside residues. A ribonucleoside 5′-phosphorimidazolide is added to a nucleoside adsorbed on montmorillonite at neutral pH with the absence of protecting groups. Approximately, 30% of the imidazolide is converted into each 2′-5′ dimer and 3′-5′ dimer with the rest hydrolyzed to the 5′-monophosphate. Experiments with many combinations have suggested the limits to which this method may be applied, including heterochiral and chimeric syntheses. This greener chemistry has enabled the synthesis of dimers from activated nucleotides themselves, activated nucleotides with nucleosides, and activated nucleotides with nucleotide 5′-monophosphates. Both homo- and heterochiral combinations of reagents have been tried. The montmorillonite-catalyzed oligomerization of 5′-activated nucleotides leads to oligomers up to 50 residues in length (Huang & Ferris, 2007) using the excellent catalyst Volclay®. However, all oligomers must necessarily begin as dimers, so we considered it important to study in detail the formation of these products under prebiotic conditions. Then, a meaningful comparison could be drawn between our syntheses and the formation of long oligomers that is part of our studies of the origins of life. In the synthesis of trimers from these dimers, we looked for alternative synthetic methods via a 5′-phosphate dimer with activated nucleotides as well as 5′-hydroxy nucleotide dimers with the same reactant. The method has shown promise in targeting trimer synthesis and the procedure lends itself to the development of combinatorial libraries. The use of enzymatic hydrolysis has played a crucial role in this work, facilitating product identity across the spectrum of products prepared. The yields of the corresponding homochiral and heterochiral dimers from A and U will require careful modeling of the reactants in their interactions with both the clay and one another to locate the source of the similarities and differences. The lack of reactivity of arabino- and xylo-nucleosides also poses interesting structural, modeling, and origins of life issues. Results with clays that catalyze long oligomer formation only poorly reveal that they too catalyze these dimer syntheses, albeit less well than Volclay.®

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem–loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through ‘kissing’ loop–loop interactions. We also show that loop–loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop–loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.

Inhibition of 5′-UTR RNA Conformational Switching in HIV-1 Using Antisense PNAs

BackgroundThe genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5′-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5′-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization.Methods and ResultsWe evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes.ConclusionsOur results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.

Regulation of Human RNase-L by the miR-29 Family Reveals a Novel Oncogenic Role in Chronic Myelogenous Leukemia

The endoribonuclease RNase-L is the terminal component of an interferon-regulated RNA decay pathway known as the 2'-5'-oligoadenylate (2-5A) system, whose established functions include antimicrobial and tumor suppressive activities. RNase-L activity requires binding of the small molecule 2-5A, leading to RNase-L dimerization and cleavage of single-stranded RNA. RNase-L expression is controlled post-transcriptionally by its 3'-untranslated region (3' UTR), which exerts a strong negative effect on RNase-L levels. MicroRNAs (miRNAs) are a class of small noncoding RNAs that repress expression of target genes by binding to regions of complementarity often in the 3' UTR. The miR-29 family acts as a tumor suppressor in several cancers, including acute and chronic myelogenous leukemia (CML), and has many oncogenic targets. We report that the miR-29 family represses RNase-L protein expression across several cell types. Using a luciferase reporter, we showed that miR-29 acts via 4 target sites within the RNASEL 3' UTR. Mutation of all sites is required for abrogation of miR-29 repression. In light of the reported tumor suppressive role of miR-29 in K562 CML cells and miR-29 repression of RNase-L in these cells, we generated K562 cells with stable RNase-L knockdown and demonstrated that loss of RNase-L inhibits proliferation in vitro as well as tumor growth in a xenograft model. Our findings identify a previously unknown miRNA regulator of RNase-L expression and support a novel oncogenic role for RNase-L in CML and potentially other hematopoietic malignancies.

Activation of PKR by RNA misfolding: HDV ribozyme dimers activate PKR

Protein Kinase R (PKR), the double-stranded RNA (dsRNA)-activated protein kinase, plays important roles in innate immunity. Previous studies have shown that PKR is activated by long stretches of dsRNA, RNA pseudoknots, and certain single-stranded RNAs; however, regulation of PKR by RNAs with globular tertiary structure has not been reported. In this study, the HDV ribozyme is used as a model of a mostly globular RNA. In addition to a catalytic core, the ribozyme contains a peripheral 13-bp pairing region (P4), which, upon shortening, affects neither the catalytic activity of the ribozyme nor its ability to crystallize. We report that the HDV ribozyme sequence alone can activate PKR. To elucidate the RNA structural basis for this, we prepared a number of HDV variants, including those with shortened or lengthened P4 pairing regions, with the anticipation that lengthening the P4 extension would yield a more potent activator since it would offer more base pairs of dsRNA. Surprisingly, the variant with a shortened P4 was the most potent activator. Through native gel mobility and enzymatic structure mapping experiments we implicate misfolded HDV ribozyme dimers as the PKR-activating species, and show that the shortened P4 leads to enhanced occupancy of the RNA dimer. These observations have implications for how RNA misfolding relates to innate immune response and human disease.

Exploring Ty1 retrotransposon RNA structure within virus-like particles

Ty1, a long terminal repeat retrotransposon of Saccharomyces, is structurally and functionally related to retroviruses. However, a differentiating aspect between these retroelements is the diversity of the replication strategies used by long terminal repeat retrotransposons. To understand the structural organization of cis-acting elements present on Ty1 genomic RNA from the GAG region that control reverse transcription, we applied chemoenzymatic probing to RNA/tRNA complexes assembled in vitro and to the RNA in virus-like particles. By comparing different RNA states, our analyses provide a comprehensive structure of the primer-binding site, a novel pseudoknot adjacent to the primer-binding sites, three regions containing palindromic sequences that may be involved in RNA dimerization or packaging and candidate protein interaction sites. In addition, we determined the impact of a novel form of transposon control based on Ty1 antisense transcripts that associate with virus-like particles. Our results support the idea that antisense RNAs inhibit retrotransposition by targeting Ty1 protein function rather than annealing with the RNA genome.