Katarzyna J. Purzycka

Automated 3D structure composition for large RNAs

Understanding the numerous functions that RNAs play in living cells depends critically on knowledge of their three-dimensional structure. Due to the difficulties in experimentally assessing structures of large RNAs, there is currently great demand for new high-resolution structure prediction methods. We present the novel method for the fully automated prediction of RNA 3D structures from a user-defined secondary structure. The concept is founded on the machine translation system. The translation engine operates on the RNA FRABASE database tailored to the dictionary relating the RNA secondary structure and tertiary structure elements. The translation algorithm is very fast. Initial 3D structure is composed in a range of seconds on a single processor. The method assures the prediction of large RNA 3D structures of high quality. Our approach needs neither structural templates nor RNA sequence alignment, required for comparative methods. This enables the building of unresolved yet native and artificial RNA structures. The method is implemented in a publicly available, user-friendly server RNAComposer. It works in an interactive mode and a batch mode. The batch mode is designed for large-scale modelling and accepts atomic distance restraints. Presently, the server is set to build RNA structures of up to 500 residues.

RNA-Puzzles Round II: assessment of RNA structure prediction programs applied to three large RNA structures

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.

The RNA Transport Element of the Murine musD Retrotransposon Requires Long-range Intramolecular Interactions for Function

Retrovirus replication requires specialized transport mechanisms to export genomic mRNA from the nucleus to the cytoplasm of the infected cell. This regulation is mediated by a combination of viral and/or cellular factors that interact with cis-acting RNA export elements linking the viral RNA to the cellular CRM1 or NXF1 nuclear export pathways. Endogenous type D murine LTR retrotransposons (musD) were reported to contain an RNA export element located upstream of the 3′-LTR. Although functionally equivalent, the musD export element, termed the musD transport element, is distinct from the other retroviral RNA export elements, such as the constitutive transport element of simian/Mason-Pfizer monkey retroviruses and the RNA transport element found in rodent intracisternal A-particle LTR retrotransposons. We demonstrate here that the minimal RNA transport element (musD transport element) of musD comprises multiple secondary structure elements that presumably serve as recognition signals for the cellular export machinery. We identified two classes of tertiary interactions, namely kissing loops and a pseudoknot. This work constitutes the first example of an RNA transport element requiring such structural motifs to mediate nuclear export.

The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1–560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization.

Ty1 retrovirus-like element Gag contains overlapping restriction factor and nucleic acid chaperone functions

Ty1 Gag comprises the capsid of virus-like particles and provides nucleic acid chaperone (NAC) functions during retrotransposition in budding yeast. A subgenomic Ty1 mRNA encodes a truncated Gag protein (p22) that is cleaved by Ty1 protease to form p18. p22/p18 strongly inhibits transposition and can be considered an element-encoded restriction factor. Here, we show that only p22 and its short derivatives restrict Ty1 mobility whereas other regions of GAG inhibit mobility weakly if at all. Mutational analyses suggest that p22/p18 is synthesized from either of two closely spaced AUG codons. Interestingly, AUG1p18 and AUG2p18 proteins display different properties, even though both contain a region crucial for RNA binding and NAC activity. AUG1p18 shows highly reduced NAC activity but specific binding to Ty1 RNA, whereas AUG2p18 shows the converse behavior. p22/p18 affects RNA encapsidation and a mutant derivative defective for RNA binding inhibits the RNA chaperone activity of the C-terminal region (CTR) of Gag-p45. Moreover, affinity pulldowns show that p18 and the CTR interact. These results support the idea that one aspect of Ty1 restriction involves inhibition of Gag-p45 NAC functions by p22/p18-Gag interactions.

The HIV-2 Rev-response element: determining secondary structure and defining folding intermediates

Interaction between the viral protein Rev and the RNA motifs known as Rev response elements (RREs) is required for transport of unspliced and partially spliced human immunodeficiency virus (HIV)-1 and HIV-2 RNAs from the nucleus to the cytoplasm during the later stages of virus replication. A more detailed understanding of these nucleoprotein complexes and the host factors with which they interact should accelerate the development of new antiviral drugs targeting cis-acting RNA regulatory signals. In this communication, the secondary structures of the HIV-2 RRE and two RNA folding precursors have been identified using the SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing methodology together with a novel mathematical approach for determining the secondary structures of RNA conformers present in a mixture. A complementary chemical probing technique was also used to support these secondary structure models, to confirm that the RRE2 RNA undergoes a folding transition and to obtain information about the relative positioning of RRE2 substructures in three dimensions. Our analysis collectively suggests that the HIV-2 RRE undergoes two conformational transitions before assuming the energetically most favorable conformer. The 3D models for the HIV-2 RRE and folding intermediates are also presented, wherein the Rev-binding stem–loops (IIB and I) are located coaxially in the former, which is in agreement with previous models for HIV-1 Rev-RRE binding.

New, extended hairpin form of the TAR-2 RNA domain points to the structural polymorphism at the 5′ end of the HIV-2 leader RNA

The HIV-2 TAR RNA domain (TAR-2) plays a key role in the trans-activation of HIV-2 transcription as it is the target for the Tat-2 protein and several cell factors. Here, we show that the TAR-2 domain exists in vitro in two global, alternative forms: a new, extended hairpin form with two conformers and the already proposed branched hairpins form. This points strongly to the structural polymorphism of the 5′ end of the HIV-2 leader RNA. The evidence comes from the non-denaturing PAGE mobility assay, 2D structure prediction, enzymatic and Pb2+- or Mg2+-induced RNA cleavages. Existence of the TAR-2 extended form was further proved by the examination of engineered TAR-2 mutants stabilized either in the branched or extended structure. The TAR-2 extended form predominates with an increasing magnesium concentration. Gel retardation assays reveal that both TAR-2 wt and its mutant, unable to form branched structure, bind Tat-2 protein with comparable, high affinity, while RNA hairpins I and II, derived from TAR-2 branched structure model, show much less protein binding. We propose that an internal loop region of the TAR-2 extended hairpin form is a potential Tat-2 binding site.

A self-encoded capsid derivative restricts Ty1 retrotransposition in Saccharomyces

Retrotransposons and retroviral insertions have molded the genomes of many eukaryotes. Since retroelements transpose via an RNA intermediate, the additive nature of the replication cycle can result in massive increases in copy number if left unchecked. Host organisms have countered with several defense systems, including domestication of retroelement genes that now act as restriction factors to minimize propagation. We discovered a novel truncated form of the Saccharomyces Ty1 retrotransposon capsid protein, dubbed p22 that inhibits virus-like particle (VLP) assembly and function. The p22 restriction factor expands the repertoire of defense proteins targeting the capsid and highlights a novel host–parasite strategy. Instead of inhibiting all transposition by domesticating the restriction gene as a distinct locus, Ty1 and budding yeast may have coevolved a relationship that allows high levels of transposition when Ty1 copy numbers are low and progressively less transposition as copy numbers rise. Here, we offer a perspective on p22 restriction, including its mode of expression, effect on VLP functions, interactions with its target, properties as a nucleic acid chaperone, similarities to other restriction factors, and future directions.

Gammaretrovirus mRNA expression is mediated by a novel, bipartite post-transcriptional regulatory element

Post-transcriptional regulatory mechanisms of several complex and simple retroviruses and retroelements have been elucidated, with the exception of the gammaretrovirus family. We found that, similar to the other retroviruses, gag gene expression of MuLV and XMRV depends on post-transcriptional regulation mediated via an RNA sequence overlapping the pro-pol open reading frame, termed the Post-Transcriptional Element (PTE). PTE function can be replaced by heterologous RNA export elements, e.g. CTE of simian type D retroviruses. Alternatively, Gag particle production is achieved using an RNA/codon optimized gag gene. PTE function is transferable and can replace HIV Rev-RRE-regulated expression of HIV gag. Analysis of PTE by SHAPE revealed a highly structured RNA comprising seven stem-loop structures, with the 5′ and 3′ stem-loops forming an essential bipartite signal. MuLV and XMRV PTE share 98% identity and have highly similar RNA structures, with changes mostly located to single-stranded regions. PTE identification strongly suggests that all retroviruses and retroelements share common strategies of post-transcriptional gene regulation to produce Gag. Expression depends on complex RNA structures embedded within retroviral mRNA, in coding regions or the 3′ untranslated region. These specific structures serve as recognition signals for either cellular or viral proteins.

The matrix domain contributes to the nucleic acid chaperone activity of HIV-2 Gag.

BackgroundThe Gag polyprotein is a multifunctional regulator of retroviral replication and major structural component of immature virions. The nucleic acid chaperone (NAC) activity is considered necessary to retroviral Gag functions, but so far, NAC activity has only been confirmed for HIV-1 and RSV Gag polyproteins. The nucleocapsid (NC) domain of Gag is proposed to be crucial for interactions with nucleic acids and NAC activity. The major function of matrix (MA) domain is targeting and binding of Gag to the plasma membrane but MA can also interact with RNA and influence NAC activity of Gag. Here, we characterize RNA binding properties and NAC activity of HIV-2 MA and Gag, lacking p6 domain (GagΔp6) and discuss potential contribution of NC and MA domains to HIV-2 GagΔp6 functions and interactions with RNA.ResultsWe found that HIV-2 GagΔp6 is a robust nucleic acid chaperone. HIV-2 MA protein promotes nucleic acids aggregation and tRNALys3 annealing in vitro. The NAC activity of HIV-2 NC is affected by salt which is in contrast to HIV-2 GagΔp6 and MA. At a physiological NaCl concentration the tRNALys3 annealing activity of HIV-2 GagΔp6 or MA is higher than HIV-2 NC. The HIV-2 NC and GagΔp6 show strong binding to the packaging signal (Ψ) of HIV-2 RNA and preference for the purine-rich sequences, while MA protein binds mainly to G residues without favouring Ψ RNA. Moreover, HIV-2 GagΔp6 and NC promote HIV-2 RNA dimerization while our data do not support MA domain participation in this process in vitro.ConclusionsWe present that contrary to HIV-1 MA, HIV-2 MA displays NAC activity and we propose that MA domain may enhance the activity of HIV-2 GagΔp6. The role of the MA domain in the NAC activity of Gag may differ significantly between HIV-1 and HIV-2. The HIV-2 NC and MA interactions with RNA are not equivalent. Even though both NC and MA can facilitate tRNALys3 annealing, MA does not participate in RNA dimerization in vitro. Our data on HIV-2 indicate that the role of the MA domain in the NAC activity of Gag differs not only between, but also within, retroviral genera.