rna-secondary-structure-prediction-using-high-throughput-shape

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Frederick National Laboratory for Cancer Research

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.

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.

Le domaine de l&#39;ARN du VIH-2 goudron (TAR-2) joue un r&ocirc;le cl&eacute; dans la trans-activation de la transcription de l&#39;HIV-2 tel qu&#39;il est la cible de la prot&eacute;ine Tat-2 et plusieurs facteurs de cellules. Ici, nous montrons que le domaine de TAR-2 existe in vitro en deux mondiaux, d&#39;autres formes : une forme nouvelle, &eacute;largie en &eacute;pingle &agrave; cheveux avec deux conform&egrave;res et le proposait d&eacute;j&agrave; ramifi&eacute;s en forme d&#39;&eacute;pingles &agrave; cheveux. Cela souligne fortement le polymorphisme structural de l&#39;extr&eacute;mit&eacute; 5&#39; du VIH-2 leader RNA. La preuve vient de l&#39;analyse de mobilit&eacute; PAGE non d&eacute;naturants, pr&eacute;diction de structure 2D, enzymatique et Pb2 +- ou Mg2 +-induites par les clivages de la RNA. Existence du goudron-2 &eacute;tendu de forme a &eacute;t&eacute; prouv&eacute; plus loin en examinant les machin&eacute; TAR-2 mutants stabilis&eacute; soit dans la structure ramifi&eacute;e ou prolong&eacute;e. Le goudron-2 &eacute;tendu formulaire pr&eacute;domine avec une concentration croissante de magn&eacute;sium. Gel retard essais r&eacute;v&egrave;lent que les deux TAR-2 wt et son mutant, incapable de former une structure ramifi&eacute;e, lient la prot&eacute;ine Tat-2 avec une haute affinit&eacute; comparable, tandis que les piques de RNA I et II, d&eacute;riv&eacute; du mod&egrave;le de structure ramifi&eacute;e de TAR-2, montrent beaucoup moins la liaison aux prot&eacute;ines. Nous proposons qu&#39;une r&eacute;gion de la boucle interne de la forme d&#39;&eacute;pingle &agrave; cheveux de TAR-2 &eacute;tendu est un site de liaison de Tat-2 potentiel.

Forme nouvelle, &eacute;largie en &eacute;pingle &agrave; cheveux du domaine ARN TAR-2 pointe vers le polymorphisme structural &agrave; l&#39;extr&eacute;mit&eacute; 5&#39; du VIH-2 leader RNA.

Il dominio di RNA di HIV-2 TAR (catrame-2) svolge un ruolo chiave nella trans-attivazione della trascrizione di HIV-2 &egrave; la destinazione per la proteina Tat-2 e diversi fattori di cella. Qui, ci mostrano che il dominio di TAR-2 esiste in vitro in due forme alternative, globale: una forma nuova, estesa tornante con due conformeri e gi&agrave; proposto ramificato forma forcine. Ci&ograve; indica fortemente il polimorfismo strutturale dell&#39;estremit&agrave; 5&#39; del leader RNA HIV-2. La prova viene dal dosaggio non-denaturante mobilit&agrave; pagina, previsione della struttura 2D, enzimatica e Pb2 +- o Mg2 +-indotta da fenditure di RNA. Esistenza del TAR-2 esteso forma fu ulteriormente dimostrato dall&#39;esame di ingegneria TAR-2 mutanti stabilizzato sia nella struttura ramificata o estesa. Il TAR-2 esteso forma predomina con una crescente concentrazione di magnesio. Gel ritardo dosaggi rivelano che sia TAR-2 wt e suo mutante, incapaci di formare la struttura ramificata, legano la proteina Tat-2 con comparabili, ad alta affinit&agrave;, mentre forcine RNA I e II, derivato dal modello di struttura ramificata del TAR-2, mostrano molto meno legame proteico. Proponiamo che una regione ciclo interno nella forma di forcina TAR-2 esteso &egrave; un potenziale sito di legame di Tat-2.

Forma nuova, estesa tornante del dominio RNA TAR-2 punti per il polimorfismo strutturale all&#39;estremit&agrave; 5&#39; del leader RNA HIV-2.

Recently, it has been reported that HIV-1 TAR RNA element releases functionally competent miRNAs upon processing by Dicer enzyme. Here, we extend the analysis of miRNA viral-encoding potential to the TAR RNA of the HIV-2. Using in vitro Dicer cleavages and computer-aided analysis we have found that the 124-mer TAR RNA domain, present at the 5' end of HIV-2 mRNAs, putatively encodes pre-miRNAs. When deduced sequences of the viral-encoded miRNAs were matched against the database of human mRNA 3'-UTRs, it appeared that two miRNA candidates may target a large number of cellular transcripts.

The HIV-2 TAR RNA domain as a potential source of viral-encoded miRNA. A reconnaissance study.

R&eacute;cemment, on a signal&eacute; que l&#39;&eacute;l&eacute;ment d&#39;ARN TAR du VIH-1 lib&egrave;re miARN fonctionnellement comp&eacute;tent lors du traitement par l&#39;enzyme Dicer. Ici, nous &eacute;tendons l&#39;analyse du potentiel viral-encodage de miRNA &agrave; l&#39;ARN TAR du VIH-2. &Agrave; l&#39;aide des clivages de Dicer in vitro et analyse assist&eacute;e par ordinateur, nous avons trouv&eacute; que le domaine d&#39;ARN TAR 124-mer, pr&eacute;sent &agrave; l&#39;extr&eacute;mit&eacute; 5&#39; des ARNm du VIH-2, code putativement pr&eacute;-miARN. Lorsque les s&eacute;quences d&eacute;duites de la miARN viraux cod&eacute; ont &eacute;t&eacute; compar&eacute;s &agrave; la base de donn&eacute;es d&#39;ADN messag&egrave;re humaine 3&#39; UTR-, il est apparu que deux candidats de miRNA peuvent cibler un grand nombre de transcriptions cellulaires.

Le domaine d&#39;ARN du VIH-2 goudron comme une source potentielle de miRNA virales cod&eacute;es. Une &eacute;tude de la reconnaissance.

Recentemente, &egrave; stato riportato che l&#39;elemento di HIV-1 RNA TAR rilascia miRNAs funzionalmente competenti all&#39;elaborazione di enzima Dicer. Qui, estendiamo l&#39;analisi del potenziale virale codifica miRNA al TAR RNA del HIV-2. Utilizzando in vitro Dicer fenditure ed analisi computerizzata abbiamo trovato che il dominio di catrame RNA 124-mer, presente all&#39;estremit&agrave; 5&#39; del mRNA di HIV-2, codifica stercore pre-miRNAs. Quando dedotta sequenze dei miRNAs virale codifica sono stati confrontati con il database di umana mRNA 3&#39;-UTRs, sembrava che due candidati di miRNA possono indirizzare un gran numero di cellulare trascrizioni.

Il dominio di RNA di HIV-2 TAR come una potenziale fonte di miRNA virale codificati. Uno studio di ricognizione.

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 RNA transport element of the murine musD retrotransposon requires long-range intramolecular interactions for function.

R&eacute;plication des r&eacute;trovirus exige des m&eacute;canismes de transport sp&eacute;cialis&eacute;s pour exporter des ARNm g&eacute;nomique du noyau vers le cytoplasme de la cellule infect&eacute;e. Ce r&egrave;glement est m&eacute;di&eacute; par une combinaison de virale et/ou des facteurs cellulaires qui interagissent avec les ARN intramol&eacute;culaires exporter des &eacute;l&eacute;ments liant l&#39;ARN viral pour les voies d&#39;exportations nucl&eacute;aires CRM1 ou NXF1 cellulaires. Des r&eacute;trotransposons LTR murines endog&egrave;nes de type D (musD) ont &eacute;t&eacute; signal&eacute;s &agrave; contenir un &eacute;l&eacute;ment d&#39;exportation RNA situ&eacute; en amont de la 3&#39;-LTR. Bien que fonctionnellement &eacute;quivalente, l&#39;&eacute;l&eacute;ment d&#39;exportation musD, qualifi&eacute; de l&#39;&eacute;l&eacute;ment de transport musD, est distinct des autres r&eacute;trovirus RNA exportation &eacute;l&eacute;ments, tels que l&#39;&eacute;l&eacute;ment de transport constitutif des r&eacute;trovirus simian/Mason-Pfizer singe et l&#39;&eacute;l&eacute;ment de transport RNA dans r&eacute;trotransposons LTR intracisternale rongeurs A particules. Nous montrons ici que l&#39;&eacute;l&eacute;ment minimal du transport RNA (&eacute;l&eacute;ment de transport musD) de musD comprend plusieurs &eacute;l&eacute;ments de structure secondaire qui vraisemblablement servent de signaux de reconnaissance pour la machinerie cellulaire d&#39;exportation. Nous avons identifi&eacute; deux classes d&#39;interactions tertiaires, &agrave; savoir baiser boucles et un pseudon&oelig;ud. Cet ouvrage constitue le premier exemple d&#39;un &eacute;l&eacute;ment de transport RNA n&eacute;cessitant ces motifs structurels d&#39;arbitrer les exportations nucl&eacute;aires.

L&#39;&eacute;l&eacute;ment de transport RNA de la murine musD r&eacute;trotransposon exige des interactions intramol&eacute;culaires &agrave; longue port&eacute;e pour la fonction.

La replica di retrovirus richiede meccanismi di trasporto specializzato per esportare genomic mRNA dal nucleo al citoplasma della cellula infetta. Questo regolamento &egrave; mediata da una combinazione di virale e/o fattori cellulari che interagiscono con cis-acting RNA esportare elementi che collegano il RNA virale per le vie di esportazione nucleare CRM1 o NXF1 cellulare. Tipo endogeno D murini retrotrasposoni LTR (musD) sono stati segnalati per contenere un elemento di esportazione di RNA si trova a monte del 3&#39;-LTR. Anche se funzionalmente equivalente, l&#39;elemento di esportazione musD, definito l&#39;elemento di trasporto musD, &egrave; distinto da altri retrovirus RNA esportazione elementi, come l&#39;elemento costitutivo del trasporto dei retrovirus simian/Mason-Pfizer monkey e l&#39;elemento di trasporto RNA trovato nel roditore intracisternal A-particella retrotrasposoni LTR. Qui dimostriamo che il minimo RNA trasporto elemento (musD trasporto) di musD &egrave; composto da pi&ugrave; elementi di struttura secondaria che presumibilmente servono come segnale di riconoscimento per i macchinari di esportazione cellulare. Abbiamo identificato due classi di interazioni terziarie, vale a dire baciare loop e un pseudoknot. Quest&#39;opera costituisce il primo esempio di un elemento di trasporto di RNA che richiedono tali motivi strutturali di mediare esportazione nucleare.

L&#39;elemento di trasporto del RNA dei retrotrasposoni murino musD richiede interazioni intramolecolari a lungo raggio per funzione.

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.

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

Dim&eacute;risation RNA est une &eacute;tape essentielle dans le cycle de vie r&eacute;troviral. Dim&eacute;risation et signaux d&#39;encapsidation, &eacute;troitement li&eacute;es au VIH-2, sont situ&eacute;s dans la r&eacute;gion leader RNA. La prot&eacute;ine de motif et de la nucl&eacute;ocapside SL1 sont importantes pour les deux proc&eacute;d&eacute;s. Dans cette &eacute;tude, nous montrons la structure du VIH-2 leader RNA (+ 1-560) captur&eacute; comme un dim&egrave;re l&acirc;che. Les r&eacute;arrangements structuraux potentiels dans le leader RNA ont &eacute;t&eacute; &eacute;tudi&eacute;s. Sous la forme de dim&egrave;re l&acirc;che, le brin de chef RNA HIV-2 existe in vitro comme un repli global unique. Deux baisers boucle interfaces dans le dim&egrave;re l&acirc;che ont &eacute;t&eacute; identifi&eacute;es : SL1/SL1 et TAR/TAR. Pour ces r&eacute;sultats est prouv&eacute; par l&#39;ARN sonder &agrave; l&#39;aide de la forme, r&eacute;actifs chimiques, enzymes, non-d&eacute;naturisation PAGE essais de mobilit&eacute;, l&#39;hybridation des oligonucl&eacute;otides antisens et analyse d&#39;un mutant de RNA. Fois TAR et SL1 comme domaines isol&eacute;s sont li&eacute;s par une prot&eacute;ine recombinante de NCp8 avec une haute affinit&eacute;, contrairement &agrave; des &eacute;pingles &agrave; cheveux en aval du SL1. Pied-impression de la complexe SL1/NCp8 indique que le site de liaison majeur correspond &agrave; la tige sup&eacute;rieure SL1. Pris ensemble, ces donn&eacute;es sugg&egrave;rent un mod&egrave;le dans lequel &eacute;pingle TAR III, le segment du SL1 proximale &agrave; la boucle et les r&ocirc;les sp&eacute;cifiques de PAL s&eacute;quence palindromique jouer dans le d&eacute;clenchement de la dim&eacute;risation.

L&#39;in vitro en vrac structure dim&egrave;re et r&eacute;arrangements du VIH-2 leader RNA.

Dimerizzazione di RNA &egrave; un passo essenziale nel ciclo di vita retrovirale. Dimerizzazione e segnali encapsidation, strettamente legate all&#39;HIV-2, si trovano nella regione del capo RNA. La proteina di motivo e nucleocapside SL1 sono considerati importanti per entrambi i processi. In questo studio, si visualizza la struttura del leader RNA HIV-2 (+ 1-560) catturato come un dimero sciolto. Potenziale riarrangiamenti strutturali entro il leader RNA sono stati studiati. In forma di dimero sciolto, il filamento leader RNA HIV-2 esiste in vitro come una singola piega globale. Due baci ciclo interfacce all&#39;interno del dimero sciolto sono stati identificati: SL1/SL1 e catrame/TAR. Prove per questi risultati sono fornita da RNA sondando usando la forma, reagenti chimici, gli enzimi, non-denaturante pagina mobilit&agrave; dosaggi, ibridazione di oligonucleotidi antisenso e analisi di un mutante di RNA. CATRAME e SL1 come domini isolati sono vincolati da proteina ricombinante NCp8 con alta affinit&agrave;, contrariamente a quanto i tornanti a valle di SL1. Piede-stampa della SL1/NCp8 complesso indica che il sito di legame principali mappe per lo stelo superiore SL1. Presi insieme, questi dati suggeriscono un modello nel quale tornante TAR III, il segmento di SL1 prossimale per il ciclo e i ruoli specifici PAL palindromo sequenza giocare nell&#39;avvio di dimerizzazione.

In vitro allentato struttura del dimero e riarrangiamenti del leader RNA HIV-2.

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.

Automated 3D structure composition for large RNAs.

Comprendre les nombreuses fonctions que jouent les ARN dans les cellules vivantes d&eacute;pend essentiellement de la connaissance de leur structure tridimensionnelle. En raison de la difficult&eacute; d&#39;&eacute;valuer exp&eacute;rimentalement des structures d&#39;ARN grand, il est actuellement tr&egrave;s en demande pour de nouvelles m&eacute;thodes de pr&eacute;diction de structure &agrave; haute r&eacute;solution. Nous pr&eacute;sentons la nouvelle m&eacute;thode de pr&eacute;vision enti&egrave;rement automatis&eacute;e des structures 3D d&#39;ARN d&#39;une structure secondaire d&eacute;finie par l&#39;utilisateur. Le concept est fond&eacute; sur le syst&egrave;me de traduction automatique. Le moteur de traduction fonctionne sur la base de donn&eacute;es de RNA FRABASE adapt&eacute; au dictionnaire concernant la structure secondaire d&#39;ARN et les &eacute;l&eacute;ments de la structure tertiaire. L&#39;algorithme de traduction est tr&egrave;s rapide. Structure 3D initiale est compos&eacute;e dans une gamme de secondes sur un seul processeur. La m&eacute;thode assure la pr&eacute;diction des grandes structures d&#39;ARN 3D de haute qualit&eacute;. Notre approche a besoin de mod&egrave;les structurels, ni alignement de s&eacute;quences de RNA, requis pour les m&eacute;thodes comparatives. Cela permet la construction de structures d&#39;ARN non r&eacute;solus encore natives et artificiels. La m&eacute;thode est impl&eacute;ment&eacute;e dans un serveur accessible au public, facile &agrave; utiliser RNAComposer. Il fonctionne en mode interactif et le mode batch. Le mode batch est con&ccedil;u pour la mod&eacute;lisation &agrave; grande &eacute;chelle et accepte les contraintes de distance atomique. Actuellement, le serveur est configur&eacute; pour construire des structures d&#39;ARN de r&eacute;sidus jusqu&#39;&agrave; 500.

Automatis&eacute; de composition de la structure 3D pour grands RNAs.

Comprendere le numerose funzioni che RNAs giocare nelle cellule viventi dipende criticamente sulla conoscenza della loro struttura tridimensionale. A causa delle difficolt&agrave; nel valutare sperimentalmente strutture di grande RNAs, c&#39;&egrave; attualmente grande domanda di nuovi metodi di predizione di struttura ad alta risoluzione. Vi presentiamo il nuovo metodo per la previsione automatizzata di strutture 3D di RNA da una struttura secondaria definita dall&#39;utente. Il concetto &egrave; fondato sul sistema di traduzione automatica. Il motore di traduzione opera sul database di RNA FRABASE su misura per il dizionario riguardanti la struttura secondaria del RNA e gli elementi della struttura terziaria. L&#39;algoritmo di traduzione &egrave; molto velocizza. Struttura 3D iniziale &egrave; composta in una vasta gamma di secondi su un singolo processore. Il metodo assicura la previsione di grandi strutture RNA 3D di alta qualit&agrave;. Il nostro approccio ha bisogno di modelli strutturali n&eacute; allineamento di sequenza di RNA, necessaria per i metodi comparativi. Ci&ograve; consente la costruzione di strutture di RNA non risolte ancora native e artificiale. Il metodo viene implementato in un server pubblicamente disponibile, facile da usare RNAComposer. Funziona in modo interattivo e una modalit&agrave; batch. La modalit&agrave; batch &egrave; stata progettata per la modellazione su larga scala e accetta i vincoli di distanza atomica. Attualmente, il server &egrave; impostato per creare strutture di RNA di fino a 500 residui.

Automatizzato composizione struttura 3D per grandi RNAs.

Ty1, eine lange terminal wiederholen Retrotransposon von Saccharomyces, ist strukturell und funktionell mit Retroviren verwandt. Ein ausschlaggebenden Aspekt zwischen diesen Retroelements ist jedoch die Vielfalt der Replikationsstrategien von long terminal repeat Retrotransposons verwendet. Zum Verst&auml;ndnis der strukturellen Organisation der Cis wirkende Elemente auf Ty1 genomische RNA aus der GAG-Region vorhanden, die reverse Transkription Steuern angewendet wir Chemoenzymatic zu sondieren, um RNA/tRNA-komplexe, die in Vitro zusammengebaut und die RNA in Virus-&auml;hnliche Partikel. Durch den Vergleich verschiedener RNA-betreffen, bieten unsere Analysen eine umfassende Struktur die Z&uuml;ndkapsel-Bindungsstelle, eine neuartige Pseudoknoten angrenzend an die Grundierung-Bindungsstellen, drei Regionen mit palindromische Sequenzen, die in RNA Dimerisierung oder Verpackung und Kandidat Protein Interaktion Websites beteiligt sein k&ouml;nnen. Dar&uuml;ber hinaus haben wir die Auswirkungen der eine neue Form von Transposon-Steuerelement basierend auf Ty1 antisense Abschriften, die Virus-&auml;hnliche Partikel zuordnen festgestellt. Unsere Ergebnisse unterst&uuml;tzen die Idee, dass antisense RNAs Retrotransposition hemmen, indem Sie gezielt Ty1 Proteinfunktion, anstatt Sie zu gl&uuml;hen mit das RNA-Genom.

Erkunden Ty1 Retrotransposon RNA Struktur innerhalb virus&auml;hnliche Partikel.

Ty1&#xFF0C;&#x917F;&#x9152;&#xFF0C;&#x957F;&#x7EC8;&#x7AEF;&#x91CD;&#x590D;&#x5EA7;&#x5B50;&#x7ED3;&#x6784;&#x4E0A;&#x548C;&#x529F;&#x80FD;&#x4E0A;&#x76F8;&#x5173;&#x7684;&#x9006;&#x8F6C;&#x5F55;&#x75C5;&#x6BD2;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x8FD9;&#x4E9B; retroelements &#x4E4B;&#x95F4;&#x7684;&#x533A;&#x5206;&#x65B9;&#x9762;&#x662F;&#x4F7F;&#x7528;&#x957F;&#x7EC8;&#x7AEF;&#x91CD;&#x590D;&#x53CD;&#x8F6C;&#x5F55;&#x8F6C;&#x5EA7;&#x5B50;&#x7684;&#x590D;&#x5236;&#x7B56;&#x7565;&#x7684;&#x591A;&#x6837;&#x6027;&#x3002;&#x82E5;&#x8981;&#x4E86;&#x89E3;&#x72EC;&#x8054;&#x4F53;&#x4EE3;&#x7406; GAG &#x533A;&#x57DF;&#x4ECE;&#x672C; Ty1 &#x57FA;&#x56E0;&#x7EC4; rna &#x63A7;&#x5236;&#x5143;&#x7D20;&#x53CD;&#x5411;&#x8F6C;&#x5F55;&#x7684;&#x7ED3;&#x6784;&#x7EC4;&#x7EC7;&#xFF0C;&#x5E94;&#x7528;&#x5316;&#x5B66;&#x9176;&#x63A2;&#x8BA8;&#x5411; RNA/tRNA &#x590D;&#x5408;&#x7269;&#x4F53;&#x5916;&#x7EC4;&#x88C5;&#x548C; RNA &#x75C5;&#x6BD2;&#x6837;&#x9897;&#x7C92;&#x3002;&#x901A;&#x8FC7;&#x6BD4;&#x8F83;&#x4E0D;&#x540C;&#x7684; RNA &#x56FD;&#x5BB6;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x5206;&#x6790;&#x63D0;&#x4F9B;&#x5168;&#x9762;&#x7684;&#x7ED3;&#x6784;&#x7684;&#x5E95;&#x6F06;&#x7ED1;&#x5B9A;&#x7AD9;&#x70B9;&#x3001; &#x6BD7;&#x90BB;&#x5F15;&#x7269;&#x7ED3;&#x5408;&#x4F4D;&#x70B9;&#x7684;&#x65B0;&#x578B; pseudoknot&#x3001; &#x542B;&#x590D;&#x53D1;&#x6027;&#x5E8F;&#x5217; RNA &#x4E8C;&#x805A;&#x6216;&#x5305;&#x88C5;&#x548C;&#x5019;&#x9009;&#x86CB;&#x767D;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7AD9;&#x70B9;&#x4E2D;&#x53EF;&#x80FD;&#x6D89;&#x53CA;&#x7684;&#x4E09;&#x4E2A;&#x533A;&#x57DF;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x786E;&#x5B9A;&#x7684;&#x8F6C;&#x5EA7;&#x5B50;&#x63A7;&#x5236;&#x57FA;&#x4E8E;&#x5173;&#x8054;&#x75C5;&#x6BD2;&#x6837;&#x9897;&#x7C92;&#x7684; Ty1 &#x53CD;&#x4E49;&#x8A8A;&#x672C;&#x5C0F;&#x8BF4;&#x7684;&#x5F62;&#x5F0F;&#x7684;&#x5F71;&#x54CD;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x652F;&#x6301;&#x53CD;&#x4E49; Rna &#x6291;&#x5236; retrotransposition&#xFF0C;&#x901A;&#x8FC7;&#x9488;&#x5BF9; Ty1 &#x86CB;&#x767D;&#x8D28;&#x529F;&#x80FD;&#xFF0C;&#x800C;&#x4E0D;&#x662F;&#x9000;&#x706B;&#x7684; RNA &#x57FA;&#x56E0;&#x7EC4;&#x7684;&#x60F3;&#x6CD5;&#x3002;

&#x63A2;&#x7D22; Ty1 &#x53CD;&#x8F6C;&#x5F55;&#x8F6C;&#x5EA7;&#x5B50; RNA &#x7ED3;&#x6784;&#x5185;&#x75C5;&#x6BD2;&#x6837;&#x9897;&#x7C92;&#x3002;

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.

Exploring Ty1 retrotransposon RNA structure within virus-like particles.

Ty1, un long terminal r&eacute;trotransposon r&eacute;p&eacute;ter des Saccharomyces, est structurellement et fonctionnellement li&eacute;es aux r&eacute;trovirus. Cependant, un aspect de diff&eacute;renciation entre ces r&eacute;tro&eacute;l&eacute;ments est la diversit&eacute; des strat&eacute;gies de r&eacute;plication utilis&eacute; par les r&eacute;trotransposons r&eacute;p&eacute;ter long terminales. Pour comprendre l&#39;organisation structurale des intramol&eacute;culaires &eacute;l&eacute;ments pr&eacute;sents sur Ty1 ARN g&eacute;nomique de la r&eacute;gion GAG qui contr&ocirc;lent la transcriptase inverse, nous avons appliqu&eacute; chimioenzymatique sonder de RNA/ARNt complexes assembl&eacute;s in vitro et de l&#39;ARN dans les Pseudo-particules virales. En comparant les diff&eacute;rents &Eacute;tats de RNA, nos analyses fournissent une structure globale de l&#39;appr&ecirc;t-accepteur, un roman pseudon&oelig;ud adjacent aux sites Appr&ecirc;t liant, trois r&eacute;gions contenant des s&eacute;quences palindromiques qui peuvent &ecirc;tre impliqu&eacute;s dans les sites d&#39;interaction prot&eacute;ine dim&eacute;risation ou emballage et candidat RNA. En outre, nous avons d&eacute;termin&eacute; l&#39;impact d&#39;une nouvelle forme de contr&ocirc;le de transposon issu des transcriptions Ty1 qui associent des Pseudo-particules virales. Nos r&eacute;sultats soutiennent l&#39;id&eacute;e que les ARN antisens inhibe r&eacute;trotransposition en ciblant la fonction des prot&eacute;ines Ty1 plut&ocirc;t que de recuit avec l&#39;ARN g&eacute;nomique.

Explorer la structure d&#39;ARN de r&eacute;trotransposons Ty1 dans Pseudo-particules virales.

Ty1, un lungo terminale retrotrasposone ripetizione di Saccharomyces, &egrave; strutturalmente e funzionalmente correlati a retrovirus. Tuttavia, un aspetto di differenziazione tra queste retroelements &egrave; la diversit&agrave; delle strategie replica utilizzato da retrotrasposoni ripetizione terminale lunga. Per comprendere l&#39;organizzazione strutturale degli elementi cis-acting presenti sul RNA genomico Ty1 dalla regione di GAG che controllano la trascrizione inversa, abbiamo applicato chemoenzymatic sondaggio complessi RNA/tRNA assemblati in vitro e il RNA in particelle virus-simili. Confrontando i diversi stati di RNA, le nostre analisi forniscono una struttura completa del sito di legame dell&#39;iniettore, un romanzo pseudoknot adiacente per i siti di legame dell&#39;iniettore, tre regioni contenenti sequenze palindromi che possono essere coinvolti in RNA candidati o imballaggio e dimerizzazione della proteina interazione siti. Inoltre, abbiamo determinato l&#39;impatto di una nuova forma di controllo trasposone basato sulle trascrizioni Ty1 che associano con particelle virus-simili. I nostri risultati supportano l&#39;idea che RNA antisenso inibisce la retrotrasposizione di targeting Ty1 della funzione delle proteine piuttosto che di ricottura con il genoma a RNA.

Esplorare la struttura di RNA di retrotrasposoni Ty1 all&#39;interno particelle virus-simili.

&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1&#x3001;&#x9577;&#x3044;&#x30BF;&#x30FC;&#x30DF;&#x30CA;&#x30EB; &#x30EA;&#x30D4;&#x30FC;&#x30C8; &#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3;&#x306E;&#x51FA;&#x82BD;&#x9175;&#x6BCD;&#x3001;&#x69CB;&#x9020;&#x7684;&#x3001;&#x6A5F;&#x80FD;&#x7684;&#x30EC;&#x30C8;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306B;&#x95A2;&#x9023;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E; retroelements &#x306E;&#x9593;&#x306E;&#x5DEE;&#x5225;&#x5316;&#x5074;&#x9762;&#x9577;&#x3044;&#x30BF;&#x30FC;&#x30DF;&#x30CA;&#x30EB;&#x306E;&#x30EA;&#x30D4;&#x30FC;&#x30C8; &#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3;&#x306B;&#x3088;&#x3063;&#x3066;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x308B;&#x30EC;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x6226;&#x7565;&#x306E;&#x591A;&#x69D8;&#x6027;&#x3067;&#x3059;&#x3002;&#x9006;&#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30AF;&#x30EA;&#x30D7;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B; cis &#x6F14;&#x6280;&#x8981;&#x7D20;&#x30AE;&#x30E3;&#x30B0;&#x9818;&#x57DF;&#x304B;&#x3089;&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1 &#x30B2;&#x30CE;&#x30E0; RNA &#x306B;&#x5B58;&#x5728;&#x306E;&#x69CB;&#x9020;&#x7D44;&#x7E54;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x5316;&#x5B66;&#x3092; in vitro &#x3067;&#x7D44;&#x307F;&#x7ACB;&#x3066; RNA/tRNA &#x8907;&#x5408;&#x4F53;&#x304A;&#x3088;&#x3073; RNA &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x69D8;&#x7C92;&#x5B50;&#x306E;&#x30D7;&#x30ED;&#x30FC;&#x30D3;&#x30F3;&#x30B0;&#x3092;&#x9069;&#x7528;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;RNA &#x306E;&#x7570;&#x306A;&#x308B;&#x72B6;&#x614B;&#x3092;&#x6BD4;&#x8F03;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x79C1;&#x305F;&#x3061;&#x306E;&#x5206;&#x6790;&#x306F;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30FC;&#x7D50;&#x5408;&#x30B5;&#x30A4;&#x30C8;&#x3001;&#x5C0F;&#x8AAC;&#x30B7;&#x30E5;&#x30FC;&#x30C9; &#x30CE;&#x30C3;&#x30C8;&#x306E;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30FC;&#x7D50;&#x5408;&#x90E8;&#x4F4D;&#x306B;&#x96A3;&#x63A5;&#x3059;&#x308B;&#x3001;RNA &#x306E;&#x30C0;&#x30A4;&#x30DE;&#x30FC;&#x5316;&#x307E;&#x305F;&#x306F;&#x5305;&#x88C5;&#x3068;&#x5019;&#x88DC;&#x8005;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x30B5;&#x30A4;&#x30C8;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x56DE;&#x6587;&#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x542B;&#x3080; 3 &#x3064;&#x306E;&#x5730;&#x57DF;&#x306E;&#x5305;&#x62EC;&#x7684;&#x306A;&#x69CB;&#x9020;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x6211;&#x3005; &#x306F;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x69D8;&#x7C92;&#x5B50;&#x3068;&#x3092;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x308B;&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1 &#x30A2;&#x30F3;&#x30C1;&#x30BB;&#x30F3;&#x30B9;&#x8EE2;&#x5199;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; &#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x306E;&#x65B0;&#x3057;&#x3044;&#x5F62;&#x614B;&#x306E;&#x5F71;&#x97FF;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30A2;&#x30F3;&#x30C1;&#x30BB;&#x30F3;&#x30B9; Rna l1 &#x306E;&#x30EC;&#x30C8;&#x30ED;&#x8EE2;&#x4F4D;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B; RNA &#x30B2;&#x30CE;&#x30E0;&#x3092;&#x30A2;&#x30CB;&#x30FC;&#x30EB;&#x3059;&#x308B;&#x306E;&#x3067;&#x306F;&#x306A;&#x304F;&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1 &#x86CB;&#x767D;&#x8CEA;&#x6A5F;&#x80FD;&#x306E;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x306B;&#x3088;&#x3063;&#x3066;&#x8003;&#x3048;&#x3092;&#x652F;&#x6301;&#x3059;&#x308B;&#x6211;&#x3005; &#x306E;&#x7D50;&#x679C;&#x3002;

&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1 &#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; RNA &#x69CB;&#x9020;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x69D8;&#x7C92;&#x5B50;&#x5185;&#x3092;&#x63A2;&#x7D22;&#x3057;&#x307E;&#x3059;&#x3002;

Ty1, Saccharomyces&#xC758; &#xAE34; &#xD130;&#xBBF8;&#xB110; &#xBC18;&#xBCF5; retrotransposon&#xB294; &#xAD6C;&#xC870;&#xC801; &#xBC0F; &#xAE30;&#xB2A5;&#xC801;&#xC73C;&#xB85C; &#xAD00;&#xB828;&#xC774; retroviruses. &#xADF8;&#xB7EC;&#xB098;, &#xC774;&#xB7EC;&#xD55C; retroelements &#xC0AC;&#xC774;&#xC758; &#xCC28;&#xBCC4;&#xD654; &#xCE21;&#xBA74; &#xC624;&#xB798; &#xD130;&#xBBF8;&#xB110; &#xBC18;&#xBCF5; retrotransposons&#xC5D0;&#xC11C; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xBCF5;&#xC81C; &#xC804;&#xB7B5;&#xC758; &#xB2E4;&#xC591;&#xC131; &#xC774;&#xB2E4;. &#xBC18;&#xC804; &#xB179;&#xC74C; &#xBC29;&#xC1A1; &#xC81C;&#xC5B4; &#xD558;&#xB294; cis &#xC5F0;&#xAE30; &#xC694;&#xC18C; Ty1 &#xAC8C;&#xB188; RNA&#xC5D0; &#xC788;&#xB294; &#xAC1C; &#xADF8; &#xC9C0;&#xC5ED;&#xC5D0;&#xC11C;&#xC758; &#xAD6C;&#xC870; &#xC870;&#xC9C1; &#xC774;&#xD574; &#xD558;&#xAE30; &#xC6B0;&#xB9AC; chemoenzymatic &#xD504;&#xB85C; &#xBE59; &#xCCB4; &#xC678;&#xC5D0;&#xC11C; &#xC870;&#xB9BD; &#xD558;&#xB294; RNA/tRNA &#xB2E8;&#xC9C0; &#xBC0F; RNA &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xAC19;&#xC740; &#xC785;&#xC790;&#xC5D0; &#xC801;&#xC6A9;. &#xB2E4;&#xB978; RNA &#xC0C1;&#xD0DC;&#xB97C; &#xBE44;&#xAD50; &#xD558; &#xC5EC; &#xC6B0;&#xB9AC;&#xC758; &#xBD84;&#xC11D; &#xB1CC;&#xAD00; &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8;, &#xC18C;&#xC124; pseudoknot &#xB1CC;&#xAD00; &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0; &#xC778;&#xC811; &#xD55C;, RNA dimerization &#xB610;&#xB294; &#xD3EC;&#xC7A5; &#xBC0F; &#xD6C4;&#xBCF4; &#xB2E8;&#xBC31;&#xC9C8; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0; &#xCC38;&#xC5EC; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xB41C; &#xC2DC;&#xD000;&#xC2A4;&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xC138; &#xC601;&#xC5ED;&#xC774;&#xC758; &#xD3EC;&#xAD04;&#xC801;&#xC778; &#xAD6C;&#xC870;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC; &#xC18C;&#xC124; &#xD615;&#xD0DC;&#xC758; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xAC19;&#xC740; &#xC785;&#xC790;&#xC5D0; &#xC5F0;&#xACB0; Ty1 &#xC13C;&#xC2A4; &#xC131;&#xC801;&#xD45C;&#xC5D0; &#xB530;&#xB77C; transposon &#xCEE8;&#xD2B8;&#xB864;&#xC758; &#xC601;&#xD5A5;&#xC744; &#xD655;&#xC778; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xC13C;&#xC2A4; RNAs RNA &#xAC8C;&#xB188;&#xACFC; &#xC5B4; &#xB2D0; &#xB9C1; &#xBCF4;&#xB2E4;&#xB294; Ty1 &#xB2E8;&#xBC31;&#xC9C8; &#xAE30;&#xB2A5;&#xC744; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xD558; &#xC5EC; retrotransposition&#xC744; &#xC5B5;&#xC81C; &#xD558;&#xB294; &#xC544;&#xC774;&#xB514;&#xC5B4;&#xB97C; &#xC9C0;&#xC6D0; &#xD569;&#xB2C8;&#xB2E4;.

&#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xAC19;&#xC740; &#xC785;&#xC790; &#xB0B4;&#xC5D0;&#xC11C; Ty1 retrotransposon RNA &#xAD6C;&#xC870;&#xB97C; &#xD0D0;&#xC0C9;&#xD569;&#xB2C8;&#xB2E4;.

Ty1, un retrotransposon repetici&oacute;n terminal larga de Saccharomyces, estructural y funcionalmente relaciona retrovirus. Sin embargo, un aspecto diferenciador entre estos retroelementos es la diversidad de las estrategias de replicaci&oacute;n utilizadas por retrotransposones repetici&oacute;n terminal larga. Para entender la organizaci&oacute;n estructural de elementos de acci&oacute;n cis presentes en Ty1 RNA gen&oacute;mico de la regi&oacute;n GAG que controlan la transcripci&oacute;n inversa, aplicamos chemoenzymatic sondeo complejos RNA/tRNA montado en vitro y del ARN en part&iacute;culas similares a los virus. Mediante la comparaci&oacute;n de diferentes Estados de RNA, nuestros an&aacute;lisis proporcionan una estructura completa del sitio de cartilla-Uni&oacute;n, una novela pseudoknot adyacente a los sitios de uni&oacute;n de la cartilla, tres regiones con secuencias palindr&oacute;mico que pueden estar involucradas en los sitios de interacci&oacute;n prote&iacute;na dimerizaci&oacute;n o empaquetado y candidato de RNA. Adem&aacute;s, se determin&oacute; el impacto de una forma nueva de transposones control basado en transcripciones antisentidas Ty1 que se asocian con part&iacute;culas similares a los virus. Nuestros resultados apoyan la idea que ARN antisentido inhibe retrotransposition dirigido a la funci&oacute;n de la prote&iacute;na Ty1 en lugar de recocido con el genoma de ARN.

Explorar la estructura de RNA de retrotransposon Ty1 dentro de part&iacute;culas similares a los virus.

Ty1 retrotransposon RNA has the potential to fold into a variety of distinct structures, mutation of which affects retrotransposition frequencies. We show here that one potential functional structure is located at the 5' end of the genome and can assume a pseudoknot conformation. Chemoenzymatic probing of wild-type and mutant mini-Ty1 RNAs supports the existence of such a structure, while molecular genetic analyses show that mutations disrupting pseudoknot formation interfere with retrotransposition, indicating that it provides a critical biological function. These defects are enhanced at higher temperatures. When these mutants are combined with compensatory changes, retrotransposition is restored, consistent with pseudoknot architecture. Analyses of mutants suggest a defect in Ty1 reverse transcription. Collectively, our data allow modeling of a three-dimensional structure for this novel critical cis-acting signal of the Ty1 genome.

Retrotransposon Ty1 RNA contains a 5'-terminal long-range pseudoknot required for efficient reverse transcription.

Ty1 r&eacute;trotransposon que RNA a le potentiel pour plier dans une vari&eacute;t&eacute; de structures distinctes, mutation qui affecte la r&eacute;trotransposition fr&eacute;quences. Nous montrons ici un potentiel fonctionnel structure se trouve &agrave; l&#39;extr&eacute;mit&eacute; 5&#39; du g&eacute;nome et peut prendre une conformation pseudon&oelig;ud. Chimioenzymatique sondage d&#39;ARN sauvage et mutantes mini-Ty1 soutient l&#39;existence d&#39;une telle structure, tandis que des analyses g&eacute;n&eacute;tiques mol&eacute;culaires montrent que des mutations perturbant la formation pseudon&oelig;ud g&ecirc;neront r&eacute;trotransposition, indiquant qu&#39;elle fournit une fonction biologique critique. Ces d&eacute;fauts ont &eacute;t&eacute; am&eacute;lior&eacute;s &agrave; des temp&eacute;ratures plus &eacute;lev&eacute;es. Ces mutants sont conjugu&eacute;es &agrave; des changements compensatoires, r&eacute;trotransposition est compatible avec l&#39;architecture pseudon&oelig;ud, restaur&eacute;e. Analyses de mutants semblent indiquer un d&eacute;faut de transcription inverse Ty1. Collectivement, nos donn&eacute;es permettent la mod&eacute;lisation d&#39;une structure en trois dimensions pour ce nouveau signal d&#39;intramol&eacute;culaires critique du g&eacute;nome Ty1.

R&eacute;trotransposon Ty1 ARN contient un pseudon&oelig;ud &agrave; longue port&eacute;e de 5&#39;-terminal requis pour la transcription inverse efficace.

Retrotrasposoni Ty1 che RNA ha il potenziale per piegare in una variet&agrave; di strutture distinte, mutazione che interessa le frequenze retrotrasposizione. Mostriamo qui che un potenziale funzionale struttura si trova all&#39;estremit&agrave; 5&#39; del genoma e pu&ograve; assumere una conformazione pseudoknot. Chemoenzymatic sondaggio del mutante e del selvaggio-tipo mini-Ty1 RNAs sostiene l&#39;esistenza di tale struttura, mentre le analisi genetiche molecolari mostrano che mutazioni interrompendo la formazione pseudoknot interferiscano con la retrotrasposizione, indicando che esso fornisce una funzione biologica fondamentale. Questi difetti vengono esaltati a temperature pi&ugrave; elevate. Quando questi mutanti sono combinati con modifiche compensative, retrotrasposizione &egrave; ripristinato, coerente con l&#39;architettura pseudoknot. Analisi di mutanti suggeriscono un difetto di trascrizione d&#39;inversione Ty1. Collettivamente, i nostri dati consentono la modellazione di una struttura tridimensionale per questo romanzo critico cis-acting segnale del genoma Ty1.

Retrotrasposoni Ty1 RNA contiene un pseudoknot a lungo raggio 5&#39;-terminale necessaria per efficiente trascrizione d&#39;inversione.

During retroviral RNA encapsidation, two full-length genomic (g) RNAs are selectively incorporated into assembling virions. Packaging involves a cis-acting packaging element (Î¨) within the 5' untranslated region of unspliced HIV-1 RNA genome. However, the mechanism(s) that selects and limits gRNAs for packaging remains uncertain. Using a dual complementation system involving bipartite HIV-1 gRNA, we observed that gRNA packaging is additionally dependent on a cis-acting RNA element, the genomic RNA packaging enhancer (GRPE), found within the gag p1-p6 domain and overlapping the Gag-Pol ribosomal frameshift signal. Deleting or disrupting the two conserved GRPE stem loops diminished gRNA packaging and infectivity >50-fold, while deleting gag sequences between Î¨ and GRPE had no effect. Downregulating the translation termination factor eRF1 produces defective virus particles containing 20 times more gRNA. Thus, only the HIV-1 RNAs employed for Gag-Pol translation may be specifically selected for encapsidation, possibly explaining the limitation of two gRNAs per virion.

A cis-acting element in retroviral genomic RNA links Gag-Pol ribosomal frameshifting to selective viral RNA encapsidation.

Au cours de l&#39;encapsidation de RNA r&eacute;trovirale, deux longs g&eacute;nomique (g) RNAs est incorpor&eacute;s s&eacute;lectivement assemblage des virions. Emballage implique un &eacute;l&eacute;ment d&#39;emballage intramol&eacute;culaires (&Psi;) au sein de la r&eacute;gion 5&#39; non traduite du g&eacute;nome d&#39;ARN du VIH-1 unspliced. Toutefois, le m&eacute;canisme qui s&eacute;lectionne et limite gRNAs pour l&#39;emballage reste incertain. En utilisant un syst&egrave;me de double compl&eacute;mentation impliquant gRNA bipartite de VIH-1, nous avons observ&eacute; que gRNA emballage repose en outre sur un &eacute;l&eacute;ment de RNA intramol&eacute;culaires, la g&eacute;nomique enhancer emballage RNA (GRPE), trouv&eacute; au sein du domaine de p1-p6 gag et chevauchant le signal de changement de phase ribosomique Gag-Pol. Suppression ou perturber les deux conserv&eacute;s GRPE tige boucles diminu&eacute;s gRNA emballage et infectiosit&eacute; &gt; 50 fois, tandis que la suppression de s&eacute;quences de gag entre &Psi; et GRPE a eu aucun effet. Downregulating la traduction r&eacute;siliation facteur eRF1 produit des particules de virus d&eacute;fectif contenant gRNA 20 fois plus. Ainsi, seulement l&#39;ARN VIH-1 employ&eacute;e pour la traduction de Gag-Pol peuvent &ecirc;tre sp&eacute;cifiquement choisis pour encapsidation, expliquant &eacute;ventuellement la limitation des deux gRNAs par virion.

Un &eacute;l&eacute;ment intramol&eacute;culaires dans l&#39;ARN g&eacute;nomique retroviral relie Gag-Pol ribosomique d&eacute;phasage s&eacute;lective encapsidation de RNA virale.

Durante encapsidation RNA retrovirale, due full-length genomico (g) RNAs selettivamente sono incorporati nel montaggio dei virioni. Imballaggio comporta un elemento cis-acting imballaggio (&Psi;) all&#39;interno della regione 5&#39; non tradotta del genoma di HIV-1 RNA unspliced. Tuttavia, i meccanismi comprendono che seleziona e limita la gRNAs per l&#39;imballaggio rimane incerto. Utilizzando un sistema dual complementazione coinvolgendo bipartito gRNA HIV-1, abbiamo osservato che gRNA imballaggi sono inoltre dipendente su un elemento del RNA di cis-acting, il genomic RNA imballaggio enhancer (GRPE), trovato all&#39;interno del dominio di p1-p6 gag e sovrapposizione del segnale ribosomiale frameshift Gag-Pol. L&#39;eliminazione o perturbare i due conservati GRPE stem loops diminuita gRNA imballaggio e infettivit&agrave; &amp;gt; 50-volte, mentre l&#39;eliminazione di sequenze gag tra &Psi; e GRPE ha avuto alcun effetto. Riducono la traduzione terminazione fattore eRF1 produce particelle di virus difettosi contenente gRNA 20 volte di pi&ugrave;. Cos&igrave;, solo il RNAs di HIV-1 impiegato per la traduzione di Gag-Pol possono essere specificatamente selezionato per encapsidation, possibilmente spiegando la limitazione di due gRNAs al virione.

Un elemento cis-acting retrovirali genomic RNA collegamenti Gag-Pol ribosomiale frameshifting a selettiva encapsidation RNA virale.

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.

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

Interaction entre la prot&eacute;ine virale Rev et les motifs de RNA appel&eacute;es &eacute;l&eacute;ments de r&eacute;ponse de Rev (RREs) est requise pour le transport d&#39;unspliced et partiellement &eacute;piss&eacute; virus d&#39;immunod&eacute;ficience humaine (VIH) -1 et VIH-2 ARN du noyau vers le cytoplasme pendant les derni&egrave;res phases de la r&eacute;plication du virus. Une compr&eacute;hension plus approfondie de ces complexes nucl&eacute;oprot&eacute;iques et les facteurs de l&#39;h&ocirc;te avec laquelle ils interagissent devrait acc&eacute;l&eacute;rer le d&eacute;veloppement de nouveaux m&eacute;dicaments antiviraux ciblant les signaux r&eacute;gulateurs de RNA intramol&eacute;culaires. Dans la pr&eacute;sente communication, les structures secondaires de la RRE VIH-2 et deux pr&eacute;curseurs de pliage de RNA ont &eacute;t&eacute; identifi&eacute;s &agrave; l&#39;aide de la substance chimique (acylation s&eacute;lective 2&#39;-hydroxyle analys&eacute; par extension d&#39;amorce) de forme m&eacute;thodologie ainsi qu&#39;une nouvelle approche math&eacute;matique permettant de d&eacute;terminer les structures secondaires des conform&egrave;res RNA pr&eacute;sents dans un m&eacute;lange de palpage. Un chimique compl&eacute;mentaire technique de palpage servait aussi pour soutenir ces mod&egrave;les de structure secondaire, pour confirmer que l&#39;ARN RRE2 subit une transition de pliage et d&#39;obtenir des informations sur le positionnement relatif des sous-structures RRE2 en trois dimensions. Collectivement, notre analyse sugg&egrave;re que le VIH-2 RRE subit deux transitions conformationnelles avant d&#39;assumer le conform&egrave;re &eacute;nerg&eacute;tiquement plus favorable. Les mod&egrave;les 3D pour le HIV-2 RRE et pliantes interm&eacute;diaires sont &eacute;galement pr&eacute;sent&eacute;es, dans lequel le Rev-liaison tige-boucle (IIB et moi) est situ&eacute;s coaxialement dans la premi&egrave;re, qui est en accord avec les mod&egrave;les pr&eacute;c&eacute;dents pour la liaison de HIV-1 Rev-RRE.

L&#39;&eacute;l&eacute;ment de HIV-2 Rev-r&eacute;ponse : d&eacute;termination d&#39;une structure secondaire et la d&eacute;finition de pliage interm&eacute;diaires.

Interazione tra la proteina virale Rev ed i motivi di RNA conosciuti come elementi di risposta Rev (RREs) &egrave; richiesto per il trasporto di unspliced e parzialmente impiombato human immunodeficiency virus (HIV) -1 e HIV-2 RNAs dal nucleo al citoplasma durante le successive fasi di replicazione del virus. Una comprensione pi&ugrave; dettagliata di questi complessi nucleoproteine e i fattori di host con cui interagiscono dovrebbe accelerare lo sviluppo di nuovi farmaci antivirali targeting cis-acting segnali di RNA regolatori. In questa comunicazione, sono state identificate le strutture secondarie di RRE l&#39;HIV-2 e due precursori pieghevoli RNA usando il prodotto chimico (selettivo 2&#39;-idrossile acilazione analizzato di estensione dell&#39;iniettore) forma metodologia insieme ad un nuovo approccio matematico per determinare le strutture secondarie di conformeri RNA presenti in una miscela di sondaggio. Un chimico complementare tecnica di sondaggio &egrave; stato utilizzato anche per sostenere questi modelli di struttura secondaria, per confermare che il RNA RRE2 subisce una transizione pieghevole e ottenere informazioni circa il posizionamento relativo di RRE2 sostruzioni in tre dimensioni. Collettivamente, la nostra analisi suggerisce che l&#39;HIV-2 RRE subisce due transizioni conformazionali prima di assumere il conformero energeticamente pi&ugrave; favorevole. I modelli 3D per l&#39;HIV-2 RRE e pieghevole intermedi sono anche presentati, in cui il Rev-associazione stem-loop (IIB e) si trovano coassialmente nel primo caso, che &egrave; in accordo con precedenti modelli per l&#39;associazione di HIV-1 Rev-RRE.

Article	Year
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. Nucleic acids research\| PubMed ID: 16738137	2006
The HIV-2 TAR RNA domain as a potential source of viral-encoded miRNA. A reconnaissance study. Nucleic acids symposium series (2004)\| PubMed ID: 18776478	2008
The RNA transport element of the murine musD retrotransposon requires long-range intramolecular interactions for function. The Journal of biological chemistry\| PubMed ID: 20978285	2010
The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA. Nucleic acids research\| PubMed ID: 21622659	2011
Automated 3D structure composition for large RNAs. Nucleic acids research\| PubMed ID: 22539264	2012
Exploring Ty1 retrotransposon RNA structure within virus-like particles. Nucleic acids research\| PubMed ID: 23093595	2012
Retrotransposon Ty1 RNA contains a 5'-terminal long-range pseudoknot required for efficient reverse transcription. RNA (New York, N.Y.)\| PubMed ID: 23329695	2013
A cis-acting element in retroviral genomic RNA links Gag-Pol ribosomal frameshifting to selective viral RNA encapsidation. Cell host & microbe\| PubMed ID: 23414758	2013
The HIV-2 Rev-response element: determining secondary structure and defining folding intermediates. Nucleic acids research\| PubMed ID: 23640333	2013

Katarzyna J. Purzycka

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