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

Insights into the single-cell reproduction cycle of members of the family Bromoviridae: lessons from the use of protoplast systems.

Mieux comprendre le cycle de reproduction de cellules individuelles des membres de la famille des Bromoviridae : le&ccedil;ons tir&eacute;es de l&#39;utilisation de syst&egrave;mes de protoplastes.

Intuizioni nel ciclo di riproduzione a cella singola dei membri della famiglia Bromoviridae: lezioni dall&#39;uso dei sistemi del protoplasto.

RNA-RNA recombination salvages viral RNAs and contributes to their genomic variability. A recombinationally-active subgenomic promoter (sgp) has been mapped in Brome mosaic bromovirus (BMV) RNA3 (Wierzchoslawski et al., 2004. J. Virol.78, 8552-8864) and mRNA-like 5' sgRNA3a was characterized (Wierzchoslawski et al., 2006. J. Virol. 80, 12357-12366). In this paper we describe sgRNA3a-mediated recombination in both in vitro and in vivo experiments. BMV replicase-directed co-copying of (-) RNA3 with wt sgRNA3a generated RNA3 recombinants in vitro, but it failed to when 3'-truncated sgRNA3a was substituted, demonstrating a role for the 3' polyA tail. Barley protoplast co-transfections revealed that (i) wt sgRNA3a recombines at the 3' and the internal sites; (ii) 3'-truncated sgRNA3as recombine more upstream; and (iii) 5'-truncated sgRNA3 recombine at a low rate. In planta co-inoculations confirmed the RNA3-sgRNA3a crossovers. In summary, the non-replicating sgRNA3a recombines with replicating RNA3, most likely via primer extension and/or internal template switching.

Recombination of 5' subgenomic RNA3a with genomic RNA3 of Brome mosaic bromovirus in vitro and in vivo.

Recombinaison d&#39;ARN-ARN r&eacute;cup&egrave;re l&#39;ARN viral et contribue &agrave; leur variabilit&eacute; g&eacute;nomique. Un promoteur actif recombinationally subg&eacute;nomique (sgp) a &eacute;t&eacute; localis&eacute; en bromovirus de mosa&iuml;que de Brome (BMV) RNA3 (Wierzchoslawski et al., 2004. J. Virol.78, 8552-8864) et ARNm type 5&#39; sgRNA3a a &eacute;t&eacute; caract&eacute;ris&eacute;e (Wierzchoslawski et al., 2006 J. Virol. 80, 12357-12366). Dans cet article, nous d&eacute;crivons recombinaison m&eacute;di&eacute;e par les sgRNA3a dans des exp&eacute;riences in vitro et in vivo. BMV r&eacute;plicase-dirig&eacute; co-copying de (-) RNA3 avec sgRNA3a wt g&eacute;n&eacute;r&eacute; RNA3 recombinants in vitro, mais elle n&#39;a pas quand sgRNA3a 3-tronqu&eacute; a &eacute;t&eacute; substitu&eacute;, d&eacute;montrant un r&ocirc;le pour la queue polyA de 3&#39;. Co-transfections de protoplaste orge a r&eacute;v&eacute;l&eacute; que (i) sgRNA3a wt se recombine &agrave; la 3&#39; et les sites internes ; (ii) sgRNA3as 3-tronqu&eacute; se recombinent plus en amont ; et (iii) tronqu&eacute; 5&#39; sgRNA3 se recombiner &agrave; un taux faible. Planta co-inoculations a confirm&eacute; les filtres RNA3-sgRNA3a. En r&eacute;sum&eacute;, le sgRNA3a non r&eacute;pliqu&eacute;es se recombine avec r&eacute;pliquant RNA3, tr&egrave;s probablement par extension d&#39;amorce et/ou le changement de mod&egrave;le interne.

Recombinaison de 5&#39; subg&eacute;nomique RNA3a avec g&eacute;nomique RNA3 de Brome mosa&iuml;que bromovirus in vitro et in vivo.

RNA-RNA ricombinazione salva i RNAs virale e contribuisce alla loro variabilit&agrave; genomica. Subgenomic recombinationally-attivo promotore (sgp) &egrave; stato mappato in Brome mosaico bromovirus (BMV) RNA3 (Wierzchoslawski et al., 2004. J. Virol.78, 8552-8864) e mRNA-come 5&#39; sgRNA3a &egrave; stato caratterizzato (Wierzchoslawski et al,. 2006 J Virol. 80, 12357-12366). In questo documento descriviamo ricombinazione mediata da sgRNA3a in esperimenti sia in vitro che in vivo. BMV co-copying replicasi-regia di (-) RNA3 con wt sgRNA3a generate RNA3 ricombinanti in vitro, ma non &egrave; riuscito a quando &egrave; stato sostituito sgRNA3a 3&#39;-troncata, dimostrando un ruolo per la coda di polyA 3&#39;. Co-transfections del protoplasto orzo ha rivelato che (i) sgRNA3a wt ricombina a 3&#39; e i siti interni; (ii) sgRNA3as 3&#39;-troncato ricombinare pi&ugrave; a Monte; e (iii) sgRNA3 5&#39;-troncata ricombinare a bassa quota. Planta co-inoculations ha confermato il crossover RNA3-sgRNA3a. In sintesi, la sgRNA3a non replicare ricombina con replica RNA3, probabilmente tramite estensione dell&#39;iniettore e/o cambio di modelli interni.

Ricombinazione di 5&#39; subgenomic RNA3a con genomic RNA3 di Brome mosaico bromovirus in vitro e in vivo.

Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (-)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (-)-strand RNA which is used as template to make the SG RNA. The third mechanism uses discontinuous RNA synthesis while making the (-)-strand RNA templates. Most SG RNAs are translated into structural proteins or proteins related to pathogenesis: however other SG RNAs regulate the transition between translation and replication, function as riboregulators of replication or translation, or support RNA-RNA recombination. In this review we discuss these functions of SG RNAs and how they influence viral replication, translation and recombination.

Subgenomic messenger RNAs: mastering regulation of (+)-strand RNA virus life cycle.

Beaucoup (+)-strand RNA virus utilisent des ARN subg&eacute;nomique (SG) comme messagers pour l&#39;expression de la prot&eacute;ine, ou pour r&eacute;guler leur cycle de vie viral. Trois m&eacute;canismes diff&eacute;rents ont &eacute;t&eacute; d&eacute;crits pour la synth&egrave;se d&#39;ARN de SG. Le premier m&eacute;canisme implique une initiation interne sur un (-)-chapelet descripteur d&#39;ARN et n&eacute;cessite un promoteur SGP interne. Le second m&eacute;canisme fait un pr&eacute;matur&eacute;ment termin&eacute;s (-)-strand RNA qui sert de mod&egrave;le pour faire de l&#39;ARN de SG. Le troisi&egrave;me m&eacute;canisme utilise la synth&egrave;se d&#39;ARN discontinue tout en faisant le (-)-chapelet mod&egrave;les RNA. La plupart SG RNAs sont traduits en prot&eacute;ines structurales ou prot&eacute;ines li&eacute;es &agrave; la pathogen&egrave;se : mais autre RNAs SG r&eacute;glementer la transition entre la traduction et la r&eacute;plication, fonctionner comme riboregulators de r&eacute;plication ou de la traduction, ou soutenir la recombinaison d&#39;ARN-ARN. Dans cette revue, nous discutons ces fonctions de SG RNAs et leur influence sur la recombinaison, la traduction et la r&eacute;plication virale.

ARN messager subg&eacute;nomique : ma&icirc;triser le r&egrave;glement du (+)-cycle de vie des virus &agrave; ARN strand.

Molti (+)-strand RNA virus utilizzano RNAs subgenomic (SG) come messaggeri per l&#39;espressione della proteina, o di regolare il loro ciclo di vita virale. Tre diversi meccanismi sono stati descritti per la sintesi di RNA SG. Il primo meccanismo coinvolge iniziazione interna su un (-)-strand modello RNA e richiede un promotore interno del PSC. Il secondo meccanismo rende un prematuramente terminato (-)-strand RNA che viene utilizzato come modello per rendere il RNA SG. Il terzo meccanismo utilizza discontinuo sintesi di RNA, mentre facendo il (-)-modelli di RNA del filo. La maggior parte delle SG RNAs vengono tradotti in proteine strutturali o proteine correlate a patogenesi: tuttavia, altro RNAs SG regolano la transizione tra traduzione e replica, funzionare come riboregulators di replica o di traduzione, o sostenere ricombinazione RNA-RNA. In questa recensione discutiamo di queste funzioni di SG RNAs e come esse influenzano la ricombinazione, la traduzione e la replicazione virale.

Subgenomic Messaggero RNAs: mastering regolamento (+)-ciclo di vita di strand RNA virus.

RNA-RNA recombination is one of the strongest forces shaping the genomes of plant RNA viruses. The detection of recombination is a challenging task that prompted the development of both in vitro and in vivo experimental systems. In the divided genome of Brome mosaic virus system, both inter- and intrasegmental crossovers are described. Other systems utilize satellite or defective interfering RNAs (DI-RNAs) of Turnip crinkle virus, Tomato bushy stunt virus, Cucumber necrosis virus, and Potato virus X. These assays identified the mechanistic details of the recombination process, revealing the role of RNA structure and proteins in the replicase-mediated copy-choice mechanism. In copy choice, the polymerase and the nascent RNA chain from which it is synthesized switch from one RNA template to another. RNA recombination was found to mediate the rearrangement of viral genes, the repair of deleterious mutations, and the acquisition of nonself sequences influencing the phylogenetics of viral taxa. The evidence for recombination, not only between related viruses but also among distantly related viruses, and even with host RNAs, suggests that plant viruses unabashedly test recombination with any genetic material at hand.

RNA-RNA recombination in plant virus replication and evolution.

Recombinaison d&#39;ARN-ARN est une des forces plus forts, fa&ccedil;onner les g&eacute;nomes des plantes, des virus &agrave; ARN. La d&eacute;tection de recombinaison est une t&acirc;che difficile qui a incit&eacute; le d&eacute;veloppement de syst&egrave;mes exp&eacute;rimentaux in vitro et in vivo. Dans le g&eacute;nome divis&eacute; du syst&egrave;me de Brome mosaic virus, inter- et ectodermales v&eacute;hicules multisegments sont d&eacute;crites. Autres syst&egrave;mes utilisent des satellites ou des ARN interf&eacute;rents d&eacute;fectueuses (DI-ARN) de navet crinkle virus Tomato bushy stunt virus, virus de la n&eacute;crose concombre et virus X de la pomme de terre. Ces essais identifi&eacute;s les d&eacute;tails m&eacute;canistes du processus de recombinaison, r&eacute;v&eacute;lant le r&ocirc;le de structure d&#39;ARN et de prot&eacute;ines dans le m&eacute;canisme de copie-choix de m&eacute;diation r&eacute;plicase. Dans le choix de la copie, la polym&eacute;rase et la cha&icirc;ne de RNA naissante, d&#39;o&ugrave; elle est synth&eacute;tis&eacute;e &agrave; passent d&#39;un descripteur d&#39;ARN &agrave; l&#39;autre. Recombinaison de RNA a &eacute;t&eacute; trouv&eacute;e &agrave; la m&eacute;diation le r&eacute;arrangement des g&egrave;nes viraux, la r&eacute;paration des mutations d&eacute;l&eacute;t&egrave;res et l&#39;acquisition de s&eacute;quences de non-soi influant sur la phylog&eacute;nie des taxons virales. Les &eacute;l&eacute;ments de preuve pour la recombinaison, non seulement entre des virus apparent&eacute;s mais aussi chez les virus lointainement apparent&eacute;s et m&ecirc;me avec h&ocirc;te RNAs, sugg&egrave;re que les virus de plantes test sans vergogne recombinaison avec le mat&eacute;riel g&eacute;n&eacute;tique &agrave; port&eacute;e de main.

Recombinaison d&#39;ARN-ARN dans la r&eacute;plication du virus v&eacute;g&eacute;taux et &eacute;volution.

Ricombinazione di RNA-RNA &egrave; una delle forze pi&ugrave; forti virus a RNA che modella il genoma della pianta. La rilevazione di ricombinazione &egrave; un compito impegnativo che ha richiesto lo sviluppo di sistemi sperimentali sia in vitro che in vivo. Nel genoma diviso del sistema di Brome mosaic virus, sia inter- e crossover intrasegmental sono descritti. Altri sistemi utilizzano satellitare o difettosi RNAs d&#39;interferenza (DI-RNAs) di rapa crinkle virus, Tomato bushy stunt virus, virus di necrosi del cetriolo e Potato virus X. Questi dosaggi identificato i dettagli meccanicistici del processo di ricombinazione, rivelando il ruolo di struttura di RNA e proteine nel meccanismo di copia-scelta replicasi-mediata. Nella scelta di copia, la polimerasi e la catena di RNA nascente da cui viene sintetizzato passare da uno stampo di RNA a altro. Ricombinazione di RNA &egrave; stato trovato a mediare il riarrangiamento dei geni virali, la riparazione di mutazioni dannose e l&#39;acquisizione di sequenze nonself influenzando la filogenesi dei taxa virale. Le prove per ricombinazione, non solo tra i virus correlate, ma anche tra virus lontanamente correlati e anche con host RNAs, suggeriscono che la pianta virus test sfacciatamente ricombinazione con qualsiasi materiale genetico a portata di mano.

Ricombinazione di RNA-RNA nella replicazione del virus vegetali e l&#39;evoluzione.

RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3' mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.

Mutations in the antiviral RNAi defense pathway modify Brome mosaic virus RNA recombinant profiles.

M&eacute;canisme de l&#39;interf&eacute;rence (ARNi) RNA cible pour la d&eacute;gradation de l&#39;ARN viral. Pour v&eacute;rifier que les produits des g&egrave;nes RNAi a contribu&eacute; &agrave; la recombinaison de RNA virale, une s&eacute;rie de mutants de Arabidopsis thaliana RNAi-d&eacute;fectueux &eacute;taient infect&eacute;s par le virus de mosa&iuml;que de Brome (BMV) ARN qui ont &eacute;t&eacute; con&ccedil;us pour prendre en charge les filtres dans le segment RNA3. Croisement simple RNA3-RNA1, RNA3-RNA2 et RNA3-RNA3 recombinants accumulent en le type sauvage (wt) et toutes les lignes de knock-out &agrave; des fr&eacute;quences comparables. Toutefois, une accumulation r&eacute;duite de 3&#39; roman recombinants de mosa&iuml;que RNA3 observa en lignes ago1, dcl2, dcl4 et rdr6 mais pas en wt Col-0 ou la ligne dcl3. Un mutant de r&eacute;plicase BMV accumul&eacute; un faible niveau de RNA3-RNA1-croisement simple recombinants dans les usines de Col-0, tandis que, dans un dcl2 dcl4 double mutant, la formation de RNA3-RNA1 et la mosa&iuml;que recombinants &eacute;tait &agrave; un niveau bas. Une infection control chez le mutant cpr5-2, un h&ocirc;te plus susceptible de BMV Arabidopsis, g&eacute;n&eacute;r&eacute; des profils semblables-&agrave;-la Col-0 de recombinants single-Croix et de mosa&iuml;que, indiquant que les profils recombinantes &eacute;taient, dans une certaine mesure, ind&eacute;pendante d&#39;un taux de r&eacute;plication virale. Aussi, les exp&eacute;riences de croissance relative a r&eacute;v&eacute;l&eacute; semblable pression de s&eacute;lection pour des recombinants parmi les lignes de l&#39;h&ocirc;te. Ainsi, les profils modifi&eacute;s de RNA recombinants ont son origine au niveau de la formation recombinant plut&ocirc;t raison de modifi&eacute; la s&eacute;lection. En conclusion, la r&eacute;plicase virale et les produits des g&egrave;nes h&ocirc;tes RNAi contribuent de fa&ccedil;ons distinctes de recombinaison d&#39;ARN BMV. Nos &eacute;tudes montrent que les m&eacute;canismes de RNAi antiviraux sont utilis&eacute;s par des virus &agrave; ARN plante pour augmenter leur variabilit&eacute;, qui rappelle des ph&eacute;nom&egrave;nes d&eacute;j&agrave; d&eacute;montr&eacute; chez les champignons.

Des mutations dans la voie de d&eacute;fense antivirale RNAi modifier les profils recombinantes de Brome mosaic virus RNA.

Meccanismo di RNA interference (RNAi) obiettivi RNA virale per la degradazione. Per verificare se i prodotti del gene di RNAi ha contribuito alla ricombinazione di RNA virale, una serie di mutanti di Arabidopsis thaliana RNAi difettosi sono stati infettati con virus del mosaico del Brome (BMV) RNAs che hanno stato progettato per supportare i crossover all&#39;interno del segmento RNA3. Singolo-croce RNA3-RNA1, RNA3-RNA2 e RNA3-RNA3 ricombinanti accumulano in wild-type (wt) e tutte le linee di knock-out a frequenza paragonabile. Tuttavia, un ridotto accumulo di romanzo 3&#39; ricombinanti mosaico RNA3 &egrave; stato osservato in linee ago1, dcl2, dcl4 e rdr6, ma non in wt Col-0 o la linea di dcl3. Un mutante replicasi BMV accumulato un basso livello di RNA3-RNA1 single-croce ricombinanti in piante Col-0 mentre, in un dcl2 dcl4 doppio mutante, la formazione di ricombinanti RNA3-RNA1 sia mosaico era ad un livello basso. Un&#39;infezione di controllo nel mutante cpr5-2, un ospite pi&ugrave; suscettibile di Arabidopsis BMV, generato simili-a-Col-0 profili di ricombinanti sia single-Croce e mosaico, indicando che profili ricombinante sono state, in qualche misura, indipendente di un tasso di replicazione virale. Inoltre, gli esperimenti di crescita relativa rivelato simile pressione di selezione per i recombinants tra le linee di host. Cos&igrave;, i profili di RNA ricombinanti alterati hanno provenuto a livello di formazione ricombinante piuttosto che causa di alterato selezione. In conclusione, il replicasi virale e i prodotti del gene di RNAi host contribuiscono in modi distinti per ricombinazione BMV RNA. I nostri studi rivelano che i meccanismi antivirali RNAi sono utilizzati da virus RNA pianta per aumentare la loro variabilit&agrave;, che ricordano dei fenomeni in precedenza dimostrato nei funghi.

Mutazioni nella via della difesa antivirale di RNAi modificare profili ricombinante di Brome mosaic virus RNA.

Understanding the nuances of RNA structure as they pertain to biological function remains a formidable challenge for retrovirus research and development of RNA-based therapeutics, an area of particular importance with respect to combating HIV infection. Although a variety of chemical and enzymatic RNA probing techniques have been successfully employed for more than 30 years, they primarily interrogate small (100-500â€‰nt) RNAs that have been removed from their biological context, potentially eliminating long-range tertiary interactions (such as kissing loops and pseudoknots) that may play a critical regulatory role. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE), pioneered recently by Merino and colleagues, represents a facile, user-friendly technology capable of interrogating RNA structure with a single reagent and, combined with automated capillary electrophoresis, can analyze an entire 10,000-nucleotide RNA genome in a matter of weeks. Despite these obvious advantages, SHAPE essentially provides a nucleotide "connectivity map," conversion of which into a 3-D structure requires a variety of complementary approaches. This paper summarizes contributions from SHAPE towards our understanding of the structure of retroviral genomes, modifications to which technology that have been developed to address some of its limitations, and future challenges.

Probing Retroviral and Retrotransposon Genome Structures: The "SHAPE" of Things to Come.

Comprendre les nuances de structure d&#39;ARN se rapportant &agrave; la fonction biologique demeure un formidable d&eacute;fi de r&eacute;trovirus recherche et d&eacute;veloppement de la th&eacute;rapeutique &agrave; base d&#39;ARN, une r&eacute;gion d&#39;une importance particuli&egrave;re en ce qui concerne la lutte contre le VIH. Bien qu&#39;une vari&eacute;t&eacute; d&#39;ARN chimique et enzymatique, sondant les techniques ont &eacute;t&eacute; utilis&eacute;es avec succ&egrave;s depuis plus de 30 ans, ils interrogent principalement RNAs petit (100-500 nt) qui ont &eacute;t&eacute; retir&eacute;s de leur contexte biologique, potentiellement &eacute;liminer des interactions &agrave; longue port&eacute;e tertiaires (comme les baisers des boucles et pseudoknots) qui peuvent jouer un r&ocirc;le de r&eacute;gulateur critique. Hydroxyle acylation s&eacute;lective 2&#39; analys&eacute;e par extension d&#39;amorce (SHAPE), mis au point r&eacute;cemment par Merino et coll&egrave;gues, repr&eacute;sente une technologie facile, facile &agrave; utiliser capable d&#39;interroger la structure de l&#39;ARN avec un seul r&eacute;actif et, combin&eacute;e &agrave; l&#39;&eacute;lectrophor&egrave;se capillaire automatis&eacute;e, permet d&#39;analyser un ensemble g&eacute;nome d&#39;ARN de 10 000 nucl&eacute;otides en quelques semaines. Malgr&eacute; ces avantages &eacute;vidents, forme fournit essentiellement un nucl&eacute;otide &laquo; plan de connectivit&eacute; &raquo; conversion dont dans un 3D structure n&eacute;cessite une vari&eacute;t&eacute; d&#39;approches compl&eacute;mentaires. Cet article r&eacute;sume les contributions de forme &agrave; notre compr&eacute;hension de la structure des g&eacute;nomes r&eacute;trovirales, modifications pour que la technologie qui ont &eacute;t&eacute; d&eacute;velopp&eacute;es pour r&eacute;pondre &agrave; certaines de ses limites et les d&eacute;fis &agrave; venir.

Sondage r&eacute;trovirale et Structures r&eacute;trotransposon g&eacute;nome : la &laquo; forme &raquo; des choses &agrave; venir.

Comprendere le sfumature della struttura del RNA come si riferiscono alla funzione biologica rimane una formidabile sfida per retrovirus ricerca e sviluppo di terapie basate su RNA, un&#39;area di particolare importanza per quanto riguarda la lotta contro l&#39;infezione da HIV. Anche se una variet&agrave; di RNA chimica ed enzimatica, tecniche di sondaggio sono stati impiegati con successo per pi&ugrave; di 30 anni, sono principalmente interrogare RNAs piccolo (100-500 nt) che sono stati rimossi dal loro contesto biologico, potenzialmente eliminando le interazioni terziarie a lungo raggio (ad esempio baciando loop e pseudoknots) che possono svolgere un ruolo critico e normativo. Selettivo 2&#39; idrossile acilazione analizzato di estensione dell&#39;iniettore (SHAPE), introdotto recentemente da Merino e colleghi, rappresenta una tecnologia facile, facile da usare e in grado di interrogare la struttura RNA con un singolo reagente e, combinato con l&#39;elettroforesi capillare automatizzato, in grado di analizzare un intero genoma RNA nucleotide 10.000 in una questione di settimane. Nonostante questi vantaggi evidenti, forma essenzialmente fornisce un nucleotide &quot;mappa di connettivit&agrave;,&quot; conversione di cui in un 3D struttura richiede una variet&agrave; di approcci complementari. Questo libro riassume contributi dalla forma verso la comprensione della struttura del genoma retrovirale, modifiche a cui la tecnologia che sono state sviluppate per affrontare alcune delle sue limitazioni e sfide future.

Sondaggio retrovirale e strutture di retrotrasposoni genoma: la &quot;forma&quot; delle cose a venire.

We have previously described the efficient homologous recombination system between 5' subgenomic RNA3a (sgRNA3a) and genomic RNA3 of Brome mosaic virus (BMV) in barley protoplasts (Sztuba-SoliÅ„ska et al., 2011a). Here, we demonstrated that sequence alterations in the coat protein (CP)-binding cis-acting RNA motifs, the Bbox region (in the intercistronic RNA3 sequence) and the RNA3 packaging element (PE, in the movement protein ORF), reduced crossover frequencies in protoplasts. Additionally, the modification of Bbox-like element in the 5' UTR region strongly debilitated crossovers. Along the lines of these observations, RNA3 mutants not expressing CP or expressing mutated CPs also reduced recombination. A series of reciprocal transfections demonstrated a functional crosstalk between the Bbox and PE elements. Altogether, our data imply the role of CP in sgRNA3a-directed recombination by either facilitating the interaction of the RNA substrates and/or by creating roadblocks for the viral replicase.

Mutations in the coat protein-binding cis-acting RNA motifs debilitate RNA recombination of Brome mosaic virus.

Nous avons d&eacute;j&agrave; d&eacute;crit le syst&egrave;me efficace de recombinaison homologue entre 5&#39; subg&eacute;nomique RNA3a (sgRNA3a) et la g&eacute;nomique virus de la mosa&iuml;que RNA3 de Brome (BMV) dans des protoplastes d&#39;orge (Sztuba-Soli&#x144;ska et al., 2011 a). Ici, nous avons d&eacute;montr&eacute; cette s&eacute;quence des alt&eacute;rations dans la prot&eacute;ine de capside (CP)-liaison des motifs de RNA intramol&eacute;culaires, la r&eacute;gion de Bbox (dans l&#39;ordre de RNA3 intercistroniques) et l&#39;&eacute;l&eacute;ment d&#39;emballage RNA3 (PE, dans la prot&eacute;ine de mouvement ORF), r&eacute;duit les fr&eacute;quences de coupure dans les protoplastes. En outre, la modification d&#39;&eacute;l&eacute;ment Bbox-comme dans la r&eacute;gion 5&#39; UTR a fortement affaibli v&eacute;hicules multisegments. Dans le sens de ces observations, mutants RNA3 pas exprimer CP ou exprimant CPs mut&eacute; &eacute;galement r&eacute;duit de recombinaison. Une s&eacute;rie de transfections r&eacute;ciproques ont d&eacute;montr&eacute; une diaphonie fonctionnelle entre les &eacute;l&eacute;ments Bbox et PE. Au total, nos donn&eacute;es impliquent le r&ocirc;le du CP &agrave; la recombinaison sgRNA3a-r&eacute;alis&eacute; par soit faciliter l&#39;interaction des substrats RNA et/ou en cr&eacute;ant des barrages routiers pour la r&eacute;plicase virale.

Des mutations dans les motifs de RNA intramol&eacute;culaires manteau-la liaison aux prot&eacute;ines affaiblissent la recombinaison du virus de mosa&iuml;que de Brome de RNA.

In precedenza abbiamo descritto il sistema efficiente ricombinazione omologa tra 5&#39; subgenomic RNA3a (sgRNA3a) e genomiche virus del mosaico RNA3 di Brome (BMV) in protoplasti di orzo (Sztuba-Soli&#x144;ska et al., 2011). Qui, abbiamo dimostrato che sequenza di alterazioni della proteina del cappotto (CP)-associazione cis-acting motivi di RNA, la regione Bbox (nella sequenza RNA3 intercistronic) e l&#39;elemento di imballaggio RNA3 (PE) nella proteina movimento ORF, ridotto delle frequenze di crossover in protoplasti. Inoltre, la modifica della Bbox-come elemento della regione 5&#39; UTR fortemente debilitati crossover. Lungo le linee di queste osservazioni, mutanti RNA3 non esprimendo CP o esprimendo CPs mutato anche ridotta ricombinazione. Una serie di reciproci transfezioni ha dimostrato una diafonia funzionale tra gli elementi della Bbox e PE. Complessivamente, i nostri dati implicano il ruolo di CP in sgRNA3a-regia di ricombinazione facilitando l&#39;interazione dei substrati RNA e/o mediante la creazione di posti di blocco per il replicasi virale.

Mutazioni nei cappotto della proteina-cis-acting RNA motivi debilitano ricombinazione di RNA del virus del mosaico del bromo.

The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (-) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb(2+) ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5'-3' terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3' terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via 'flipping' mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.

Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome.

Le g&eacute;nome du virus (DENV) la Dengue contient plusieurs &eacute;l&eacute;ments d&#39;intramol&eacute;culaires requis pour la r&eacute;plication et la traduction. Des &eacute;tudes ant&eacute;rieures ont indiqu&eacute; qu&#39;un minigenome subg&eacute;nomique 719-nt (DENV-MINI) est un mod&egrave;le efficace pour la traduction et la synth&egrave;se du brin RNA (-) in vitro. Nous avons effectu&eacute; une analyse structurale d&eacute;taill&eacute;e d&#39;ARN DENV-MINI, combinant des techniques chimiques acylation, Pb(2+) induite par l&#39;ion hydrolyse et mutagen&egrave;se. Nos r&eacute;sultats mettent en &eacute;vidence prot&eacute;ine ind&eacute;pendant 5&#39;-3&#39; terminales interactions impliquant l&#39;hybridation entre des motifs intramol&eacute;culaires reconnues. Sonder les analyses identifi&eacute;s en tandem halt&egrave;re structures (DBs) au sein de l&#39;extr&eacute;mit&eacute; 3&#39; espac&eacute;es de r&eacute;gions monocat&eacute;naires, des boucles internes et des &eacute;pingles &agrave; cheveux avec des motifs de type GNRA incorpor&eacute;s. Analyse des motifs conserv&eacute;s et albums boucles (TLs) de ces halt&egrave;res et leurs interactions propos&eacute;es avec les r&eacute;gions en aval pseudon&oelig;ud (PK), pr&eacute;dit un pseudon&oelig;ud type H impliquant TL1 de la DB 5&#39; et dans la r&eacute;gion compl&eacute;mentaire, PK2. Comme perturbant l&#39;interaction TL1/PK2, par l&#39;interm&eacute;diaire de &laquo; retournement &raquo; des mutations de PK2, d&eacute;j&agrave; att&eacute;nu&eacute; r&eacute;plication DENV, ce pseudon&oelig;ud peut-&ecirc;tre participer &agrave; la r&eacute;gulation de la synth&egrave;se de RNA. Mod&eacute;lisation implicite que ce motif pourrait fonctionner comme &eacute;l&eacute;ment structurel et r&eacute;glementaire autonome. De plus, nos &eacute;tudes ciblant les &eacute;l&eacute;ments de la 3&#39; DB et sa r&eacute;gion compl&eacute;mentaire PK1 a indiqu&eacute; que la communication entre 5&#39;-3&#39; r&eacute;gions terminales d&eacute;pend fortement de composition structure et la s&eacute;quence de la r&eacute;gion de cyclisation de 5&#39;.

Complexit&eacute; de la structure du virus de la Dengue non traduite de r&eacute;gions : motifs de RNA intramol&eacute;culaires et interactions pseudon&oelig;ud modulant la fonctionnalit&eacute; du g&eacute;nome viral.

Il genoma del virus (DENV) Dengue contiene pi&ugrave; elementi cis-acting, necessari per la traduzione e la replica. Precedenti studi ha indicato che un minigenome subgenomic 719-nt (DENV-MINI) &egrave; un modello efficace per la traduzione e la sintesi di strand RNA (-) in vitro. Abbiamo effettuato una dettagliata analisi strutturale di RNA DENV-MINI, combinando tecniche chimiche acilazione, Pb(2+) indotta da ioni idrolisi e mutagenesi luogo-diretta. I nostri risultati evidenziano indipendente dalla proteina 5&#39;-3&#39; terminale interazioni che coinvolgono l&#39;ibridazione tra riconosciuti motivi cis-acting. Sondaggio analisi identificati tandem manubrio strutture (DBs) entro il 3&#39; capolinea distanziati da regioni a singolo filamento, loop interno e forcine con motivi simil-GNRA incorporati. Analisi dei motivi conservati e top loop (TLs) di questi manubri e proposti interazioni con le regioni a valle pseudoknot (PK), ha previsto un pseudoknot H-tipo che coinvolgono TL1 5&#39; DB e la regione complementare, PK2. Come interrompere l&#39;interazione TL1/PK2, via &#39;capovolgimento&#39; mutazioni di PK2, attenuato in precedenza replica DENV, questo pseudoknot possono partecipare nella regolazione della sintesi di RNA. La modellizzazione al computer implicita che questo motivo potrebbe funzionare come elemento strutturale/regolamentazione autonoma. Inoltre, i nostri studi targeting elementi di 3&#39; DB e la sua regione complementari PK1 indicarono che la comunicazione tra regioni 5&#39;-3&#39; terminale dipende fortemente dalla composizione di struttura e sequenza della regione 5&#39; ciclizzazione.

Complessit&agrave; strutturale del virus Dengue untranslated regioni: cis-acting RNA motivi e interazioni pseudoknot modulante funzionalit&agrave; del genoma 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.

Joanna Sztuba-Solinska

RT Biochemistry Section,
HIV Drug Resistance Program,
RT Biochemistry Section, HIV Drug Resistance Program

Frederick National Laboratory for Cancer Research

RNA Secondary Structure Prediction Using High-throughput SHAPE

Joanna Sztuba-Solinska

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}RT Biochemistry Section,HIV Drug Resistance Program,RT Biochemistry Section, HIV Drug Resistance Program

Frederick National Laboratory for Cancer Research

RNA Secondary Structure Prediction Using High-throughput SHAPE

RT Biochemistry Section,
HIV Drug Resistance Program,
RT Biochemistry Section, HIV Drug Resistance Program