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Sample records for em rna para

  1. RNA editing of the Drosophila para Na(+) channel transcript. Evolutionary conservation and developmental regulation.

    PubMed Central

    Hanrahan, C J; Palladino, M J; Ganetzky, B; Reenan, R A

    2000-01-01

    Post-transcriptional editing of pre-mRNAs through the action of dsRNA adenosine deaminases results in the modification of particular adenosine (A) residues to inosine (I), which can alter the coding potential of the modified transcripts. We describe here three sites in the para transcript, which encodes the major voltage-activated Na(+) channel polypeptide in Drosophila, where RNA editing occurs. The occurrence of RNA editing at the three sites was found to be developmentally regulated. Editing at two of these sites was also conserved across species between the D. melanogaster and D. virilis. In each case, a highly conserved region was found in the intron downstream of the editing site and this region was shown to be complementary to the region of the exonic editing site. Thus, editing at these sites would appear to involve a mechanism whereby the edited exon forms a base-paired secondary structure with the distant conserved noncoding sequences located in adjacent downstream introns, similar to the mechanism shown for A-to-I RNA editing of mammalian glutamate receptor subunits (GluRs). For the third site, neither RNA editing nor the predicted RNA secondary structures were evolutionarily conserved. Transcripts from transgenic Drosophila expressing a minimal editing site construct for this site were shown to faithfully undergo RNA editing. These results demonstrate that Na(+) channel diversity in Drosophila is increased by RNA editing via a mechanism analogous to that described for transcripts encoding mammalian GluRs. PMID:10880477

  2. Astronomia para/com crianças carentes em Limeira

    NASA Astrophysics Data System (ADS)

    Bretones, P. S.; Oliveira, V. C.

    2003-08-01

    Em 2001, o Instituto Superior de Ciências Aplicadas (ISCA Faculdades de Limeira) iniciou um projeto pelo qual o Observatório do Morro Azul empreendeu uma parceria com o Centro de Promoção Social Municipal (CEPROSOM), instituição mantida pela Prefeitura Municipal de Limeira para atender crianças e adolescentes carentes. O CEPROSOM contava com dois projetos: Projeto Centro de Convivência Infantil (CCI) e Programa Criança e Adolescente (PCA), que atendiam crianças e adolescentes em Centros Comunitários de diversas áreas da cidade. Esses projetos têm como prioridades estabelecer atividades prazerosas para as crianças no sentido de retirá-las das ruas. Assim sendo, as crianças passaram a ter mais um tipo de atividade - as visitas ao observatório. Este painel descreve as várias fases do projeto, que envolveu: reuniões de planejamento, curso de Astronomia para as orientadoras dos CCIs e PCAs, atividades relacionadas a visitas das crianças ao Observatório, proposta de construção de gnômons e relógios de Sol nos diversos Centros Comunitários de Limeira e divulgação do projeto na imprensa. O painel inclui discussões sobre a aprendizagem de crianças carentes, relatos que mostram a postura das orientadoras sobre a pertinência do ensino de Astronomia, relatos do monitor que fez o atendimento no Observatório e o que o número de crianças atendidas representou para as atividades da instituição desde o início de suas atividades e, em particular, em 2001. Os resultados são baseados na análise de relatos das orientadoras e do monitor do Observatório, registros de visitas e matérias da imprensa local. Conclui com uma avaliação do que tal projeto representou para as Instituições participantes. Para o Observatório, em particular, foi feita uma análise com relação às outras modalidades de atendimentos que envolvem alunos de escolas e público em geral. Também é abordada a questão do compromisso social do Observatório na educação do

  3. Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM.

    PubMed

    Li, Wen; Agirrezabala, Xabier; Lei, Jianlin; Bouakaz, Lamine; Brunelle, Julie L; Ortiz-Meoz, Rodrigo F; Green, Rachel; Sanyal, Suparna; Ehrenberg, Måns; Frank, Joachim

    2008-12-17

    The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNA(Phe), Trp-tRNA(Trp), or Leu-tRNA(LeuI). The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same 'loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon-anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection. PMID:19020518

  4. OV-Wav: um novo pacote para análise multiescalar em astronomia

    NASA Astrophysics Data System (ADS)

    Pereira, D. N. E.; Rabaça, C. R.

    2003-08-01

    Wavelets e outras formas de análise multiescalar têm sido amplamente empregadas em diversas áreas do conhecimento, sendo reconhecidamente superiores a técnicas mais tradicionais, como as análises de Fourier e de Gabor, em certas aplicações. Embora a teoria dos wavelets tenha começado a ser elaborada há quase trinta anos, seu impacto no estudo de imagens astronômicas tem sido pequeno até bem recentemente. Apresentamos um conjunto de programas desenvolvidos ao longo dos últimos três anos no Observatório do Valongo/UFRJ que possibilitam aplicar essa poderosa ferramenta a problemas comuns em astronomia, como a remoção de ruído, a detecção hierárquica de fontes e a modelagem de objetos com perfis de brilho arbitrários em condições não ideais. Este pacote, desenvolvido para execução em plataforma IDL, teve sua primeira versão concluída recentemente e está sendo disponibilizado à comunidade científica de forma aberta. Mostramos também resultados de testes controlados ao quais submetemos os programas, com a sua aplicação a imagens artificiais, com resultados satisfatórios. Algumas aplicações astrofísicas foram estudadas com o uso do pacote, em caráter experimental, incluindo a análise da componente de luz difusa em grupos compactos de galáxias de Hickson e o estudo de subestruturas de nebulosas planetárias no espaço multiescalar.

  5. Uma grade de perfis teóricos para estrelas massivas em transição

    NASA Astrophysics Data System (ADS)

    Nascimento, C. M. P.; Machado, M. A.

    2003-08-01

    Na XXVIII Reunião Anual da Sociedade Astronômica Brasileira (2002) apresentamos uma grade de perfis calculados de acordo com os pontos da trajetória evolutiva de metalicidade solar, Z = 0.02 e taxa de perda de massa () padrão, para estrelas com massa inicial de 25, 40, 60, 85 e 120 massas solares. Estes perfis foram calculados com o auxílio de um código numérico adequado para descrever os ventos de objetos massivos, supondo simetria esférica, estacionaridade e homogeneidade. No presente trabalho, apresentamos a complementação da grade com os perfis teóricos relativos às trajetórias de Z = 0.02 com taxa de perda de massa dobrada em relação a padrão (2´), e de metalicidade Z = 0.008. Para cada ponto das três trajetórias obtemos os perfis teóricos de Ha, Hb, Hg e Hd, e como esperado eles se apresentam em pura emissão, pura absorção ou em P-Cygni. Para valores de taxa de perda de massa muito baixos (~10-7) não há formação de linhas, o que é visto nos primeiros pontos em todas as trajetórias. Em geral, para um mesmo ponto a componente de emissão diminui e a absorção aumenta de Ha para Hd. É verificado que as trajetórias com Z = 0.02 e padrão possuem menos circuitos (loops) do que as com metalicidade Z = 0.02 e 2´ padrão, e seus perfis são, em geral, menos intensos. Em relação a trajetória de Z = 0.008, verifica-se menos circuitos e maior variação em luminosidade, e seus perfis mostram-se em, algumas trajetórias, mais intensos. Verificamos também que, pontos distintos em uma mesma trajetória, apresentam perfis diferentes para valores similares de luminosidade e temperatura efetiva. Sendo assim, uma grade de perfis teóricos parece ser útil para fornecer uma informação preliminar sobre o estágio evolutivo de uma estrela massiva.

  6. Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM

    PubMed Central

    Li, Wen; Agirrezabala, Xabier; Lei, Jianlin; Bouakaz, Lamine; Brunelle, Julie L; Ortiz-Meoz, Rodrigo F; Green, Rachel; Sanyal, Suparna; Ehrenberg, Måns; Frank, Joachim

    2008-01-01

    The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNAPhe, Trp-tRNATrp, or Leu-tRNALeuI. The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same ‘loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon–anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection. PMID:19020518

  7. Vínculos observacionais para o processo-S em estrelas gigantes de Bário

    NASA Astrophysics Data System (ADS)

    Smiljanic, R. H. S.; Porto de Mello, G. F.; da Silva, L.

    2003-08-01

    Estrelas de bário são gigantes vermelhas de tipo GK que apresentam excessos atmosféricos dos elementos do processo-s. Tais excessos são esperados em estrelas na fase de pulsos térmicos do AGB (TP-AGB). As estrelas de bário são, no entanto, menos massivas e menos luminosas que as estrelas do AGB, assim, não poderiam ter se auto-enriquecido. Seu enriquecimento teria origem em uma estrela companheira, inicialmente mais massiva, que evolui pelo TP-AGB, se auto-enriquece com os elementos do processo-s e transfere material contaminado para a atmosfera da atual estrela de bário. A companheira evolui então para anã branca deixando de ser observada diretamente. As estrelas de bário são, portanto, úteis como testes observacionais para teorias de nucleossíntese pelo processo-s, convecção e perda de massa. Análises detalhadas de abundância com dados de alta qualidade para estes objetos são ainda escassas na literatura. Neste trabalho construímos modelos de atmosferas e, procedendo a uma análise diferencial, determinamos parâmetros atmosféricos e evolutivos de uma amostra de dez gigantes de bário e quatro normais. Determinamos seus padrões de abundância para Na, Mg, Al, Si, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Sr, Y, Zr, Ba, La, Ce, Nd, Sm, Eu e Gd, concluindo que algumas estrelas classificadas na literatura como gigantes de bário são na verdade gigantes normais. Comparamos dois padrões médios de abundância, para estrelas com grandes excessos e estrelas com excessos moderados, com modelos teóricos de enriquecimento pelo processo-s. Os dois grupos de estrelas são ajustados pelos mesmos parâmetros de exposição de nêutrons. Tal resultado sugere que a ocorrência do fenômeno de bário com diferentes intensidades não se deve a diferentes exposições de nêutrons. Discutimos ainda efeitos nucleossintéticos, ligados ao processo-s, sugeridos na literatura para os elementos Cu, Mn, V e Sc.

  8. Para amino benzoic acid-derived self-assembled biocompatible nanoparticles for efficient delivery of siRNA

    PubMed Central

    Reddy, Teegala Lakshminarayan; Krishnarao, P Sivarama; Rao, Garikapati Koteswara; Bhimireddy, Eswar; Venkateswarlu, P; Mohapatra, Debendra K; Yadav, JS; Bhadra, Utpal; Pal Bhadra, Manika

    2015-01-01

    A number of diseases can result from abnormal gene expression. One of the approaches for treating such diseases is gene therapy to inhibit expression of a particular gene in a specific cell population by RNA interference. Use of efficient delivery vehicles increases the safety and success of gene therapy. Here we report the development of functionalized biocompatible fluorescent nanoparticles from para amino benzoic acid nanoparticles for efficient delivery of short interfering RNA (siRNA). These nanoparticles were non-toxic and did not interfere with progression of the cell cycle. The intrinsic fluorescent nature of these nanoparticles allows easy tracking and an opportunity for diagnostic applications. Human Bcl-2 siRNA was complexed with these nanoparticles to inhibit expression in cells at both the transcriptional and translational levels. Our findings indicated high gene transfection efficiency. These biocompatible nanoparticles allow targeted delivery of siRNA, providing an efficient vehicle for gene delivery. PMID:26491299

  9. BSSDATA - um programa otimizado para filtragem de dados em radioastronomia solar

    NASA Astrophysics Data System (ADS)

    Martinon, A. R. F.; Sawant, H. S.; Fernandes, F. C. R.; Stephany, S.; Preto, A. J.; Dobrowolski, K. M.

    2003-08-01

    A partir de 1998, entrou em operação regular no INPE, em São José dos Campos, o Brazilian Solar Spectroscope (BSS). O BSS é dedicado às observações de explosões solares decimétricas com alta resolução temporal e espectral, com a principal finalidade de investigar fenômenos associados com a liberação de energia dos "flares" solares. Entre os anos de 1999 e 2002, foram catalogadas, aproximadamente 340 explosões solares classificadas em 8 tipos distintos, de acordo com suas características morfológicas. Na análise detalhada de cada tipo, ou grupo, de explosões solares deve-se considerar a variação do fluxo do sol calmo ("background"), em função da freqüência e a variação temporal, além da complexidade das explosões e estruturas finas registradas superpostas ao fundo variável. Com o intuito de realizar tal análise foi desenvolvido o programa BSSData. Este programa, desenvolvido em linguagem C++, é constituído de várias ferramentas que auxiliam no tratamento e análise dos dados registrados pelo BSS. Neste trabalho iremos abordar as ferramentas referentes à filtragem do ruído de fundo. As rotinas do BSSData para filtragem de ruído foram testadas nos diversos grupos de explosões solares ("dots", "fibra", "lace", "patch", "spikes", "tipo III" e "zebra") alcançando um bom resultado na diminuição do ruído de fundo e obtendo, em conseqüência, dados onde o sinal torna-se mais homogêneo ressaltando as áreas onde existem explosões solares e tornando mais precisas as determinações dos parâmetros observacionais de cada explosão. Estes resultados serão apresentados e discutidos.

  10. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  11. Cryo-EM near-atomic structure of a dsRNA fungal virus shows ancient structural motifs preserved in the dsRNA viral lineage

    PubMed Central

    Luque, Daniel; Gómez-Blanco, Josué; Garriga, Damiá; Brilot, Axel F.; González, José M.; Havens, Wendy M.; Carrascosa, José L.; Trus, Benes L.; Verdaguer, Nuria; Ghabrial, Said A.; Castón, José R.

    2014-01-01

    Viruses evolve so rapidly that sequence-based comparison is not suitable for detecting relatedness among distant viruses. Structure-based comparisons suggest that evolution led to a small number of viral classes or lineages that can be grouped by capsid protein (CP) folds. Here, we report that the CP structure of the fungal dsRNA Penicillium chrysogenum virus (PcV) shows the progenitor fold of the dsRNA virus lineage and suggests a relationship between lineages. Cryo-EM structure at near-atomic resolution showed that the 982-aa PcV CP is formed by a repeated α-helical core, indicative of gene duplication despite lack of sequence similarity between the two halves. Superimposition of secondary structure elements identified a single “hotspot” at which variation is introduced by insertion of peptide segments. Structural comparison of PcV and other distantly related dsRNA viruses detected preferential insertion sites at which the complexity of the conserved α-helical core, made up of ancestral structural motifs that have acted as a skeleton, might have increased, leading to evolution of the highly varied current structures. Analyses of structural motifs only apparent after systematic structural comparisons indicated that the hallmark fold preserved in the dsRNA virus lineage shares a long (spinal) α-helix tangential to the capsid surface with the head-tailed phage and herpesvirus viral lineage. PMID:24821769

  12. Accommodation of tmRNA-SmpB into stalled ribosomes: a cryo-EM study.

    PubMed

    Weis, Felix; Bron, Patrick; Rolland, Jean-Paul; Thomas, Daniel; Felden, Brice; Gillet, Reynald

    2010-02-01

    In eubacteria, translation of defective messenger RNAs (mRNAs) produces truncated polypeptides that stall on the ribosome. A quality control mechanism referred to as trans-translation is performed by transfer-messenger RNA (tmRNA), a specialized RNA acting as both a tRNA and an mRNA, associated with small protein B (SmpB). So far, a clear view of the structural movements of both the protein and RNA necessary to perform accommodation is still lacking. By using a construct containing the tRNA-like domain as well as the extended helix H2 of tmRNA, we present a cryo-electron microscopy study of the process of accommodation. The structure suggests how tmRNA and SmpB move into the ribosome decoding site after the release of EF-Tu.GDP. While two SmpB molecules are bound per ribosome in a preaccommodated state, our results show that during accommodation the SmpB protein interacting with the small subunit decoding site stays in place while the one interacting with the large subunit moves away. Relative to canonical translation, an additional movement is observed due to the rotation of H2. This suggests that the larger movement required to resume translation on a tmRNA internal open reading frame starts during accommodation. PMID:20038631

  13. Functional conformations of the L11–ribosomal RNA complex revealed by correlative analysis of cryo-EM and molecular dynamics simulations

    PubMed Central

    Li, Wen; Sengupta, Jayati; Rath, Bimal K.; Frank, Joachim

    2006-01-01

    The interaction between the GTPase-associated center (GAC) and the aminoacyl-tRNA·EF-Tu·GTP ternary complex is of crucial importance in the dynamic process of decoding and tRNA accommodation. The GAC includes protein L11 and helices 43–44 of 23S rRNA (referred to as L11–rRNA complex). In this study, a method of fitting based on a systematic comparison between cryo-electron microscopy (cryo-EM) density maps and structures obtained by molecular dynamics simulations has been developed. This method has led to the finding of atomic models of the GAC that fit the EM maps with much improved cross-correlation coefficients compared with the fitting of the X-ray structure. Two types of conformations of the L11–rRNA complex, produced by the simulations, match the cryo-EM maps representing the states either bound or unbound to the aa-tRNA·EF-Tu·GTP ternary complex. In the bound state, the N-terminal domain of L11 is extended from its position in the crystal structure, and the base of nucleotide A1067 in the 23S ribosomal RNA is flipped out. This position of the base allows the RNA to reach the elbow region of the aminoacyl-tRNA when the latter is bound in the A/T site. In the unbound state, the N-terminal domain of L11 is rotated only slightly, and A1067 of the RNA is flipped back into the less-solvent-exposed position, as in the crystal structure. By matching our experimental cryo-EM maps with much improved cross-correlation coefficients compared to the crystal structure, these two conformations prove to be strong candidates of the two functional states. PMID:16682558

  14. Código para imageamento indireto de estrelas em sistemas binarios: simulação de variações elipsoidais e do perfil das linhas

    NASA Astrophysics Data System (ADS)

    Souza, T. R.; Baptista, R.

    2003-08-01

    As estrelas secundárias em variáveis cataclí smicas (VCs) e binárias-x de baixa massa (BXBMs) são cruciais para o entendimento da origem, evolução e comportamento destas binárias interagentes. Elas são estrelas magneticamente ativas submetidas a condições ambientais extremas [e.g., estão muito próximas de uma fonte quente e irradiante; têm rotação extremamente rápida e forma distorcida; estão perdendo massa a taxas de 10-8-10-10 M¤/ano] que contribuem para que suas propriedades sejam distintas das de estrelas de mesma massa na seqüência principal. Por outro lado, o padrão de irradiação na face da secundária fornece informação sobre a geometria das estruturas de acréscimo em torno da estrela primária. Assim, a obtenção de imagens da superfície destas estrelas é de grande interesse astrofísico. A Tomografia Roche usa as variações no perfil das linhas de emissão/absorção da estrela secundária em função da fase orbital para mapear a distribuição de brilho em sua superfície. Neste trabalho apresentamos os resultados iniciais do desenvolvimento de um programa para o mapeamento da distribuição de brilho na superfí cie das estrelas secundárias em VCs e BXBMs com técnicas de astro-tomografia. Presentemente temos em operação um código que simula as variações no perfil das linhas em conseqüência de efeito Doppler resultante da combinação de rotação e translação de uma estrela em forma de lobo de Roche em torno do centro de massa da binária, em função da distribuição de brilho na superfície desta estrela. O código igualmente produz a curva de luz resultante das variações de aspecto da estrela em função da fase orbital (variações elipsoidais).

  15. Implication of microRNA regulation in para-phenylenediamine-induced cell death and senescence in normal human hair dermal papilla cells.

    PubMed

    Lee, Ok-Kyu; Cha, Hwa Jun; Lee, Myung Joo; Lim, Kyung Mi; Jung, Jae Wook; Ahn, Kyu Joong; An, In-Sook; An, Sungkwan; Bae, Seunghee

    2015-07-01

    Para-phenylenediamine (PPD) is a major component of hair coloring and black henna products. Although it has been largely demonstrated that PPD induces allergic reactions and increases the risk of tumors in the kidney, liver, thyroid gland and urinary bladder, the effect on dermal papilla cells remains to be elucidated. Therefore, the current study evaluated the effects of PPD on growth, cell death and senescence using cell-based assays and microRNA (miRNA) microarray in normal human hair dermal papilla cells (nHHDPCs). Cell viability and cell cycle analyses demonstrated that PPD exhibited a significant cytotoxic effect on nHHDPCs through inducing cell death and G2 phase cell cycle arrest in a dose-dependent manner. It was additionally observed that treatment of nHHDPCs with PPD induced cellular senescence by promoting cellular oxidative stress. In addition, the results of the current study indicated that these PPD-mediated effects were involved in the alteration of miRNA expression profiles. Treatment of nHHDPCs with PPD altered the expression levels of 74 miRNAs by ≥ 2-fold (16 upregulated and 58 downregulated miRNAs). Further bioinformatics analysis determined that these identified miRNA target genes were likely to be involved in cell growth, cell cycle arrest, cell death, senescence and the induction of oxidative stress. In conclusion, the observations of the current study suggested that PPD was able to induce several cytotoxic effects through alteration of miRNA expression levels in nHHDPCs. PMID:25776079

  16. Examinations of tRNA Range of Motion Using Simulations of Cryo-EM Microscopy and X-Ray Data

    PubMed Central

    Caulfield, Thomas R.; Devkota, Batsal; Rollins, Geoffrey C.

    2011-01-01

    We examined tRNA flexibility using a combination of steered and unbiased molecular dynamics simulations. Using Maxwell's demon algorithm, molecular dynamics was used to steer X-ray structure data toward that from an alternative state obtained from cryogenic-electron microscopy density maps. Thus, we were able to fit X-ray structures of tRNA onto cryogenic-electron microscopy density maps for hybrid states of tRNA. Additionally, we employed both Maxwell's demon molecular dynamics simulations and unbiased simulation methods to identify possible ribosome-tRNA contact areas where the ribosome may discriminate tRNAs during translation. Herein, we collected >500 ns of simulation data to assess the global range of motion for tRNAs. Biased simulations can be used to steer between known conformational stop points, while unbiased simulations allow for a general testing of conformational space previously unexplored. The unbiased molecular dynamics data describes the global conformational changes of tRNA on a sub-microsecond time scale for comparison with steered data. Additionally, the unbiased molecular dynamics data was used to identify putative contacts between tRNA and the ribosome during the accommodation step of translation. We found that the primary contact regions were H71 and H92 of the 50S subunit and ribosomal proteins L14 and L16. PMID:21716650

  17. Uso de modelos mecânicos em curso informal de astronomia para deficientes visuais. Resgate de uma experiência

    NASA Astrophysics Data System (ADS)

    Tavares, E. T., Jr.; Klafke, J. C.

    2003-08-01

    O presente trabalho propõe-se a resgatar uma experiência que teve lugar no Planetário de São Paulo nos anos 60. Em 1962, o Sr. Acácio, então com 37 anos, deficiente visual desde os 27, passou a assistir às aulas ministradas pelo Prof. Aristóteles Orsini aos integrantes do corpo de servidores do Planetário. O Sr. Acácio era o único deficiente da turma e, embora possuísse conhecimentos básicos e relativamente avançados de matemática, enfrentava dificuldades na compreensão e acompanhamento da exposição, como também em estudos posteriores. Com o intuito de auxiliá-lo na superação desses problemas, o Prof. Orsini solicitou a construção de modelos mecânicos que, através do sentido do tato, permitissem o acompanhamento das aulas e a transposição do modelo para o "constructo" mental. Essa prática mostrou-se tão eficaz que facilitou sobejamente o aprendizado da matéria pelo sujeito. O Sr. Acácio passou a integrar o corpo de professores do Planetário/Escola Municipal de Astrofísica, tendo ficado responsável pelo curso de "Introdução à Astronomia" por vários anos. Além disso, a experiência foi tão bem sucedida que alguns dos modelos tiveram seus elementos constitutivos pintados diferencialmente para serem utilizados em cursos regulares do Planetário, tornando-se parte integrante do conjunto de recursos didáticos da instituição. É pensando nessa eficácia, tanto em seu objetivo original permitir o aprendizado de um deficiente visual quanto no subsidiário recurso didático sistemático da instituição que decidimos resgatar essa experiência. Estribados nela, acreditamos ser extremamente produtivo, em termos educacionais, o aperfeiçoamento dos modelos originais, agora resgatados e restaurados, e a criação de outros que pudessem ser utilizados no ensino dessa ciência a deficientes visuais.

  18. Re-analysis of cryoEM data on HCV IRES bound to 40S subunit of human ribosome integrated with recent structural information suggests new contact regions between ribosomal proteins and HCV RNA

    PubMed Central

    Joseph, Agnel Praveen; Bhat, Prasanna; Das, Saumitra; Srinivasan, Narayanaswamy

    2014-01-01

    In this study, we combine available high resolution structural information on eukaryotic ribosomes with low resolution cryo-EM data on the Hepatitis C Viral RNA (IRES) human ribosome complex. Aided further by the prediction of RNA-protein interactions and restrained docking studies, we gain insights on their interaction at the residue level. We identified the components involved at the major and minor contact regions, and propose that there are energetically favorable local interactions between 40S ribosomal proteins and IRES domains. Domain II of the IRES interacts with ribosomal proteins S5 and S25 while the pseudoknot and the downstream domain IV region bind to ribosomal proteins S26, S28 and S5. We also provide support using UV cross-linking studies to validate our proposition of interaction between the S5 and IRES domains II and IV. We found that domain IIIe makes contact with the ribosomal protein S3a (S1e). Our model also suggests that the ribosomal protein S27 interacts with domain IIIc while S7 has a weak contact with a single base RNA bulge between junction IIIabc and IIId. The interacting residues are highly conserved among mammalian homologs while IRES RNA bases involved in contact do not show strict conservation. IRES RNA binding sites for S25 and S3a show the best conservation among related viral IRESs. The new contacts identified between ribosomal proteins and RNA are consistent with previous independent studies on RNA-binding properties of ribosomal proteins reported in literature, though information at the residue level is not available in previous studies. PMID:25268799

  19. RNA Interference

    MedlinePlus

    ... NIGMS Home > Science Education > RNA Interference Fact Sheet RNA Interference Fact Sheet Tagline (Optional) Middle/Main Content Area What is RNA interference? RNA interference (RNAi) is a natural process ...

  20. RNA topology

    PubMed Central

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases. PMID:23603781

  1. Uncoating of common cold virus is preceded by RNA switching as determined by X-ray and cryo-EM analyses of the subviral A-particle

    PubMed Central

    Pickl-Herk, Angela; Luque, Daniel; Vives-Adrián, Laia; Querol-Audí, Jordi; Garriga, Damià; Trus, Benes L.; Verdaguer, Nuria; Blaas, Dieter; Castón, José R.

    2013-01-01

    During infection, viruses undergo conformational changes that lead to delivery of their genome into host cytosol. In human rhinovirus A2, this conversion is triggered by exposure to acid pH in the endosome. The first subviral intermediate, the A-particle, is expanded and has lost the internal viral protein 4 (VP4), but retains its RNA genome. The nucleic acid is subsequently released, presumably through one of the large pores that open at the icosahedral twofold axes, and is transferred along a conduit in the endosomal membrane; the remaining empty capsids, termed B-particles, are shuttled to lysosomes for degradation. Previous structural analyses revealed important differences between the native protein shell and the empty capsid. Nonetheless, little is known of A-particle architecture or conformation of the RNA core. Using 3D cryo-electron microscopy and X-ray crystallography, we found notable changes in RNA–protein contacts during conversion of native virus into the A-particle uncoating intermediate. In the native virion, we confirmed interaction of nucleotide(s) with Trp38 of VP2 and identified additional contacts with the VP1 N terminus. Study of A-particle structure showed that the VP2 contact is maintained, that VP1 interactions are lost after exit of the VP1 N-terminal extension, and that the RNA also interacts with residues of the VP3 N terminus at the fivefold axis. These associations lead to formation of a well-ordered RNA layer beneath the protein shell, suggesting that these interactions guide ordered RNA egress. PMID:24277846

  2. Compilação de dados atômicos e moleculares do UV ao IV próximo para uso em síntese espectral

    NASA Astrophysics Data System (ADS)

    Coelho, P.; Barbuy, B.; Melendez, J.; Allen, D. M.; Castilho, B.

    2003-08-01

    Espectros sintéticos são utéis em uma grande variedade de aplicações, desde análise de abundâncias em espectros estelares de alta resolução ao estudo de populações estelares em espectros integrados. A confiabilidade de um espectro sintético depende do modelo de atmosfera adotado, do código de formação de linhas e da qualidade dos dados atômicos e moleculares que são determinantes no cálculo das opacidades da fotosfera. O nosso grupo no departamento de Astronomia no IAG tem utilizado espectros sintéticos há mais de 15 anos, em aplicações voltadas principalmente para a análise de abundâncias de estrelas G, K e M e populações estelares velhas. Ao longo desse tempo, as listas de linhas vieram sendo construídas e atualizadas continuamente, e alguns acréscimos recentes podem ser citados: Castilho (1999, átomos e moléculas no UV), Schiavon (1998, bandas moleculares de TiO) e Melendez (2001, átomos e moléculas no IV próximo). Com o intuito de calcular uma grade de espectros do UV ao IV próximo para uso no estudo de populações estelares velhas, se fazia necessário compilar e homogeneizar as diversas listas em apenas uma lista atômica e uma molecular. Nesse processo, a nova lista compilada foi correlacionada com outras bases de dados (NIST, Kurucz Database, O' Brian et al. 1991) para atualização dos parâmetros que caracterizam a transição atômica (comprimento de onda, log gf e potencial de excitação). Adicionalmente as constantes de interação C6 foram calculadas segundo a teoria de Anstee & O'Mara (1995) e artigos posteriores. As bandas moleculares de CH e CN foram recalculadas com o programa LIFBASE (Luque & Crosley 1999). Nesse poster estão detalhados os procedimentos citados acima, as comparações entre espectros calculados com as novas listas e espectros observados em alta resolução do Sol e de Arcturus, e uma análise do impacto decorrente da utilização de diferentes modelos de atmosfera no espectro sintético. Ao

  3. RNA genetics

    SciTech Connect

    Domingo, E. ); Holland, J.J. . Dept. of Biology); Ahlquist, P. . Dept. of Plant Pathology)

    1988-01-01

    This book contains the proceedings on RNA genetics: Retroviruses, Viroids, and RNA recombination, Volume 2. Topics covered include: Replication of retrovirus genomes, Hepatitis B virus replication, and Evolution of RNA viruses.

  4. RNA genetics

    SciTech Connect

    Domingo, E.; Holland, J.J.; Ahlquist, P.

    1988-01-01

    These three volumes comprise reference on RNA genomes. The replication, mutation, recombination-assortment, and extreme evolutionary variability of RNA viruses and related RNA replicons is emphasized. The replication mechanisms of positive, negative, and double-stranded RNA viruses of animals and plants are featured.

  5. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  6. RNA helicases

    PubMed Central

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes. PMID:23093803

  7. Moving RNA moves RNA forward.

    PubMed

    Peng, Lina; Li, Yujiao; Zhang, Lan; Yu, Wenqiang

    2013-10-01

    Cell communication affects all aspects of cell structure and behavior, such as cell proliferation, differentiation, division, and coordination of various physiological functions. The moving RNA in plants and mammalian cells indicates that nucleic acid could be one of the various types of messengers for cell communication. The microvesicle is a critical pathway that mediates RNA moving and keeps moving RNA stable in body fluids. When moving miRNA enters the target cell, it functions by altering the gene expression profile and significantly inhibiting mRNA translation in recipient cells. Thus, moving RNA may act as a long-range modulator during development, organogenesis, and tumor metastasis. PMID:24008386

  8. RNA epigenetics

    PubMed Central

    Liu, Nian; Pan, Tao

    2014-01-01

    Summary Mammalian messenger and long non-coding RNA contain tens of thousands of post-transcriptional chemical modifications. Among these, the N6-methyl-adenosine (m6A) modification is the most abundant and can be removed by specific mammalian enzymes. M6A modification is recognized by families of RNA binding proteins that affect many aspects of mRNA function. mRNA/lncRNA modification represents another layer of epigenetic regulation of gene expression, analogous to DNA methylation and histone modification. PMID:24768686

  9. RNA helicases

    PubMed Central

    Ranji, Arnaz

    2010-01-01

    RNA helicases serve multiple roles at the virus-host interface. In some situations, RNA helicases are essential host factors to promote viral replication; however, in other cases they serve as a cellular sensor to trigger the antiviral state in response to viral infection. All family members share the conserved ATP-dependent catalytic core linked to different substrate recognition and protein-protein interaction domains. These flanking domains can be shuffled between different helicases to achieve functional diversity. This review summarizes recent studies, This review summarizes recent studies of RNA helicases in virus biology. First, RNA helicases are catalysts of progressive RNA-protein rearrangements that begin at gene transcription and culminate in release of infectious virus. Second, RNA helicases can act as a scaffold for alternative protein-protein interactions that can defeat the antiviral state. The mounting fundamental understanding of RNA helicases is being used to develop selective and efficacious drugs against human and animal pathogens. The analysis of RNA helicases in virus model systems continues to provide insights into virology, cell biology and immunology and has provided fresh perspective to continue unraveling the complexity of virus-host interactions. PMID:21173576

  10. RNA. Introduction.

    PubMed

    Bao, Marie Z; Kruger, Robert P; Rivas, Fabiola; Smith, Orla; Szewczak, Lara

    2009-02-20

    Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells. PMID:19263588

  11. RNA Research

    NASA Technical Reports Server (NTRS)

    1998-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. It is widely believed that this RNA World was extensive and therefore a sophisticated nucleic acid replication machinery would presumably predate the translation machinery which would not be needed until later stages in the development of life. This view of an extended RNA World is not necessarily correct. From the point of view of exobiology, the difference in these two views mainly affects the significance of studies of the extent of catalysis possible by RNA- In either case, the origin of the translation machinery and the principles of RNA evolution remain central problems in exobiology. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modem organisms came to exist by the time of the last common ancestor (as detected by 16S RRNA sequence studies). Third, the RNAs that comprise the ribosome are themselves likely of very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.

  12. Structural characterization of mRNA-tRNA translocation intermediates

    PubMed Central

    Agirrezabala, Xabier; Liao, Hstau Y.; Schreiner, Eduard; Fu, Jie; Ortiz-Meoz, Rodrigo F.; Schulten, Klaus; Green, Rachel; Frank, Joachim

    2012-01-01

    Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation. PMID:22467828

  13. Structural characterization of mRNA-tRNA translocation intermediates.

    PubMed

    Agirrezabala, Xabier; Liao, Hstau Y; Schreiner, Eduard; Fu, Jie; Ortiz-Meoz, Rodrigo F; Schulten, Klaus; Green, Rachel; Frank, Joachim

    2012-04-17

    Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation. PMID:22467828

  14. Intermediate States During mRNA-tRNA Translocation

    PubMed Central

    Frank, Joachim

    2012-01-01

    Recent studies support the notion that the pre-translocation (PRE) ribosomal complex functions, at least in part, as a Brownian machine, stochastically fluctuating among multiple conformations and transfer RNA (tRNA) binding configurations. Apart from the relatively more energetically stable conformational states of the PRE complex, termed macrostate I (MS I) and macrostate II (MS II), several additional intermediate states have been recently discovered. Structural and kinetic analyses of these states, made possible by cryogenic electron microscopy (cryo-EM), X-ray crystallography, and single-molecule fluorescence resonance energy transfer (smFRET), have provided important insights into the translocation process, which is now understood to proceed, at least in the first step of the process, as a Brownian machine that is transiently stabilized in the “productive” MS II conformation by the binding of the translocase elongation factor G (EF-G). PMID:22906732

  15. RAID: a comprehensive resource for human RNA-associated (RNA-RNA/RNA-protein) interaction.

    PubMed

    Zhang, Xiaomeng; Wu, Deng; Chen, Liqun; Li, Xiang; Yang, Jinxurong; Fan, Dandan; Dong, Tingting; Liu, Mingyue; Tan, Puwen; Xu, Jintian; Yi, Ying; Wang, Yuting; Zou, Hua; Hu, Yongfei; Fan, Kaili; Kang, Juanjuan; Huang, Yan; Miao, Zhengqiang; Bi, Miaoman; Jin, Nana; Li, Kongning; Li, Xia; Xu, Jianzhen; Wang, Dong

    2014-07-01

    Transcriptomic analyses have revealed an unexpected complexity in the eukaryote transcriptome, which includes not only protein-coding transcripts but also an expanding catalog of noncoding RNAs (ncRNAs). Diverse coding and noncoding RNAs (ncRNAs) perform functions through interaction with each other in various cellular processes. In this project, we have developed RAID (http://www.rna-society.org/raid), an RNA-associated (RNA-RNA/RNA-protein) interaction database. RAID intends to provide the scientific community with all-in-one resources for efficient browsing and extraction of the RNA-associated interactions in human. This version of RAID contains more than 6100 RNA-associated interactions obtained by manually reviewing more than 2100 published papers, including 4493 RNA-RNA interactions and 1619 RNA-protein interactions. Each entry contains detailed information on an RNA-associated interaction, including RAID ID, RNA/protein symbol, RNA/protein categories, validated method, expressing tissue, literature references (Pubmed IDs), and detailed functional description. Users can query, browse, analyze, and manipulate RNA-associated (RNA-RNA/RNA-protein) interaction. RAID provides a comprehensive resource of human RNA-associated (RNA-RNA/RNA-protein) interaction network. Furthermore, this resource will help in uncovering the generic organizing principles of cellular function network. PMID:24803509

  16. RNA as an Enzyme.

    ERIC Educational Resources Information Center

    Cech, Thomas R.

    1986-01-01

    Reviews current findings that explain RNA's function as an enzyme in addition to being an informational molecule. Highlights recent research efforts and notes changes in the information base on RNA activity. Includes models and diagrams of RNA activity. (ML)

  17. Benchmarking RNA-Seq quantification tools

    PubMed Central

    Chandramohan, R.; Wu, Po-Yen; Phan, J.H.; Wang, M.D.

    2016-01-01

    RNA-Seq, a deep sequencing technique, promises to be a potential successor to microarraysfor studying the transcriptome. One of many aspects of transcriptomics that are of interest to researchers is gene expression estimation. With rapid development in RNA-Seq, there are numerous tools available to estimate gene expression, each producing different results. However, we do not know which of these tools produces the most accurate gene expression estimates. In this study we have addressed this issue using Cufflinks, IsoEM, HTSeq, and RSEM to quantify RNA-Seq expression profiles. Comparing results of these quantification tools, we observe that RNA-Seq relative expression estimates correlate with RT-qPCR measurements in the range of 0.85 to 0.89, with HTSeq exhibiting the highest correlation. But, in terms of root-mean-square deviation of RNA-Seq relative expression estimates from RT-qPCR measurements, we find HTSeq to produce the greatest deviation. Therefore, we conclude that, though Cufflinks, RSEM, and IsoEM might not correlate as well as HTSeq with RT-qPCR measurements, they may produce expression values with higher accuracy. PMID:24109770

  18. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation.

    PubMed

    Martin, Franck; Ménétret, Jean-François; Simonetti, Angelita; Myasnikov, Alexander G; Vicens, Quentin; Prongidi-Fix, Lydia; Natchiar, S Kundhavai; Klaholz, Bruno P; Eriani, Gilbert

    2016-01-01

    Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon. PMID:27554013

  19. Ribosomal 18S rRNA base pairs with mRNA during eukaryotic translation initiation

    PubMed Central

    Martin, Franck; Ménétret, Jean-François; Simonetti, Angelita; Myasnikov, Alexander G.; Vicens, Quentin; Prongidi-Fix, Lydia; Natchiar, S. Kundhavai; Klaholz, Bruno P.; Eriani, Gilbert

    2016-01-01

    Eukaryotic mRNAs often contain a Kozak sequence that helps tether the ribosome to the AUG start codon. The mRNA of histone H4 (h4) does not undergo classical ribosome scanning but has evolved a specific tethering mechanism. The cryo-EM structure of the rabbit ribosome complex with mouse h4 shows that the mRNA forms a folded, repressive structure at the mRNA entry site on the 40S subunit next to the tip of helix 16 of 18S ribosomal RNA (rRNA). Toe-printing and mutational assays reveal that an interaction exists between a purine-rich sequence in h4 mRNA and a complementary UUUC sequence of helix h16. Together the present data establish that the h4 mRNA harbours a sequence complementary to an 18S rRNA sequence which tethers the mRNA to the ribosome to promote proper start codon positioning, complementing the interactions of the 40S subunit with the Kozak sequence that flanks the AUG start codon. PMID:27554013

  20. Structure of a mitochondrial ribosome with minimal RNA.

    PubMed

    Sharma, Manjuli R; Booth, Timothy M; Simpson, Larry; Maslov, Dmitri A; Agrawal, Rajendra K

    2009-06-16

    The Leishmania tarentolae mitochondrial ribosome (Lmr) is a minimal ribosomal RNA (rRNA)-containing ribosome. We have obtained a cryo-EM map of the Lmr. The map reveals several features that have not been seen in previously-determined structures of eubacterial or eukaryotic (cytoplasmic or organellar) ribosomes to our knowledge. Comparisons of the Lmr map with X-ray crystallographic and cryo-EM maps of the eubacterial ribosomes and a cryo-EM map of the mammalian mitochondrial ribosome show that (i) the overall structure of the Lmr is considerably more porous, (ii) the topology of the intersubunit space is significantly different, with fewer intersubunit bridges, but more tunnels, and (iii) several of the functionally-important rRNA regions, including the alpha-sarcin-ricin loop, have different relative positions within the structure. Furthermore, the major portions of the mRNA channel, the tRNA passage, and the nascent polypeptide exit tunnel contain Lmr-specific proteins, suggesting that the mechanisms for mRNA recruitment, tRNA interaction, and exiting of the nascent polypeptide in Lmr must differ markedly from the mechanisms deduced for ribosomes in other organisms. Our study identifies certain structural features that are characteristic solely of mitochondrial ribosomes and other features that are characteristic of both mitochondrial and chloroplast ribosomes (i.e., organellar ribosomes). PMID:19497863

  1. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

    T7-RNA Polymerase grown on STS-81. Structure-Function Relationships of RNA Polymerase: DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. Principal Investigator's include Dr. Dan Carter, Dr. B.C. Wang, and Dr. John Rose of New Century Pharmaceuticals.

  2. Analyzing MiRNA-LncRNA Interactions.

    PubMed

    Paraskevopoulou, Maria D; Hatzigeorgiou, Artemis G

    2016-01-01

    Long noncoding RNAs (lncRNAs) are noncoding transcripts usually longer than 200 nts that have recently emerged as one of the largest and significantly diverse RNA families. The biological role and functions of lncRNAs are still mostly uncharacterized. Their target-mimetic, sponge/decoy function on microRNAs was recently uncovered. miRNAs are a class of noncoding RNA species (~22 nts) that play a central role in posttranscriptional regulation of protein coding genes by mRNA cleavage, direct translational repression and/or mRNA destabilization. LncRNAs can act as miRNA sponges, reducing their regulatory effect on mRNAs. This function introduces an extra layer of complexity in the miRNA-target interaction network. This chapter focuses on the study of miRNA-lncRNA interactions with either in silico or experimentally supported analyses. The proposed methodologies can be appropriately adapted in order to become the backbone of advanced multistep functional miRNA analyses. PMID:26721498

  3. Stranded Whole Transcriptome RNA-Seq for All RNA Types

    PubMed Central

    Yan, Pearlly X.; Fang, Fang; Buechlein, Aaron; Ford, James B.; Tang, Haixu; Huang, Tim H.; Burow, Matthew E.; Liu, Yunlong; Rusch, Douglas B.

    2015-01-01

    Stranded whole transcriptome RNA-Seq described in this unit captures quantitative expression data for all types of RNA including, but not limited to miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (large non-coding intergenic RNA), SRP RNA (signal recognition particle RNA), tRNA (transfer RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA). The size and nature of these types of RNA are irrelevant to the approach described here. Barcoded libraries for multiplexing on the Illumina platform are generated with this approach but it can be applied to other platforms with a few modifications. PMID:25599667

  4. Absence of knots in known RNA structures.

    PubMed

    Micheletti, Cristian; Di Stefano, Marco; Orland, Henri

    2015-02-17

    The ongoing effort to detect and characterize physical entanglement in biopolymers has so far established that knots are present in many globular proteins and also, abound in viral DNA packaged inside bacteriophages. RNA molecules, however, have not yet been systematically screened for the occurrence of physical knots. We have accordingly undertaken the systematic profiling of the several thousand RNA structures present in the Protein Data Bank (PDB). The search identified no more than three deeply knotted RNA molecules. These entries are rRNAs of about 3,000 nt solved by cryo-EM. Their genuine knotted state is, however, doubtful based on the detailed structural comparison with homologs of higher resolution, which are all unknotted. Compared with the case of proteins and viral DNA, the observed incidence of knots in available RNA structures is, therefore, practically negligible. This fact suggests that either evolutionary selection or thermodynamic and kinetic folding mechanisms act toward minimizing the entanglement of RNA to an extent that is unparalleled by other types of biomolecules. A possible general strategy for designing synthetic RNA sequences capable of self-tying in a twist-knot fold is finally proposed. PMID:25646433

  5. Replication of Tobamovirus RNA.

    PubMed

    Ishibashi, Kazuhiro; Ishikawa, Masayuki

    2016-08-01

    Tobacco mosaic virus and other tobamoviruses have served as models for studying the mechanisms of viral RNA replication. In tobamoviruses, genomic RNA replication occurs via several steps: (a) synthesis of viral replication proteins by translation of the genomic RNA; (b) translation-coupled binding of the replication proteins to a 5'-terminal region of the genomic RNA; (c) recruitment of the genomic RNA by replication proteins onto membranes and formation of a complex with host proteins TOM1 and ARL8; (d) synthesis of complementary (negative-strand) RNA in the complex; and (e) synthesis of progeny genomic RNA. This article reviews current knowledge on tobamovirus RNA replication, particularly regarding how the genomic RNA is specifically selected as a replication template and how the replication proteins are activated. We also focus on the roles of the replication proteins in evading or suppressing host defense systems. PMID:27296148

  6. RNA-Catalyzed RNA Ligation on an External RNA Template

    NASA Technical Reports Server (NTRS)

    McGinness, Kathleen E.; Joyce, Gerald F.

    2002-01-01

    Variants of the hc ligase ribozyme, which catalyzes ligation of the 3' end of an RNA substrate to the 5' end of the ribozyme, were utilized to evolve a ribozyme that catalyzes ligation reactions on an external RNA template. The evolved ribozyme catalyzes the joining of an oligonucleotide 3'-hydroxyl to the 5'-triphosphate of an RNA hairpin molecule. The ribozyme can also utilize various substrate sequences, demonstrating a largely sequence-independent mechanism for substrate recognition. The ribozyme also carries out the ligation of two oligonucleotides that are bound at adjacent positions on a complementary template. Finally, it catalyzes addition of mononucleoside '5-triphosphates onto the '3 end of an oligonucleotide primer in a template-dependent manner. The development of ribozymes that catalyze polymerase-type reactions contributes to the notion that an RNA world could have existed during the early history of life on Earth.

  7. Fast Prediction of RNA-RNA Interaction

    NASA Astrophysics Data System (ADS)

    Salari, Raheleh; Backofen, Rolf; Sahinalp, S. Cenk

    Regulatory antisense RNAs are a class of ncRNAs that regulate gene expression by prohibiting the translation of an mRNA by establishing stable interactions with a target sequence. There is great demand for efficient computational methods to predict the specific interaction between an ncRNA and its target mRNA(s). There are a number of algorithms in the literature which can predict a variety of such interactions - unfortunately at a very high computational cost. Although some existing target prediction approaches are much faster, they are specialized for interactions with a single binding site.

  8. Combinatorics of RNA-RNA interaction.

    PubMed

    Li, Thomas J X; Reidys, Christian M

    2012-02-01

    RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including their generating function, singularity analysis as well as explicit recurrence relations. In particular, our results imply simple asymptotic formulas for the number of joint structures. PMID:21541694

  9. On the Formation of a Study Group to the Realization of Workshops for Teachers: Astronomy in Basic Education in Umuarama-Pr (Spanish Title: De la Formación de un Grupo de Estudios a la Realización de los Talleres Para los Profesores: la Astronomía en la Educación Básica en Umuarama-Pr ) Da Formação de um Grupo de Estudos À Realização de Oficinas Para Professores: a Astronomia na Educação Básica em Umuarama-Pr

    NASA Astrophysics Data System (ADS)

    Belusso, Diane; Akira Sakai, Otávio

    2013-12-01

    In this article, we aimed to present the activities developed by the Astronomy Study Group (ASG) to contribute to the dissemination and improvement of the astronomy teaching-learning. The results of a research carried out in schools of Umuarama-PR are shown, with the intention of checking the students' knowledge and interest in relation to Astronomy. It is reported the realization of workshops for Science teachers linked to the Education Regional Nucleus. The research and the workshop execution promoted the direct contact of the study group with the community; the results were used to diagnose the state of astronomy teaching-learning, in the basic education in Umuarama-PR. En este artículo se intenta presentar las actividades desarrolladas por el Grupo de Estudios de Astronomía (GEA) y contribuir para la divulgación y mejoría de la enseñanza-aprendizaje de la Astronomía. Se presentan los resultados de una investigación realizada en las escuelas de Umuarama-PR, con la intención de determinar el grado de conocimiento y el interés de los estudiantes en relación a la astronomía. Se relata la realización de talleres de capacitación para los profesores de ciencias vinculados al Núcleo Regional del Educación. La ejecución de la investigación y de los talleres promovió el contacto directo del grupo de estudios con la comunidad; los resultados sirvieron de diagnóstico de la enseñanza aprendizaje de la astronomía en la educación básica en Umuarama-PR. Neste artigo, objetiva-se apresentar as atividades desenvolvidas pelo Grupo de Estudos de Astronomia (GEA) e contribuir para a divulgação e melhoria do ensino-aprendizagem de astronomia. São apresentados os resultados de uma pesquisa realizada nas escolas de Umuarama-PR, com o intuito de averiguar o conhecimento e o interesse dos estudantes em relação à astronomia. Relata-se a realização de oficinas de capacitação para professores de ciências vinculados ao Núcleo Regional de Educação. A

  10. Cytoplasmic Z-RNA

    SciTech Connect

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-09-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.

  11. mRNA imprinting

    PubMed Central

    2011-01-01

    Following its synthesis in the nucleus, mRNA undergoes various stages that are critical for the proper synthesis, localization and possibly functionality of its encoded protein. Recently, we have shown that two RNA polymerase II (Pol II) subunits, Rpb4p and Rpb7p, associate with the nascent transcript co-transcriptionally. This “mRNA imprinting” lasts throughout the mRNA lifetime and is required for proper regulation of all major stages that the mRNA undergoes. Other possible cases of co-transcriptional imprinting are discussed. Since mRNAs can be transported from the synthesizing cell to other cells, we propose that mRNA imprinting can also affect the phenotype of the recipient cells. This can be viewed as “mRNA-based epigenetics.” PMID:21686103

  12. RNA Viruses Infecting Pest Insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA viruses are viruses whose genetic material is ribonucleic acid (RNA). RNA viruses may be double or single-stranded based on the type of RNA they contain. Single-stranded RNA viruses can be further grouped into negative sense or positive-sense viruses according to the polarity of their RNA. Fur...

  13. Click-EM for imaging metabolically tagged nonprotein biomolecules.

    PubMed

    Ngo, John T; Adams, Stephen R; Deerinck, Thomas J; Boassa, Daniela; Rodriguez-Rivera, Frances; Palida, Sakina F; Bertozzi, Carolyn R; Ellisman, Mark H; Tsien, Roger Y

    2016-06-01

    EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes. PMID:27110681

  14. Strategies in RNA crystallography.

    PubMed

    Reyes, Francis E; Garst, Andrew D; Batey, Robert T

    2009-01-01

    A number of RNAs ranging from small helices to large megadalton ribonucleoprotein complexes have been solved to atomic resolution using X-ray crystallography. As with proteins, RNA crystallography involves a number of screening trials in which the concentration of macromolecule, precipitant, salt, and temperature are varied, an approach known as searching "condition space." In contrast to proteins, the nature of base pairing in nucleic acids creates predictable secondary structure that facilitates the rational design of RNA variants, allowing "sequence space" to be screened in parallel. This chapter reviews RNA-specific techniques and considerations for RNA crystallography and presents a complete workflow used by our laboratory for solving RNA structures starting with initial library construction, methods to investigate and improve RNA crystal quality, and finally phase determination and structure solution. PMID:20946787

  15. RNA Sequencing in Schizophrenia

    PubMed Central

    Li, Xin; Teng, Shaolei

    2015-01-01

    Schizophrenia (SCZ) is a serious psychiatric disorder that affects 1% of general population and places a heavy burden worldwide. The underlying genetic mechanism of SCZ remains unknown, but studies indicate that the disease is associated with a global gene expression disturbance across many genes. Next-generation sequencing, particularly of RNA sequencing (RNA-Seq), provides a powerful genome-scale technology to investigate the pathological processes of SCZ. RNA-Seq has been used to analyze the gene expressions and identify the novel splice isoforms and rare transcripts associated with SCZ. This paper provides an overview on the genetics of SCZ, the advantages of RNA-Seq for transcriptome analysis, the accomplishments of RNA-Seq in SCZ cohorts, and the applications of induced pluripotent stem cells and RNA-Seq in SCZ research. PMID:27053919

  16. Multifunctional RNA Nanoparticles

    PubMed Central

    2015-01-01

    Our recent advancements in RNA nanotechnology introduced novel nanoscaffolds (nanorings); however, the potential of their use for biomedical applications was never fully revealed. As presented here, besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous embedment of assorted RNA aptamers, fluorescent dyes, proteins, as well as recently developed RNA–DNA hybrids aimed to conditionally activate multiple split functionalities inside cells. PMID:25267559

  17. Visualizing the global secondary structure of a viral RNA genome with cryo-electron microscopy

    PubMed Central

    Garmann, Rees F.; Gopal, Ajaykumar; Athavale, Shreyas S.; Knobler, Charles M.; Gelbart, William M.; Harvey, Stephen C.

    2015-01-01

    The lifecycle, and therefore the virulence, of single-stranded (ss)-RNA viruses is regulated not only by their particular protein gene products, but also by the secondary and tertiary structure of their genomes. The secondary structure of the entire genomic RNA of satellite tobacco mosaic virus (STMV) was recently determined by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE). The SHAPE analysis suggested a single highly extended secondary structure with much less branching than occurs in the ensemble of structures predicted by purely thermodynamic algorithms. Here we examine the solution-equilibrated STMV genome by direct visualization with cryo-electron microscopy (cryo-EM), using an RNA of similar length transcribed from the yeast genome as a control. The cryo-EM data reveal an ensemble of branching patterns that are collectively consistent with the SHAPE-derived secondary structure model. Thus, our results both elucidate the statistical nature of the secondary structure of large ss-RNAs and give visual support for modern RNA structure determination methods. Additionally, this work introduces cryo-EM as a means to distinguish between competing secondary structure models if the models differ significantly in terms of the number and/or length of branches. Furthermore, with the latest advances in cryo-EM technology, we suggest the possibility of developing methods that incorporate restraints from cryo-EM into the next generation of algorithms for the determination of RNA secondary and tertiary structures. PMID:25752599

  18. EM International. Volume 1

    SciTech Connect

    Not Available

    1993-07-01

    It is the intent of EM International to describe the Office of Environmental Restoration and Waste Management`s (EM`s) various roles and responsibilities within the international community. Cooperative agreements and programs, descriptions of projects and technologies, and synopses of visits to international sites are all highlighted in this semiannual journal. Focus on EM programs in this issue is on international collaboration in vitrification projects. Technology highlights covers: in situ sealing for contaminated sites; and remote sensors for toxic pollutants. Section on profiles of countries includes: Arctic contamination by the former Soviet Union, and EM activities with Germany--cooperative arrangements.

  19. RNA based evolutionary optimization

    NASA Astrophysics Data System (ADS)

    Schuster, Peter

    1993-12-01

    The notion of an RNA world has been introduced for a prebiotic scenario that is dominated by RNA molecules and their properties, in particular their capabilities to act as templates for reproduction and as catalysts for several cleavage and ligation reactions of polynucleotides and polypeptides. This notion is used here also for simple experimental assays which are well suited to study evolution in the test tube. In molecular evolution experiments fitness is determined in essence by the molecular structures of RNA molecules. Evidence is presented for adaptation to environment in cell-free media. RNA based molecular evolution experiments have led to interesting spin-offs in biotechnology, commonly called ‘applied molecular evolution’, which make use of Darwinian trial-and-error strategies in order to synthesize new pharmacological compounds and other advanced materials on a biological basis. Error-propagation in RNA replication leads to formation of mutant spectra called ‘quasispecies’. An increase in the error rate broadens the mutant spectrum. There exists a sharply defined threshold beyond which heredity breaks down and evolutionary adaptation becomes impossible. Almost all RNA viruses studied so far operate at conditions close to this error threshold. Quasispecies and error thresholds are important for an understanding of RNA virus evolution, and they may help to develop novel antiviral strategies. Evolution of RNA molecules can be studied and interpreted by considering secondary structures. The notion of sequence space introduces a distance between pairs of RNA sequences which is tantamount to counting the minimal number of point mutations required to convert the sequences into each other. The mean sensitivity of RNA secondary structures to mutation depends strongly on the base pairing alphabet: structures from sequences which contain only one base pair (GC or AU are much less stable against mutation than those derived from the natural (AUGC) sequences

  20. Ab initio RNA folding

    NASA Astrophysics Data System (ADS)

    Cragnolini, Tristan; Derreumaux, Philippe; Pasquali, Samuela

    2015-06-01

    RNA molecules are essential cellular machines performing a wide variety of functions for which a specific three-dimensional structure is required. Over the last several years, the experimental determination of RNA structures through x-ray crystallography and NMR seems to have reached a plateau in the number of structures resolved each year, but as more and more RNA sequences are being discovered, the need for structure prediction tools to complement experimental data is strong. Theoretical approaches to RNA folding have been developed since the late nineties, when the first algorithms for secondary structure prediction appeared. Over the last 10 years a number of prediction methods for 3D structures have been developed, first based on bioinformatics and data-mining, and more recently based on a coarse-grained physical representation of the systems. In this review we are going to present the challenges of RNA structure prediction and the main ideas behind bioinformatic approaches and physics-based approaches. We will focus on the description of the more recent physics-based phenomenological models and on how they are built to include the specificity of the interactions of RNA bases, whose role is critical in folding. Through examples from different models, we will point out the strengths of physics-based approaches, which are able not only to predict equilibrium structures, but also to investigate dynamical and thermodynamical behavior, and the open challenges to include more key interactions ruling RNA folding.

  1. Ab initio RNA folding.

    PubMed

    Cragnolini, Tristan; Derreumaux, Philippe; Pasquali, Samuela

    2015-06-17

    RNA molecules are essential cellular machines performing a wide variety of functions for which a specific three-dimensional structure is required. Over the last several years, the experimental determination of RNA structures through x-ray crystallography and NMR seems to have reached a plateau in the number of structures resolved each year, but as more and more RNA sequences are being discovered, the need for structure prediction tools to complement experimental data is strong. Theoretical approaches to RNA folding have been developed since the late nineties, when the first algorithms for secondary structure prediction appeared. Over the last 10 years a number of prediction methods for 3D structures have been developed, first based on bioinformatics and data-mining, and more recently based on a coarse-grained physical representation of the systems. In this review we are going to present the challenges of RNA structure prediction and the main ideas behind bioinformatic approaches and physics-based approaches. We will focus on the description of the more recent physics-based phenomenological models and on how they are built to include the specificity of the interactions of RNA bases, whose role is critical in folding. Through examples from different models, we will point out the strengths of physics-based approaches, which are able not only to predict equilibrium structures, but also to investigate dynamical and thermodynamical behavior, and the open challenges to include more key interactions ruling RNA folding. PMID:25993396

  2. CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains.

    PubMed

    Wright, Patrick R; Georg, Jens; Mann, Martin; Sorescu, Dragos A; Richter, Andreas S; Lott, Steffen; Kleinkauf, Robert; Hess, Wolfgang R; Backofen, Rolf

    2014-07-01

    CopraRNA (Comparative prediction algorithm for small RNA targets) is the most recent asset to the Freiburg RNA Tools webserver. It incorporates and extends the functionality of the existing tool IntaRNA (Interacting RNAs) in order to predict targets, interaction domains and consequently the regulatory networks of bacterial small RNA molecules. The CopraRNA prediction results are accompanied by extensive postprocessing methods such as functional enrichment analysis and visualization of interacting regions. Here, we introduce the functionality of the CopraRNA and IntaRNA webservers and give detailed explanations on their postprocessing functionalities. Both tools are freely accessible at http://rna.informatik.uni-freiburg.de. PMID:24838564

  3. CopraRNA and IntaRNA: predicting small RNA targets, networks and interaction domains

    PubMed Central

    Wright, Patrick R.; Georg, Jens; Mann, Martin; Sorescu, Dragos A.; Richter, Andreas S.; Lott, Steffen; Kleinkauf, Robert; Hess, Wolfgang R.; Backofen, Rolf

    2014-01-01

    CopraRNA (Comparative prediction algorithm for small RNA targets) is the most recent asset to the Freiburg RNA Tools webserver. It incorporates and extends the functionality of the existing tool IntaRNA (Interacting RNAs) in order to predict targets, interaction domains and consequently the regulatory networks of bacterial small RNA molecules. The CopraRNA prediction results are accompanied by extensive postprocessing methods such as functional enrichment analysis and visualization of interacting regions. Here, we introduce the functionality of the CopraRNA and IntaRNA webservers and give detailed explanations on their postprocessing functionalities. Both tools are freely accessible at http://rna.informatik.uni-freiburg.de. PMID:24838564

  4. Generation of siRNA Nanosheets for Efficient RNA Interference

    PubMed Central

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-01-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances. PMID:27120975

  5. lncRNA/MicroRNA interactions in the vasculature

    PubMed Central

    Ballantyne, MD; McDonald, RA

    2016-01-01

    MicroRNA (miRNA) have gained widespread attention for their role in diverse vascular processes including angiogenesis, apoptosis, proliferation, and migration. Despite great understanding of miRNA expression and function, knowledge of long noncoding RNA (lncRNA) molecular mechanisms still remains limited. The influence of miRNA on lncRNA function, and the converse, is now beginning to emerge. lncRNA may regulate miRNA function by acting as endogenous sponges to regulate gene expression and miRNA have been shown to bind and regulate lncRNA stability. A detailed understanding of the molecular and cellular effects of lncRNA‐miRNA‐mediated interactions in vascular pathophysiology could pave the way for new diagnostic markers and therapeutic approaches, but first there is a requirement for a more detailed understanding of the impact of such regulatory networks. PMID:26910520

  6. Generation of siRNA Nanosheets for Efficient RNA Interference

    NASA Astrophysics Data System (ADS)

    Kim, Hyejin; Lee, Jae Sung; Lee, Jong Bum

    2016-04-01

    After the discovery of small interference RNA (siRNA), nanostructured siRNA delivery systems have been introduced to achieve an efficient regulation of the target gene expression. Here we report a new siRNA-generating two dimensional nanostructure in a formation of nanosized sheet. Inspired by tunable mechanical and functional properties of the previously reported RNA membrane, siRNA nanosized sheets (siRNA-NS) with multiple Dicer cleavage sites were prepared. The siRNA-NS has two dimensional structure, providing a large surface area for Dicer to cleave the siRNA-NS for the generation of functional siRNAs. Furthermore, downregulation of the cellular target gene expression was achieved by delivery of siRNA-NS without chemical modification of RNA strands or conjugation to other substances.

  7. Shapes of Interacting RNA Complexes

    PubMed Central

    Fu, Benjamin M.M.

    2014-01-01

    Abstract Shapes of interacting RNA complexes are studied using a filtration via their topological genus. A shape of an RNA complex is obtained by (iteratively) collapsing stacks and eliminating hairpin loops. This shape projection preserves the topological core of the RNA complex, and for fixed topological genus there are only finitely many such shapes. Our main result is a new bijection that relates the shapes of RNA complexes with shapes of RNA structures. This allows for computing the shape polynomial of RNA complexes via the shape polynomial of RNA structures. We furthermore present a linear time uniform sampling algorithm for shapes of RNA complexes of fixed topological genus. PMID:25075750

  8. Multi-target siRNA: Therapeutic Strategy for Hepatocellular Carcinoma

    PubMed Central

    Li, Tiejun; Xue, Yuwen; Wang, Guilan; Gu, Tingting; Li, Yunlong; Zhu, York Yuanyuan; Chen, Li

    2016-01-01

    Multiple targets RNAi strategy is a preferred way to treat multigenic diseases, especially cancers. In the study, multi-target siRNAs were designed to inhibit NET-1, EMS1 and VEGF genes in hepatocellular carcinoma (HCC) cells. And multi-target siRNAs showed better silencing effects on NET-1, EMS1 and VEGF, compared with single target siRNA. Moreover, multi-target siRNA showed greater suppression effects on proliferation, migration, invasion, angiogenesis and induced apoptosis in HCC cells. The results suggested that multi-target siRNA might be a preferred strategy for cancer therapy and NET-1, EMS1 and VEGF could be effective targets for HCC treatments. PMID:27390607

  9. Minotaur is critical for primary piRNA biogenesis

    PubMed Central

    Vagin, Vasily V.; Yu, Yang; Jankowska, Anna; Luo, Yicheng; Wasik, Kaja A.; Malone, Colin D.; Harrison, Emily; Rosebrock, Adam; Wakimoto, Barbara T.; Fagegaltier, Delphine; Muerdter, Felix; Hannon, Gregory J.

    2013-01-01

    Piwi proteins and their associated small RNAs are essential for fertility in animals. In part, this is due to their roles in guarding germ cell genomes against the activity of mobile genetic elements. piRNA populations direct Piwi proteins to silence transposon targets and, as such, form a molecular code that discriminates transposons from endogenous genes. Information ultimately carried by piRNAs is encoded within genomic loci, termed piRNA clusters. These give rise to long, single-stranded, primary transcripts that are processed into piRNAs. Despite the biological importance of this pathway, neither the characteristics that define a locus as a source of piRNAs nor the mechanisms that catalyze primary piRNA biogenesis are well understood. We searched an EMS-mutant collection annotated for fertility phenotypes for genes involved in the piRNA pathway. Twenty-seven homozygous sterile strains showed transposon-silencing defects. One of these, which strongly impacted primary piRNA biogenesis, harbored a causal mutation in CG5508, a member of the Drosophila glycerol-3-phosphate O-acetyltransferase (GPAT) family. These enzymes catalyze the first acylation step on the path to the production of phosphatidic acid (PA). Though this pointed strongly to a function for phospholipid signaling in the piRNA pathway, a mutant form of CG5508, which lacks the GPAT active site, still functions in piRNA biogenesis. We have named this new biogenesis factor Minotaur. PMID:23788724

  10. Specific ligation to double-stranded RNA for analysis of cellular RNA::RNA interactions.

    PubMed

    Faridani, Omid R; McInerney, Gerald M; Gradin, Katarina; Good, Liam

    2008-09-01

    Double-stranded RNA (dsRNA) is formed in cells as intra- and intermolecular RNA interactions and is involved in a range of biological processes including RNA metabolism, RNA interference and translation control mediated by natural antisense RNA and microRNA. Despite this breadth of activities, few molecular tools are available to analyse dsRNA as native hybrids. We describe a two-step ligation method for enzymatic joining of dsRNA adaptors to any dsRNA molecule in its duplex form without a need for prior sequence or termini information. The method is specific for dsRNA and can ligate various adaptors to label, map or amplify dsRNA sequences. When combined with reverse transcription-polymerase chain reaction, the method is sensitive and can detect low nanomolar concentrations of dsRNA in total RNA. As examples, we mapped dsRNA/single-stranded RNA junctions within Escherichia coli hok mRNA and the human immunodeficiency virus TAR element using RNA from bacteria and mammalian cells. PMID:18628292

  11. Structure and assembly of the essential RNA ring component of a viral DNA packaging motor.

    PubMed

    Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R; Anderson, Dwight L; Jardine, Paul J; Grimes, Shelley; Ke, Ailong

    2011-05-01

    Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage 29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 Å resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of 29 DNA. PMID:21471452

  12. Structure and assembly of the essential RNA ring component of a viral DNA packaging motor

    SciTech Connect

    Ding, Fang; Lu, Changrui; Zhao, Wei; Rajashankar, Kanagalaghatta R.; Anderson, Dwight L.; Jardine, Paul J.; Grimes, Shelley; Ke, Ailong

    2011-07-25

    Prohead RNA (pRNA) is an essential component in the assembly and operation of the powerful bacteriophage {psi}29 DNA packaging motor. The pRNA forms a multimeric ring via intermolecular base-pairing interactions between protomers that serves to guide the assembly of the ring ATPase that drives DNA packaging. Here we report the quaternary structure of this rare multimeric RNA at 3.5 {angstrom} resolution, crystallized as tetrameric rings. Strong quaternary interactions and the inherent flexibility helped rationalize how free pRNA is able to adopt multiple oligomerization states in solution. These characteristics also allowed excellent fitting of the crystallographic pRNA protomers into previous prohead/pRNA cryo-EM reconstructions, supporting the presence of a pentameric, but not hexameric, pRNA ring in the context of the DNA packaging motor. The pentameric pRNA ring anchors itself directly to the phage prohead by interacting specifically with the fivefold symmetric capsid structures that surround the head-tail connector portal. From these contacts, five RNA superhelices project from the pRNA ring, where they serve as scaffolds for binding and assembly of the ring ATPase, and possibly mediate communication between motor components. Construction of structure-based designer pRNAs with little sequence similarity to the wild-type pRNA were shown to fully support the packaging of {psi}29 DNA.

  13. Biology Today: Respect for RNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C., Ed.

    1991-01-01

    The high points of the story of RNA are presented. The functions of RNA within the cell, how these functions are carried out, and how they evolved are described. The topics of splicing, self-splicing, RNA editing, transcription and translation, and antisense RNA are discussed. (KR)

  14. Topology of RNA-RNA interaction structures.

    PubMed

    Andersen, Jørgen E; Huang, Fenix W D; Penner, Robert C; Reidys, Christian M

    2012-07-01

    The topological filtration of interacting RNA complexes is studied, and the role is analyzed of certain diagrams called irreducible shadows, which form suitable building blocks for more general structures. We prove that, for two interacting RNAs, called interaction structures, there exist for fixed genus only finitely many irreducible shadows. This implies that, for fixed genus, there are only finitely many classes of interaction structures. In particular, the simplest case of genus zero already provides the formalism for certain types of structures that occur in nature and are not covered by other filtrations. This case of genus zero interaction structures is already of practical interest, is studied here in detail, and is found to be expressed by a multiple context-free grammar that extends the usual one for RNA secondary structures. We show that, in O(n(6)) time and O(n(4)) space complexity, this grammar for genus zero interaction structures provides not only minimum free energy solutions but also the complete partition function and base pairing probabilities. PMID:22731621

  15. Alignments of RNA structures.

    PubMed

    Blin, Guillaume; Denise, Alain; Dulucq, Serge; Herrbach, Claire; Touzet, Hélène

    2010-01-01

    We describe a theoretical unifying framework to express the comparison of RNA structures, which we call alignment hierarchy. This framework relies on the definition of common supersequences for arc-annotated sequences and encompasses the main existing models for RNA structure comparison based on trees and arc-annotated sequences with a variety of edit operations. It also gives rise to edit models that have not been studied yet. We provide a thorough analysis of the alignment hierarchy, including a new polynomial-time algorithm and an NP-completeness proof. The polynomial-time algorithm involves biologically relevant edit operations such as pairing or unpairing nucleotides. It has been implemented in a software, called gardenia, which is available at the Web server http://bioinfo.lifl.fr/RNA/gardenia. PMID:20431150

  16. Pyrite footprinting of RNA

    SciTech Connect

    Schlatterer, Joerg C.; Wieder, Matthew S.; Jones, Christopher D.; Pollack, Lois; Brenowitz, Michael

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer RNA structure is mapped by pyrite mediated {sup {center_dot}}OH footprinting. Black-Right-Pointing-Pointer Repetitive experiments can be done in a powdered pyrite filled cartridge. Black-Right-Pointing-Pointer High {sup {center_dot}}OH reactivity of nucleotides imply dynamic role in Diels-Alderase catalysis. -- Abstract: In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals ({sup {center_dot}}OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS{sub 2}) can produce sufficient {sup {center_dot}}OH to footprint DNA. The 49-nt Diels-Alder RNA enzyme catalyzes the C-C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme's active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels-Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to {sup {center_dot}}OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop's flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated {sup {center_dot}}OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.

  17. Editor meets silencer: crosstalk between RNA editing and RNA interference

    PubMed Central

    Nishikura, Kazuko

    2010-01-01

    The most prevalent type of RNA editing is mediated by ADAR (adenosine deaminase acting on RNA) enzymes, which convert adenosines to inosines (a process known as A→I RNA editing) in double-stranded (ds)RNA substrates. A→I RNA editing was long thought to affect only selected transcripts by altering the proteins they encode. However, genome-wide screening has revealed numerous editing sites within inverted Alu repeats in introns and untranslated regions. Also, recent evidence indicates that A→I RNA editing crosstalks with RNA-interference pathways, which, like A→I RNA editing, involve dsRNAs. A→I RNA editing therefore seems to have additional functions, including the regulation of retrotransposons and gene silencing, which adds a new urgency to the challenges of fully understanding ADAR functions. PMID:17139332

  18. RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

    PubMed Central

    Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

    2015-01-01

    MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. PMID:26674414

  19. Genome-scale identification of miRNA-mRNA and miRNA-lncRNA interactions in domestic animals.

    PubMed

    Li, A; Zhang, J; Zhou, Z; Wang, L; Sun, X; Liu, Y

    2015-12-01

    Domestic animals show considerable genetic diversity. Previous studies suggested that animal phenotypes were affected by miRNA-mRNA interplay, but these studies focused mainly on the analysis of one or several miRNA-mRNA interactions. However, in this study, we investigated miRNA-mRNA and miRNA-lncRNA interactions on a genomic scale using miranda and targetscan algorithms. There has been strong directional artificial selection practiced during the domestication of animals. Thus, we investigated SNPs that were located in miRNAs and miRNA binding sites and found that several SNPs located in 3'-UTRs of mRNAs had the potential to affect miRNA-mRNA interactions. In addition, a database, named miRBond, was developed to provide visualization, analysis and downloading of the resulting datasets. Our results open the way to further experimental verification of miRNA-mRNA and miRNA-lncRNA interactions as well as the influence of SNPs upon such interplay. PMID:26360131

  20. [Ribosomal RNA Evolution

    NASA Technical Reports Server (NTRS)

    1997-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modern organisms came to exist by the time of the last common ancestor (as detected by 16S rRNA sequence studies). Third, the ribosomal RNAs themselves likely had a very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.During the past year we have ave conducted a comparative study of four completely sequenced bacterial genoames. We have focused initially on conservation of gene order. The second component of the project continues to build on the model system for studying the validity of variant 5S rRNA sequences in the vicinity of the modern Vibrio proteolyticus 5S rRNA that we established earlier. This system has made it possible to conduct a detailed and extensive analysis of a local portion of the sequence space. These core methods have been used to construct numerous mutants during the last several years. Although it has been a secondary focus, this work has continued over the last year such that we now have in excess of 125 V. proteolyticus derived constructs which have been made and characterized. We have also continued high resolution NMR work on RNA oligomers originally initiated by G. Kenneth Smith who was funded by a NASA Graduate Student Researcher's Fellowship Award until May of 1996. Mr. Smith

  1. Direct Characterization of Transcription Elongation by RNA Polymerase I.

    PubMed

    Ucuncuoglu, Suleyman; Engel, Krysta L; Purohit, Prashant K; Dunlap, David D; Schneider, David A; Finzi, Laura

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  2. Direct Characterization of Transcription Elongation by RNA Polymerase I

    PubMed Central

    Ucuncuoglu, Suleyman; Engel, Krysta L.; Purohit, Prashant K.; Dunlap, David D.; Schneider, David A.

    2016-01-01

    RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of transcription in a growing cell. Despite this fundamental role that directly impacts cell growth and proliferation, the kinetics of transcription by Pol I are poorly understood. This study provides direct characterization of S. Cerevisiae Pol I transcription elongation using tethered particle microscopy (TPM). Pol I was shown to elongate at an average rate of approximately 20 nt/s. However, the maximum speed observed was, in average, about 60 nt/s, comparable to the rate calculated based on the in vivo number of active genes, the cell division rate and the number of engaged polymerases observed in EM images. Addition of RNA endonucleases to the TPM elongation assays enhanced processivity. Together, these data suggest that additional transcription factors contribute to efficient and processive transcription elongation by RNA polymerase I in vivo. PMID:27455049

  3. RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

    PubMed Central

    Liao, C L; Lai, M M

    1992-01-01

    Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur

  4. Transcriptome assembly and quantification from Ion Torrent RNA-Seq data

    PubMed Central

    2014-01-01

    Background High throughput RNA sequencing (RNA-Seq) can generate whole transcriptome information at the single transcript level providing a powerful tool with multiple interrelated applications including transcriptome reconstruction and quantification. The sequences of novel transcripts can be reconstructed from deep RNA-Seq data, but this is computationally challenging due to sequencing errors, uneven coverage of expressed transcripts, and the need to distinguish between highly similar transcripts produced by alternative splicing. Another challenge in transcriptomic analysis comes from the ambiguities in mapping reads to transcripts. Results We present MaLTA, a method for simultaneous transcriptome assembly and quantification from Ion Torrent RNA-Seq data. Our approach explores transcriptome structure and incorporates a maximum likelihood model into the assembly and quantification procedure. A new version of the IsoEM algorithm suitable for Ion Torrent RNA-Seq reads is used to accurately estimate transcript expression levels. The MaLTA-IsoEM tool is publicly available at: http://alan.cs.gsu.edu/NGS/?q=malta Conclusions Experimental results on both synthetic and real datasets show that Ion Torrent RNA-Seq data can be successfully used for transcriptome analyses. Experimental results suggest increased transcriptome assembly and quantification accuracy of MaLTA-IsoEM solution compared to existing state-of-the-art approaches. PMID:25082147

  5. Mapping RNA-RNA interactome and RNA structure in vivo by MARIO.

    PubMed

    Nguyen, Tri C; Cao, Xiaoyi; Yu, Pengfei; Xiao, Shu; Lu, Jia; Biase, Fernando H; Sridhar, Bharat; Huang, Norman; Zhang, Kang; Zhong, Sheng

    2016-01-01

    The pervasive transcription of our genome presents a possibility of revealing new genomic functions by investigating RNA interactions. Current methods for mapping RNA-RNA interactions have to rely on an 'anchor' protein or RNA and often require molecular perturbations. Here we present the MARIO (Mapping RNA interactome in vivo) technology to massively reveal RNA-RNA interactions from unperturbed cells. We mapped tens of thousands of endogenous RNA-RNA interactions from mouse embryonic stem cells and brain. We validated seven interactions by RNA antisense purification and one interaction using single-molecule RNA-FISH. The experimentally derived RNA interactome is a scale-free network, which is not expected from currently perceived promiscuity in RNA-RNA interactions. Base pairing is observed at the interacting regions between long RNAs, including transposon transcripts, suggesting a class of regulatory sequences acting in trans. In addition, MARIO data reveal thousands of intra-molecule interactions, providing in vivo data on high-order RNA structures. PMID:27338251

  6. The RNA interference revolution.

    PubMed

    Lenz, G

    2005-12-01

    The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular mechanism present in the majority of eukaryotic cells and access to a potent and specific new method for gene silencing. PMID:16302089

  7. Shaping tRNA

    ERIC Educational Resources Information Center

    Priano, Christine

    2013-01-01

    This model-building activity provides a quick, visual, hands-on tool that allows students to examine more carefully the cloverleaf structure of a typical tRNA molecule. When used as a supplement to lessons that involve gene expression, this exercise reinforces several concepts in molecular genetics, including nucleotide base-pairing rules, the…

  8. RNA in the Loop

    PubMed Central

    Kung, Johnny T.Y.; Lee, Jeannie T.

    2013-01-01

    Long noncoding RNAs (lncRNAs) have been implicated in a variety of biological roles, particularly as cis or trans gene expression regulators. Reporting recently in Nature, Lai et al. (2013) show that a class of gene-activating lncRNAs combines two gene regulation paradigms: enhancer-directed chromosomal looping and RNA-mediated protein effector recruitment. PMID:23537627

  9. Divergent RNA transcription

    PubMed Central

    Naughton, Catherine; Corless, Samuel; Gilbert, Nick

    2013-01-01

    New approaches using biotinylated-psoralen as a probe for investigating DNA structure have revealed new insights into the relationship between DNA supercoiling, transcription and chromatin compaction. We explore a hypothesis that divergent RNA transcription generates negative supercoiling at promoters facilitating initiation complex formation and subsequent promoter clearance. PMID:23863199

  10. Messenger RNA (mRNA) nanoparticle tumour vaccination

    NASA Astrophysics Data System (ADS)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  11. Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling

    PubMed Central

    McDonald, Richard I.; Guilinger, John P.; Mukherji, Shankar; Curtis, Edward A.; Lee, Won I.; Liu, David R.

    2014-01-01

    Probes that form covalent bonds with RNA molecules based on their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind to the ASH1 mRNA. PMID:25306441

  12. Mammalian synthetic circuits with RNA binding proteins delivered by RNA

    PubMed Central

    Wroblewska, Liliana; Kitada, Tasuku; Endo, Kei; Siciliano, Velia; Stillo, Breanna; Saito, Hirohide; Weiss, Ron

    2015-01-01

    Synthetic regulatory circuits encoded on RNA rather than DNA could provide a means to control cell behavior while avoiding potentially harmful genomic integration in therapeutic applications. We create post-transcriptional circuits using RNA-binding proteins, which can be wired in a plug-and-play fashion to create networks of higher complexity. We show that the circuits function in mammalian cells when encoded on modified mRNA or self-replicating RNA. PMID:26237515

  13. Protein-RNA networks revealed through covalent RNA marks.

    PubMed

    Lapointe, Christopher P; Wilinski, Daniel; Saunders, Harriet A J; Wickens, Marvin

    2015-12-01

    Protein-RNA networks are ubiquitous and central in biological control. We present an approach termed RNA Tagging that enables the user to identify protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or cross-linking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA via high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The results showed that although RNA-binding proteins productively bind specific RNAs to control their function, they also 'sample' RNAs without exerting a regulatory effect. We used the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. RNA Tagging is well suited to detect and analyze protein-RNA networks in vivo. PMID:26524240

  14. Quantitative Model of microRNA-mRNA interaction

    NASA Astrophysics Data System (ADS)

    Noorbakhsh, Javad; Lang, Alex; Mehta, Pankaj

    2012-02-01

    MicroRNAs are short RNA sequences that regulate gene expression and protein translation by binding to mRNA. Experimental data reveals the existence of a threshold linear output of protein based on the expression level of microRNA. To understand this behavior, we propose a mathematical model of the chemical kinetics of the interaction between mRNA and microRNA. Using this model we have been able to quantify the threshold linear behavior. Furthermore, we have studied the effect of internal noise, showing the existence of an intermediary regime where the expression level of mRNA and microRNA has the same order of magnitude. In this crossover regime the mRNA translation becomes sensitive to small changes in the level of microRNA, resulting in large fluctuations in protein levels. Our work shows that chemical kinetics parameters can be quantified by studying protein fluctuations. In the future, studying protein levels and their fluctuations can provide a powerful tool to study the competing endogenous RNA hypothesis (ceRNA), in which mRNA crosstalk occurs due to competition over a limited pool of microRNAs.

  15. Protein-RNA networks revealed through covalent RNA marks

    PubMed Central

    Lapointe, Christopher P.; Wilinski, Daniel; Saunders, Harriet A. J.; Wickens, Marvin

    2015-01-01

    Protein-RNA networks are ubiquitous and central in biological control. We present an approach, termed “RNA Tagging,” that identifies protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or crosslinking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA using high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The method revealed that while RNA-binding proteins productively bind specific RNAs to control their function, they also “sample” RNAs without exerting a regulatory effect. We exploited the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. The RNA Tagging approach is well-suited to detect and analyze protein-RNA networks in vivo. PMID:26524240

  16. RNA-RNA interaction prediction using genetic algorithm

    PubMed Central

    2014-01-01

    Background RNA-RNA interaction plays an important role in the regulation of gene expression and cell development. In this process, an RNA molecule prohibits the translation of another RNA molecule by establishing stable interactions with it. In the RNA-RNA interaction prediction problem, two RNA sequences are given as inputs and the goal is to find the optimal secondary structure of two RNAs and between them. Some different algorithms have been proposed to predict RNA-RNA interaction structure. However, most of them suffer from high computational time. Results In this paper, we introduce a novel genetic algorithm called GRNAs to predict the RNA-RNA interaction. The proposed algorithm is performed on some standard datasets with appropriate accuracy and lower time complexity in comparison to the other state-of-the-art algorithms. In the proposed algorithm, each individual is a secondary structure of two interacting RNAs. The minimum free energy is considered as a fitness function for each individual. In each generation, the algorithm is converged to find the optimal secondary structure (minimum free energy structure) of two interacting RNAs by using crossover and mutation operations. Conclusions This algorithm is properly employed for joint secondary structure prediction. The results achieved on a set of known interacting RNA pairs are compared with the other related algorithms and the effectiveness and validity of the proposed algorithm have been demonstrated. It has been shown that time complexity of the algorithm in each iteration is as efficient as the other approaches. PMID:25114714

  17. Ribosomal Protein S14 of Saccharomyces cerevisiae Regulates Its Expression by Binding to RPS14B Pre-mRNA and to 18S rRNA

    PubMed Central

    Fewell, Sheara W.; Woolford, John L.

    1999-01-01

    Production of ribosomal protein S14 in Saccharomyces cerevisiae is coordinated with the rate of ribosome assembly by a feedback mechanism that represses expression of RPS14B. Three-hybrid assays in vivo and filter binding assays in vitro demonstrate that rpS14 directly binds to an RNA stem-loop structure in RPS14B pre-mRNA that is necessary for RPS14B regulation. Moreover, rpS14 binds to a conserved helix in 18S rRNA with approximately five- to sixfold-greater affinity. These results support the model that RPS14B regulation is mediated by direct binding of rpS14 either to its pre-mRNA or to rRNA. Investigation of these interactions with the three-hybrid system reveals two regions of rpS14 that are involved in RNA recognition. D52G and E55G mutations in rpS14 alter the specificity of rpS14 for RNA, as indicated by increased affinity for RPS14B RNA but reduced affinity for the rRNA target. Deletion of the C terminus of rpS14, where multiple antibiotic resistance mutations map, prevents binding of rpS14 to RNA and production of functional 40S subunits. The emetine-resistant protein, rpS14-EmRR, which contains two mutations near the C terminus of rpS14, does not bind either RNA target in the three-hybrid or in vitro assays. This is the first direct demonstration that an antibiotic resistance mutation alters binding of an r protein to rRNA and is consistent with the hypothesis that antibiotic resistance mutations can result from local alterations in rRNA structure. PMID:9858605

  18. RNA Interference in Ticks

    PubMed Central

    Kocan, Katherine M.; Blouin, Edmour; de la Fuente, José

    2011-01-01

    Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5. RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10. RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines

  19. lncRNA-RNA Interactions across the Human Transcriptome

    PubMed Central

    Szcześniak, Michał Wojciech; Makałowska, Izabela

    2016-01-01

    Long non-coding RNAs (lncRNAs) represent a numerous class of non-protein coding transcripts longer than 200 nucleotides. There is possibility that a fraction of lncRNAs are not functional and represent mere transcriptional noise but a growing body of evidence shows they are engaged in a plethora of molecular functions and contribute considerably to the observed diversification of eukaryotic transcriptomes and proteomes. Still, however, only ca. 1% of lncRNAs have well established functions and much remains to be done towards decipherment of their biological roles. One of the least studied aspects of lncRNAs biology is their engagement in gene expression regulation through RNA-RNA interactions. By hybridizing with mate RNA molecules, lncRNAs could potentially participate in modulation of pre-mRNA splicing, RNA editing, mRNA stability control, translation activation, or abrogation of miRNA-induced repression. Here, we implemented a similarity-search based method for transcriptome-wide identification of RNA-RNA interactions, which enabled us to find 18,871,097 lncRNA-RNA base-pairings in human. Further analyses showed that the interactions could be involved in processing, stability control and functions of 57,303 transcripts. An extensive use of RNA-Seq data provided support for approximately one third of the interactions, at least in terms of the two RNA components being co-expressed. The results suggest that lncRNA-RNA interactions are broadly used to regulate and diversify the human transcriptome. PMID:26930590

  20. Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

    PubMed Central

    Li, Wen; Liu, Zheng; Koripella, Ravi Kiran; Langlois, Robert; Sanyal, Suparna; Frank, Joachim

    2015-01-01

    During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome. PMID:26229983

  1. 'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target.

    PubMed

    Lustig, Yaniv; Wachtel, Chaim; Safro, Mark; Liu, Li; Michaeli, Shulamit

    2010-01-01

    In this study we describe a novel method to investigate the RNA-RNA interactions between a small RNA and its target that we termed 'RNA walk'. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT-PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by 'RNA walk' and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that 'RNA walk, is a generic method to map target RNA small RNAs interactions in vivo. PMID:19854950

  2. Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

    SciTech Connect

    Bodkin, D.K.

    1985-01-01

    RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to (5'/sup 32/P)-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52/sup 0/C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share greater than or equal to 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups.

  3. Amplification of RNA by an RNA polymerase ribozyme.

    PubMed

    Horning, David P; Joyce, Gerald F

    2016-08-30

    In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life-the replication of genetic information and its conversion into functional molecules-can now be accomplished with RNA in the complete absence of proteins. PMID:27528667

  4. RNA-SSPT: RNA Secondary Structure Prediction Tools.

    PubMed

    Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

    2013-01-01

    The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes. PMID:24250115

  5. Current preclinical small interfering RNA (siRNA)-based conjugate systems for RNA therapeutics.

    PubMed

    Lee, Soo Hyeon; Kang, Yoon Young; Jang, Hyo-Eun; Mok, Hyejung

    2016-09-01

    Recent promising clinical results of RNA therapeutics have drawn big attention of academia and industries to RNA therapeutics and their carrier systems. To improve their feasibility in clinics, systemic evaluations of currently available carrier systems under clinical trials and preclinical studies are needed. In this review, we focus on recent noticeable preclinical studies and clinical results regarding siRNA-based conjugates for clinical translations. Advantages and drawbacks of siRNA-based conjugates are discussed, compared to particle-based delivery systems. Then, representative siRNA-based conjugates with aptamers, peptides, carbohydrates, lipids, polymers, and nanostructured materials are introduced. To improve feasibility of siRNA conjugates in preclinical studies, several considerations for the rational design of siRNA conjugates in terms of cleavability, immune responses, multivalent conjugations, and mechanism of action are also presented. Lastly, we discuss lessons from previous preclinical and clinical studies related to siRNA conjugates and perspectives of their clinical applications. PMID:26514375

  6. Visualizing nuclear export of different classes of RNA by electron microscopy.

    PubMed

    Panté, N; Jarmolowski, A; Izaurralde, E; Sauder, U; Baschong, W; Mattaj, I W

    1997-05-01

    Export of RNA from the cell nucleus to the cytoplasm occurs through nuclear pore complexes (NPCs). To examine nuclear export of RNA, we have gold-labeled different types of RNA (i.e., mRNA, tRNA, U snRNAs), and followed their export by electron microscopy (EM) after their microinjection into Xenopus oocyte nuclei. By changing the polarity of the negatively charged colloidal gold, complexes with mRNA, tRNA, and U1 snRNA can be formed efficiently, and gold-tagged RNAs are exported to the cytoplasm with kinetics and specific saturation behavior similar to that of unlabeled RNAs. U6 snRNA conjugates, in contrast, remain in the nucleus, as does naked U6 snRNA. During export, RNA-gold was found distributed along the central axis of the NPC, within the nuclear basket, or accumulated at the nuclear and cytoplasmic periphery of the central gated channel, but not associated with the cytoplasmic fibrils. In an attempt to identify the initial NPC docking site(s) for RNA, we have explored various conditions that either yield docking of import ligands to the NPC or inhibit the export of nuclear RNAs. Surprisingly, we failed to observe docking of RNA destined for export at the nuclear periphery of the NPC under any of these conditions. Instead, each condition in which export of any of the RNA-gold conjugates was inhibited caused accumulation of gold particles scattered uniformly throughout the nucleoplasm. These results point to the existence of steps in export involving mobilization of the export substrate from the nucleoplasm to the NPC. PMID:9149231

  7. The Impact of mRNA Structure on Guide RNA Targeting in Kinetoplastid RNA Editing

    PubMed Central

    Reifur, Larissa; Yu, Laura E.; Cruz-Reyes, Jorge; vanHartesvelt, Michelle; Koslowsky, Donna J.

    2010-01-01

    Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process. In this study, we selected four mRNAs with distinct target structures and investigated how sequence and structure affected efficient gRNA targeting. Two of the mRNAs, including the ATPase subunit 6 and ND7-550 (5′ end of NADH dehydrogenase subunit 7) that have open, accessible anchor binding sites show very efficient gRNA targeting. Electrophoretic mobility shift assays indicate that the cognate gRNA for ND7-550 had 10-fold higher affinity for its mRNA than the A6 pair. Surface plasmon resonance studies indicate that the difference in affinity was due to a four-fold faster association rate. As expected, mRNAs with considerable structure surrounding the anchor binding sites were less accessible and had very low affinity for their cognate gRNAs. In vitro editing assays indicate that efficient pairing is crucial for gRNA directed cleavage. However, only the A6 substrate showed gRNA-directed cleavage at the correct editing site. This suggests that different gRNA/mRNA pairs may require different “sets” of accessory factors for efficient editing. By characterizing a number of different gRNA/mRNA interactions, we may be able to define a “bank” of RNA editing substrates with different putative chaperone and other co-factor requirements. This will allow the more efficient identification and characterization of transcript specific RNA editing accessory proteins. PMID:20808932

  8. Barotrauma em peixes em usinas hidrelétricas: ferramentas para o estudo

    SciTech Connect

    Do Vale Beirao, Bernardo; Castelo Branco Marciano, Natlia; de Souza Dias, Luma; Carvalho Falco, Ricardo; Wander Dias, Edson; Leite Fabrino, Daniela; Barreira Martinez, Carlos; Martins Da Silva, Luiz Gustavo; Walker, Ricardo W.; Brown, Richard S.; Deng, Zhiqun

    2015-09-30

    The main source of electric power generation in Brazil comes from hydropower plants, nevertheless, the installed power is expected to raise 56.8%, reaching a total of 116,000 MW at the year 2020. The increase at the hydroelectric sector will be responsible for a series of fish community impacts. One of the impacts over the fish community is related to fish kills due to downstream passage through turbines or fish entrance at the draft tube from the tailrace. Usually when there is a maneuver and the turbine stops, fish get attracted and enter the draft tube and, just as the downstream passage through a turbine, when the turbine starts, a rapid decompression occurs and can cause barotrauma. When such events happen, according to Boyle’s law (P1V1=P2V2), swim bladder volume expands at the same rate that the pressure decreases, which can lead to the organ’s rupture.

  9. Semiautomated improvement of RNA alignments

    PubMed Central

    Andersen, Ebbe S.; Lind-Thomsen, Allan; Knudsen, Bjarne; Kristensen, Susie E.; Havgaard, Jakob H.; Torarinsson, Elfar; Larsen, Niels; Zwieb, Christian; Sestoft, Peter; Kjems, Jørgen; Gorodkin, Jan

    2007-01-01

    We have developed a semiautomated RNA sequence editor (SARSE) that integrates tools for analyzing RNA alignments. The editor highlights different properties of the alignment by color, and its integrated analysis tools prevent the introduction of errors when doing alignment editing. SARSE readily connects to external tools to provide a flexible semiautomatic editing environment. A new method, Pcluster, is introduced for dividing the sequences of an RNA alignment into subgroups with secondary structure differences. Pcluster was used to evaluate 574 seed alignments obtained from the Rfam database and we identified 71 alignments with significant prediction of inconsistent base pairs and 102 alignments with significant prediction of novel base pairs. Four RNA families were used to illustrate how SARSE can be used to manually or automatically correct the inconsistent base pairs detected by Pcluster: the mir-399 RNA, vertebrate telomase RNA (vert-TR), bacterial transfer-messenger RNA (tmRNA), and the signal recognition particle (SRP) RNA. The general use of the method is illustrated by the ability to accommodate pseudoknots and handle even large and divergent RNA families. The open architecture of the SARSE editor makes it a flexible tool to improve all RNA alignments with relatively little human intervention. Online documentation and software are available at http://sarse.ku.dk. PMID:17804647

  10. Bacteriophage T5 transfer RNA

    SciTech Connect

    Hunt, C.; Desai, S.M.; Vaughan, J.; Weiss, S.B.

    1980-04-10

    Previous studies from this laboratory have provided a high resolution map for 16 tRNA genes located on the continuous heavy DNA strand of bacteriophage T5 DNA. All of the T5 tRNA genes were located in three clusters within the DNA C segment, except for tRNA/sup Arg/, which mapped on the left end of the DNA D segment. In this report, we present evidence for the presence of eight additional T5 tRNA species, five of which are located in two new loci within the DNA C segment. We also describe a two-dimensional gel electrophoresis system for the separation and isolation of T5 tRNA species from crude infected RNA preparations. The gel electrophoresis system separates tRNA isoacceptors specific for different amino acids; evidence is presented that the isoacceptors for isoleucine, histidine, and serine are coded by different T5 genes.

  11. Flavivirus RNA synthesis in vitro.

    PubMed

    Padmanabhan, Radhakrishnan; Takhampunya, Ratree; Teramoto, Tadahisa; Choi, Kyung H

    2015-12-01

    Establishment of in vitro systems to study mechanisms of RNA synthesis for positive strand RNA viruses have been very useful in the past and have shed light on the composition of protein and RNA components, optimum conditions, the nature of the products formed, cis-acting RNA elements and trans-acting protein factors required for efficient synthesis. In this review, we summarize our current understanding regarding the requirements for flavivirus RNA synthesis in vitro. We describe details of reaction conditions, the specificity of template used by either the multi-component membrane-bound viral replicase complex or by purified, recombinant RNA-dependent RNA polymerase. We also discuss future perspectives to extend the boundaries of our knowledge. PMID:26272247

  12. The tmRNA website

    DOE PAGESBeta

    Hudson, Corey M.; Williams, Kelly P.

    2014-11-05

    We report that the transfer-messenger RNA (tmRNA) and its partner protein SmpB act together in resolving problems arising when translating bacterial ribosomes reach the end of mRNA with no stop codon. Their genes have been found in nearly all bacterial genomes and in some organelles. The tmRNA Website serves tmRNA sequences, alignments and feature annotations, and has recently moved to http: //bioinformatics.sandia.gov/tmrna/. New features include software used to find the sequences, an update raising the number of unique tmRNA sequences from 492 to 1716, and a database of SmpB sequences which are served along with the tmRNA sequence from themore » same organism.« less

  13. The tmRNA website

    SciTech Connect

    Hudson, Corey M.; Williams, Kelly P.

    2014-11-05

    We report that the transfer-messenger RNA (tmRNA) and its partner protein SmpB act together in resolving problems arising when translating bacterial ribosomes reach the end of mRNA with no stop codon. Their genes have been found in nearly all bacterial genomes and in some organelles. The tmRNA Website serves tmRNA sequences, alignments and feature annotations, and has recently moved to http: //bioinformatics.sandia.gov/tmrna/. New features include software used to find the sequences, an update raising the number of unique tmRNA sequences from 492 to 1716, and a database of SmpB sequences which are served along with the tmRNA sequence from the same organism.

  14. Global Mapping of Human RNA-RNA Interactions.

    PubMed

    Sharma, Eesha; Sterne-Weiler, Tim; O'Hanlon, Dave; Blencowe, Benjamin J

    2016-05-19

    The majority of the human genome is transcribed into non-coding (nc)RNAs that lack known biological functions or else are only partially characterized. Numerous characterized ncRNAs function via base pairing with target RNA sequences to direct their biological activities, which include critical roles in RNA processing, modification, turnover, and translation. To define roles for ncRNAs, we have developed a method enabling the global-scale mapping of RNA-RNA duplexes crosslinked in vivo, "LIGation of interacting RNA followed by high-throughput sequencing" (LIGR-seq). Applying this method in human cells reveals a remarkable landscape of RNA-RNA interactions involving all major classes of ncRNA and mRNA. LIGR-seq data reveal unexpected interactions between small nucleolar (sno)RNAs and mRNAs, including those involving the orphan C/D box snoRNA, SNORD83B, that control steady-state levels of its target mRNAs. LIGR-seq thus represents a powerful approach for illuminating the functions of the myriad of uncharacterized RNAs that act via base-pairing interactions. PMID:27184080

  15. Transfer RNA and human disease

    PubMed Central

    Abbott, Jamie A.; Francklyn, Christopher S.; Robey-Bond, Susan M.

    2014-01-01

    Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are “hotspots” for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease. PMID:24917879

  16. Transfer RNA and human disease.

    PubMed

    Abbott, Jamie A; Francklyn, Christopher S; Robey-Bond, Susan M

    2014-01-01

    Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease. PMID:24917879

  17. RNA Thermodynamic Structural Entropy

    PubMed Central

    Garcia-Martin, Juan Antonio; Clote, Peter

    2015-01-01

    Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner’99 and Turner’04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http

  18. Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro.

    PubMed

    Reichert, Vienna L; Choi, Mehee; Petrillo, Jessica E; Gehrke, Lee

    2007-07-20

    Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals. PMID:17400272

  19. RNA Binding Proteins in the miRNA Pathway

    PubMed Central

    Connerty, Patrick; Ahadi, Alireza; Hutvagner, Gyorgy

    2015-01-01

    microRNAs (miRNAs) are short ~22 nucleotides (nt) ribonucleic acids which post-transcriptionally regulate gene expression. miRNAs are key regulators of all cellular processes, and the correct expression of miRNAs in an organism is crucial for proper development and cellular function. As a result, the miRNA biogenesis pathway is highly regulated. In this review, we outline the basic steps of miRNA biogenesis and miRNA mediated gene regulation focusing on the role of RNA binding proteins (RBPs). We also describe multiple mechanisms that regulate the canonical miRNA pathway, which depends on a wide range of RBPs. Moreover, we hypothesise that the interaction between miRNA regulation and RBPs is potentially more widespread based on the analysis of available high-throughput datasets. PMID:26712751

  20. Structure of an RNA polymerase II preinitiation complex

    PubMed Central

    Murakami, Kenji; Tsai, Kuang-Lei; Kalisman, Nir; Bushnell, David A.; Asturias, Francisco J.; Kornberg, Roger D.

    2015-01-01

    The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6–11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein–protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription. PMID:26483468

  1. Structure of an RNA polymerase II preinitiation complex.

    PubMed

    Murakami, Kenji; Tsai, Kuang-Lei; Kalisman, Nir; Bushnell, David A; Asturias, Francisco J; Kornberg, Roger D

    2015-11-01

    The structure of a 33-protein, 1.5-MDa RNA polymerase II preinitiation complex (PIC) was determined by cryo-EM and image processing at a resolution of 6-11 Å. Atomic structures of over 50% of the mass were fitted into the electron density map in a manner consistent with protein-protein cross-links previously identified by mass spectrometry. The resulting model of the PIC confirmed the main conclusions from previous cryo-EM at lower resolution, including the association of promoter DNA only with general transcription factors and not with the polymerase. Electron density due to DNA was identifiable by the grooves of the double helix and exhibited sharp bends at points downstream of the TATA box, with an important consequence: The DNA at the downstream end coincides with the DNA in a transcribing polymerase. The structure of the PIC is therefore conducive to promoter melting, start-site scanning, and the initiation of transcription. PMID:26483468

  2. mRNA transcript therapy.

    PubMed

    Weissman, Drew

    2015-02-01

    mRNA is the central molecule of all forms of life. It is generally accepted that current life on Earth descended from an RNA world. mRNA, after its first therapeutic description in 1992, has recently come into increased focus as a method to deliver genetic information. The recent solution to the two main difficulties in using mRNA as a therapeutic, immune stimulation and potency, has provided the basis for a wide range of applications. While mRNA-based cancer immunotherapies have been in clinical trials for a few years, novel approaches; including, in vivo delivery of mRNA to replace or supplement proteins, mRNA-based generation of pluripotent stem cells, or genome engineering using mRNA-encoded meganucleases are beginning to be realized. This review presents the current state of mRNA drug technologies and potential applications, as well as discussing the challenges and prospects in mRNA development and drug discovery. PMID:25359562

  3. Subgenomic mRNA regulation by a distal RNA element in a (+)-strand RNA virus.

    PubMed Central

    Zhang, G; Slowinski, V; White, K A

    1999-01-01

    Subgenomic (sg) mRNAs are synthesized by (+)-strand RNA viruses to allow for efficient translation of products encoded 3' in their genomes. This strategy also provides a means for regulating the expression of such products via modulation of sg mRNA accumulation. We have studied the mechanism by which sg mRNAs levels are controlled in tomato bushy stunt virus, a small (+)-strand RNA virus which synthesizes two sg mRNAs during infections. Neither the viral capsid nor movement proteins were found to play any significant role in modulating the accumulation levels of either sg mRNA. Deletion analysis did, however, identify a 12-nt-long RNA sequence located approximately 1,000 nt upstream from the site of initiation of sg mRNA2 synthesis that was required specifically for accumulation of sg mRNA2. Further analysis revealed a potential base-pairing interaction between this sequence and a sequence located just 5' to the site of initiation for sg mRNA2 synthesis. Mutant genomes in which this interaction was either disrupted or maintained were analyzed and the results indicated a positive correlation between the predicted stability of the base-pairing interaction and the efficiency of sg mRNA2 accumulation. The functional significance of the long-distance interaction was further supported by phylogenetic sequence analysis which revealed conservation of base-pairing interactions of similar stability and relative position in the genomes of different tombusviruses. It is proposed that the upstream sequence represents a cis-acting RNA element which facilitates sg mRNA accumulation by promoting efficient synthesis of sg mRNA2 via a long-distance RNA-RNA interaction. PMID:10199571

  4. The RNA shapes studio

    PubMed Central

    Janssen, Stefan; Giegerich, Robert

    2015-01-01

    Motivation: Abstract shape analysis, first proposed in 2004, allows one to extract several relevant structures from the folding space of an RNA sequence, preferable to focusing in a single structure of minimal free energy. We report recent extensions to this approach. Results: We have rebuilt the original RNAshapes as a repository of components that allows us to integrate several established tools for RNA structure analysis: RNAshapes, RNAalishapes and pknotsRG, including its recent extension pKiss. As a spin-off, we obtain heretofore unavailable functionality: e. g. with pKiss, we can now perform abstract shape analysis for structures holding pseudoknots up to the complexity of kissing hairpin motifs. The new tool pAliKiss can predict kissing hairpin motifs from aligned sequences. Along with the integration, the functionality of the tools was also extended in manifold ways. Availability and implementation: As before, the tool is available on the Bielefeld Bioinformatics server at http://bibiserv.cebitec.uni-bielefeld.de/rnashapesstudio. Contact: bibi-help@cebitec.uni-bielefeld.de PMID:25273103

  5. RNA-Seq and find: entering the RNA deep field

    PubMed Central

    2011-01-01

    Initial high-throughput RNA sequencing (RNA-Seq) experiments have revealed a complex and dynamic transcriptome, but because it samples transcripts in proportion to their abundances, assessing the extent and nature of low-level transcription using this technique has been difficult. A new assay, RNA CaptureSeq, addresses this limitation of RNA-Seq by enriching for low-level transcripts with cDNA tiling arrays prior to high-throughput sequencing. This approach reveals a plethora of transcripts that have been previously dismissed as 'noise', and hints at single-cell transcription fingerprints that may be crucial in defining cellular function in normal and disease states. PMID:22113004

  6. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    PubMed

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme. PMID:25161314

  7. antaRNA: ant colony-based RNA sequence design

    PubMed Central

    Kleinkauf, Robert; Mann, Martin; Backofen, Rolf

    2015-01-01

    Motivation: RNA sequence design is studied at least as long as the classical folding problem. Although for the latter the functional fold of an RNA molecule is to be found, inverse folding tries to identify RNA sequences that fold into a function-specific target structure. In combination with RNA-based biotechnology and synthetic biology, reliable RNA sequence design becomes a crucial step to generate novel biochemical components. Results: In this article, the computational tool antaRNA is presented. It is capable of compiling RNA sequences for a given structure that comply in addition with an adjustable full range objective GC-content distribution, specific sequence constraints and additional fuzzy structure constraints. antaRNA applies ant colony optimization meta-heuristics and its superior performance is shown on a biological datasets. Availability and implementation: http://www.bioinf.uni-freiburg.de/Software/antaRNA Contact: backofen@informatik.uni-freiburg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26023105

  8. Tiempo para un cambio

    NASA Astrophysics Data System (ADS)

    Woltjer, L.

    1987-06-01

    En la reunion celebrada en diciembre dei ano pasado informe al Consejo de mi deseo de terminar mi contrato como Director General de la ESO una vez que fuera aprobado el proyecto dei VLT, que se espera sucedera hacia fines de este aAo. Cuando fue renovada mi designacion hace tres aAos, el Consejo conocia mi intencion de no completar los cinco aAos dei contrato debido a mi deseo de disponer de mas tiempo para otras actividades. Ahora, una vez terminada la fase preparatoria para el VLT, Y habiendose presentado el proyecto formalmente al Consejo el dia 31 de marzo, y esperando su muy probable aprobacion antes dei termino de este ano, me parece que el 10 de enero de 1988 presenta una excelente fecha para que se produzca un cambio en la administracion de la ESO.

  9. Plant snoRNA database

    PubMed Central

    Brown, John W. S.; Echeverria, Manuel; Qu, Liang-Hu; Lowe, Todd M.; Bachellerie, Jean-Pierre; Hüttenhofer, Alexander; Kastenmayer, James P.; Green, Pamela J.; Shaw, Paul; Marshall, Dave F.

    2003-01-01

    The Plant snoRNA database (http://www.scri.sari.ac.uk/plant_snoRNA/) provides information on small nucleolar RNAs from Arabidopsis and eighteen other plant species. Information includes sequences, expression data, methylation and pseudouridylation target modification sites, initial gene organization (polycistronic, single gene and intronic) and the number of gene variants. The Arabidopsis information is divided into box C/D and box H/ACA snoRNAs, and within each of these groups, by target sites in rRNA, snRNA or unknown. Alignments of orthologous genes and gene variants from different plant species are available for many snoRNA genes. Plant snoRNA genes have been given a standard nomenclature, designed wherever possible, to provide a consistent identity with yeast and human orthologues. PMID:12520043

  10. mRNA-mRNA duplexes that autoelicit Staufen1-mediated mRNA decay.

    PubMed

    Gong, Chenguang; Tang, Yalan; Maquat, Lynne E

    2013-10-01

    We report a new mechanism by which human mRNAs cross-talk: an Alu element in the 3' untranslated region (3' UTR) of one mRNA can base-pair with a partially complementary Alu element in the 3' UTR of a different mRNA, thereby creating a Staufen1 (STAU1)-binding site (SBS). STAU1 binding to a 3'-UTR SBS was previously shown to trigger STAU1-mediated mRNA decay (SMD) by directly recruiting the ATP-dependent RNA helicase UPF1, which is also a key factor in the mechanistically related nonsense-mediated mRNA decay (NMD) pathway. In the case of a 3'-UTR SBS created by mRNA-mRNA base-pairing, we show that SMD targets both mRNAs in the duplex, provided that both mRNAs are translated. If only one mRNA is translated, then it alone is targeted for SMD. We demonstrate the functional importance of mRNA-mRNA-triggered SMD in cell migration and invasion. PMID:24056942

  11. Hyperexpansion of RNA Bacteriophage Diversity

    PubMed Central

    Krishnamurthy, Siddharth R.; Janowski, Andrew B.; Zhao, Guoyan; Barouch, Dan; Wang, David

    2016-01-01

    Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent. PMID:27010970

  12. Hyperexpansion of RNA Bacteriophage Diversity.

    PubMed

    Krishnamurthy, Siddharth R; Janowski, Andrew B; Zhao, Guoyan; Barouch, Dan; Wang, David

    2016-03-01

    Bacteriophage modulation of microbial populations impacts critical processes in ocean, soil, and animal ecosystems. However, the role of bacteriophages with RNA genomes (RNA bacteriophages) in these processes is poorly understood, in part because of the limited number of known RNA bacteriophage species. Here, we identify partial genome sequences of 122 RNA bacteriophage phylotypes that are highly divergent from each other and from previously described RNA bacteriophages. These novel RNA bacteriophage sequences were present in samples collected from a range of ecological niches worldwide, including invertebrates and extreme microbial sediment, demonstrating that they are more widely distributed than previously recognized. Genomic analyses of these novel bacteriophages yielded multiple novel genome organizations. Furthermore, one RNA bacteriophage was detected in the transcriptome of a pure culture of Streptomyces avermitilis, suggesting for the first time that the known tropism of RNA bacteriophages may include gram-positive bacteria. Finally, reverse transcription PCR (RT-PCR)-based screening for two specific RNA bacteriophages in stool samples from a longitudinal cohort of macaques suggested that they are generally acutely present rather than persistent. PMID:27010970

  13. Compact intermediates in RNA folding

    SciTech Connect

    Woodson, S.A.

    2011-12-14

    Large noncoding RNAs fold into their biologically functional structures via compact yet disordered intermediates, which couple the stable secondary structure of the RNA with the emerging tertiary fold. The specificity of the collapse transition, which coincides with the assembly of helical domains, depends on RNA sequence and counterions. It determines the specificity of the folding pathways and the magnitude of the free energy barriers to the ensuing search for the native conformation. By coupling helix assembly with nascent tertiary interactions, compact folding intermediates in RNA also play a crucial role in ligand binding and RNA-protein recognition.

  14. Synthetic biology with RNA motifs.

    PubMed

    Saito, Hirohide; Inoue, Tan

    2009-02-01

    Structural motifs in naturally occurring RNAs and RNPs can be employed as new molecular parts for synthetic biology to facilitate the development of novel devices and systems that modulate cellular functions. In this review, we focus on the following: (i) experimental evolution techniques of RNA molecules in vitro and (ii) their applications for regulating gene expression systems in vivo. For experimental evolution, new artificial RNA aptamers and RNA enzymes (ribozymes) have been selected in vitro. These functional RNA molecules are likely to be applicable in the reprogramming of existing gene regulatory systems. Furthermore, they may be used for designing hypothetical RNA-based living systems in the so-called RNA world. For the regulation of gene expressions in living cells, the development of new riboswitches allows us to modulate the target gene expression in a tailor-made manner. Moreover, recently RNA-based synthetic genetic circuits have been reported by employing functional RNA molecules, expanding the repertory of synthetic biology with RNA motifs. PMID:18775792

  15. emGain: Determination of EM gain of CCD

    NASA Astrophysics Data System (ADS)

    Daigle, Olivier; Carignan, Claude; Blais-Ouellette, Sebastien

    2012-01-01

    The determination of the EM gain of the CCD is best done by fitting the histogram of many low-light frames. Typically, the dark+CIC noise of a 30ms frame itself is a sufficient amount of signal to determine accurately the EM gain with about 200 512x512 frames. The IDL code emGain takes as an input a cube of frames and fit the histogram of all the pixels with the EM stage output probability function. The function returns the EM gain of the frames as well as the read-out noise and the mean signal level of the frames.

  16. Bringing RNA into View: RNA and Its Roles in Biology.

    ERIC Educational Resources Information Center

    Atkins, John F.; Ellington, Andrew; Friedman, B. Ellen; Gesteland, Raymond F.; Noller, Harry F.; Pasquale, Stephen M.; Storey, Richard D.; Uhlenbeck, Olke C.; Weiner, Alan M.

    This guide presents a module for college students on ribonucleic acid (RNA) and its role in biology. The module aims to integrate the latest research and its findings into college-level biology and provide an opportunity for students to understand biological processes. Four activities are presented: (1) "RNA Structure: Tapes to Shapes"; (2) "RNA…

  17. What is an RNA? A top layer for RNA classification

    PubMed Central

    Brosius, Jürgen; Raabe, Carsten A.

    2016-01-01

    ABSTRACT Every ribonucleic acid begins its cellular life as a transcript. If the transcript or its processing product has a function it should be regarded an RNA. Nonfunctional transcripts, by-products from processing, degradation intermediates, even those originating from (functional) RNAs, and non-functional products of transcriptional gene regulation accomplished via the act of transcription, as well as stochastic (co)transcripts could simply be addressed as transcripts (class 0). The copious functional RNAs (class I), often maturing after one or more processing steps, already are systematized into ever expanding sub-classifications ranging from micro RNAs to rRNAs. Established sub-classifications addressing a wide functional diversity remain unaffected. mRNAs (class II) are distinct from any other RNA by virtue of their potential to be translated into (poly)peptide(s) on ribosomes. We are not proposing a novel RNA classification, but wish to add a basic concept with existing terminology (transcript, RNA, and mRNA) that should serve as an additional framework for carefully delineating RNA function from an avalanche of RNA sequencing data. At the same time, this top level hierarchical model should illuminate important principles of RNA evolution and biology thus heightening our awareness that in biology boundaries and categorizations are typically fuzzy. PMID:26818079

  18. The pivotal regulatory landscape of RNA modifications.

    PubMed

    Li, Sheng; Mason, Christopher E

    2014-01-01

    Posttranscriptionally modified nucleosides in RNA play integral roles in the cellular control of biological information that is encoded in DNA. The modifications of RNA span all three phylogenetic domains (Archaea, Bacteria, and Eukarya) and are pervasive across RNA types, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and (less frequently) small nuclear RNA (snRNA) and microRNA (miRNA). Nucleotide modifications are also one of the most evolutionarily conserved properties of RNAs, and the sites of modification are under strong selective pressure. However, many of these modifications, as well as their prevalence and impact, have only recently been discovered. Here, we examine both labile and permanent modifications, from simple methylation to complex transcript alteration (RNA editing and intron retention); detail the models for their processing; and highlight remaining questions in the field of the epitranscriptome. PMID:24898039

  19. RCARE: RNA Sequence Comparison and Annotation for RNA Editing

    PubMed Central

    2015-01-01

    The post-transcriptional sequence modification of transcripts through RNA editing is an important mechanism for regulating protein function and is associated with human disease phenotypes. The identification of RNA editing or RNA-DNA difference (RDD) sites is a fundamental step in the study of RNA editing. However, a substantial number of false-positive RDD sites have been identified recently. A major challenge in identifying RDD sites is to distinguish between the true RNA editing sites and the false positives. Furthermore, determining the location of condition-specific RDD sites and elucidating their functional roles will help toward understanding various biological phenomena that are mediated by RNA editing. The present study developed RNA-sequence comparison and annotation for RNA editing (RCARE) for searching, annotating, and visualizing RDD sites using thousands of previously known editing sites, which can be used for comparative analyses between multiple samples. RCARE also provides evidence for improving the reliability of identified RDD sites. RCARE is a web-based comparison, annotation, and visualization tool, which provides rich biological annotations and useful summary plots. The developers of previous tools that identify or annotate RNA-editing sites seldom mention the reliability of their respective tools. In order to address the issue, RCARE utilizes a number of scientific publications and databases to find specific documentations respective to a particular RNA-editing site, which generates evidence levels to convey the reliability of RCARE. Sequence-based alignment files can be converted into VCF files using a Python script and uploaded to the RCARE server for further analysis. RCARE is available for free at http://www.snubi.org/software/rcare/. PMID:26043858

  20. dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing.

    PubMed Central

    Palladino, M J; Keegan, L P; O'Connell, M A; Reenan, R A

    2000-01-01

    We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR. The dADAR locus has been localized to the 2B6-7 region of the X chromosome and the complete genomic sequence organization is reported here. dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B. Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates. Transcripts of the dADAR locus originate from two regulated promoters. In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs. dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated. PMID:10917596

  1. Molecular structures of unbound and transcribing RNA polymerase III

    PubMed Central

    Hoffmann, Niklas A.; Jakobi, Arjen J.; Moreno-Morcillo, Maria; Glatt, Sebastian; Kosinski, Jan; Hagen, Wim J. H.; Sachse, Carsten; Müller, Christoph W.

    2015-01-01

    Transcription of genes encoding small structured RNAs such as tRNAs, spliceosomal U6 snRNA and ribosomal 5S RNA is carried out by RNA polymerase III (Pol III), the largest yet structurally least characterized eukaryotic RNA polymerase. The cryo-EM structures of the S. cerevisiae Pol III elongating complex at 3.9 Å resolution and the apo Pol III enzyme in two different conformations at 4.6 and 4.7 Å resolution, respectively, allow for the first time to build a 17-subunit atomic model of Pol III. The reconstructions reveal the precise orientation of the C82/C34/C31 heterotrimer in close proximity to the stalk. The C53/C37 heterodimer positions residues involved in transcription termination close to the non-template DNA strand. In the apo Pol III structures, the stalk adopts different orientations coupled with closed and open conformations of the clamp. Our results provide novel insights into Pol III-specific transcription and the adaptation of Pol III towards its small transcriptional targets. PMID:26605533

  2. Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles

    PubMed Central

    2015-01-01

    Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use. PMID:25521794

  3. Improvement of DNA minicircle production by optimization of the secondary structure of the 5'-UTR of ParA resolvase.

    PubMed

    Šimčíková, Michaela; Alves, Cláudia P A; Brito, Liliana; Prather, Kristala L J; Prazeres, Duarte M F; Monteiro, Gabriel A

    2016-08-01

    The use of minicircles in gene therapy applications is dependent on the availability of high-producer cell systems. In order to improve the performance of minicircle production in Escherichia coli by ParA resolvase-mediated in vivo recombination, we focus on the 5' untranslated region (5'-UTR) of parA messenger RNA (mRNA). The arabinose-inducible PBAD/araC promoter controls ParA expression and strains with improved arabinose uptake are used. The 27-nucleotide-long 5'-UTR of parA mRNA was optimized using a predictive thermodynamic model. An analysis of original and optimized mRNA subsequences predicted a decrease of 8.6-14.9 kcal/mol in the change in Gibbs free energy upon assembly of the 30S ribosome complex with the mRNA subsequences, indicating a more stable mRNA-rRNA complex and enabling a higher (48-817-fold) translation initiation rate. No effect of the 5'-UTR was detected when ParA was expressed from a low-copy number plasmid (∼14 copies/cell), with full recombination obtained within 2 h. However, when the parA gene was inserted in the bacterial chromosome, a faster and more effective recombination was obtained with the optimized 5'-UTR. Interestingly, the amount of this transcript was 2.6-3-fold higher when compared with the transcript generated from the original sequence, highlighting that 5'-UTR affects the level of the transcript. A Western blot analysis confirmed that E. coli synthesized higher amounts of ParA with the new 5'-UTR (∼1.8 ± 0.7-fold). Overall, these results show that the improvements made in the 5'-UTR can lead to a more efficient translation and hence to faster and more efficient minicircle generation. PMID:27147534

  4. Bifurcations in the interplay of messenger RNA, protein and nonprotein coding RNA

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2008-07-01

    The interplay of messenger RNA (mRNA), protein, produced via translation of this RNA, and nonprotein coding RNA (ncRNA) may include regulation of the ncRNA production by protein and (i) ncRNA-protein association resulting in suppression of the protein regulatory activity or (ii) ncRNA-mRNA association resulting in degradation of the miRNA-mRNA complex. The kinetic models describing these two scenarios are found to predict bistability provided that protein suppresses the ncRNA formation.

  5. Exploration of RNA structure spaces

    NASA Technical Reports Server (NTRS)

    Fox, G. E.

    1991-01-01

    In order to understand the structure of real structure spaces, we are studying the 5S rRNA structure space experimentally. A plasmid containing a synthetic 5S rRNA gene, two rRNA promoters, and transcription terminators has been assembled. Assays are conducted to determine if the foreign 5S rRNA is expressed, and to see whether or not it is incorporated into ribosomes. Evolutionary competition is used to determine the relative fitness of strains containing the foreign 5S rRNA and a control 5S rRNA. By using site directed mutagenesis, a number of mutants can be made in order to study the boundaries of the structure space and how sharply defined they are. By making similar studies in the vicinity of structure space, it will be possible to determine how homogeneous the 5S rRNA structure space is. Useable experimental protocols have been developed, and a number of mutants have already been studied. Initial results suggest an explanation of why single stranded regions of the RNA are less subject to mutation than double stranded regions.

  6. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

    EPA Science Inventory

    This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

  7. Catalysis and prebiotic RNA synthesis

    NASA Technical Reports Server (NTRS)

    Ferris, James P.

    1993-01-01

    The essential role of catalysis for the origins of life is discussed. The status of the prebiotic synthesis of 2',5'- and 3'5'-linked oligomers of RNA is reviewed. Examples of the role of metal ion and mineral catalysis in RNA oligomer formation are discussed.

  8. Self-assembling RNA square

    SciTech Connect

    Dibrov, Sergey M.; McLean, Jaime; Parsons, Jerod; Hermann, Thomas

    2011-12-22

    The three-dimensional structures of noncoding RNA molecules reveal recurring architectural motifs that have been exploited for the design of artificial RNA nanomaterials. Programmed assembly of RNA nanoobjects from autonomously folding tetraloop-receptor complexes as well as junction motifs has been achieved previously through sequence-directed hybridization of complex sets of long oligonucleotides. Due to size and complexity, structural characterization of artificial RNA nanoobjects has been limited to low-resolution microscopy studies. Here we present the design, construction, and crystal structure determination at 2.2 {angstrom} of the smallest yet square-shaped nanoobject made entirely of double-stranded RNA. The RNA square is comprised of 100 residues and self-assembles from four copies each of two oligonucleotides of 10 and 15 bases length. Despite the high symmetry on the level of secondary structure, the three-dimensional architecture of the square is asymmetric, with all four corners adopting distinct folding patterns. We demonstrate the programmed self-assembly of RNA squares from complex mixtures of corner units and establish a concept to exploit the RNA square as a combinatorial nanoscale platform.

  9. Small RNA Deep Sequencing Reveals Role for Arabidopsis thaliana RNA-Dependent RNA Polymerases in Viral siRNA Biogenesis

    PubMed Central

    Qi, Xiaopeng; Bao, Forrest Sheng; Xie, Zhixin

    2009-01-01

    RNA silencing functions as an important antiviral defense mechanism in a broad range of eukaryotes. In plants, biogenesis of several classes of endogenous small interfering RNAs (siRNAs) requires RNA-dependent RNA Polymerase (RDR) activities. Members of the RDR family proteins, including RDR1and RDR6, have also been implicated in antiviral defense, although a direct role for RDRs in viral siRNA biogenesis has yet to be demonstrated. Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profile in wild-type plants as well as rdr mutants by applying small RNA deep sequencing technology. Over 100,000 TMV-Cg-specific small RNA reads, mostly of 21- (78.4%) and 22-nucleotide (12.9%) in size and originating predominately (79.9%) from the genomic sense RNA strand, were captured at an early infection stage, yielding the first high-resolution small RNA map for a plant virus. The TMV-Cg genome harbored multiple, highly reproducible small RNA-generating hot spots that corresponded to regions with no apparent local hairpin-forming capacity. Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis. In addition, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing. Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP α), respectively, yielded a positive result in cleavage validation by 5′RACE assays. Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity. PMID:19308254

  10. Structure of the RNA claw of the DNA packaging motor of bacteriophage Φ29.

    PubMed

    Harjes, Elena; Kitamura, Aya; Zhao, Wei; Morais, Marc C; Jardine, Paul J; Grimes, Shelley; Matsuo, Hiroshi

    2012-10-01

    Bacteriophage DNA packaging motors translocate their genomic DNA into viral heads, compacting it to near-crystalline density. The Bacillus subtilis phage 29 has a unique ring of RNA (pRNA) that is an essential component of its motor, serving as a scaffold for the packaging ATPase. Previously, deletion of a three-base bulge (18-CCA-20) in the pRNA A-helix was shown to abolish packaging activity. Here, we solved the structure of this crucial bulge by nuclear magnetic resonance (NMR) using a 27mer RNA fragment containing the bulge (27b). The bulge actually involves five nucleotides (17-UCCA-20 and A100), as U17 and A100 are not base paired as predicted. Mutational analysis showed these newly identified bulge residues are important for DNA packaging. The bulge introduces a 33-35° bend in the helical axis, and inter-helical motion around this bend appears to be restricted. A model of the functional 120b pRNA was generated using a 27b NMR structure and the crystal structure of the 66b prohead-binding domain. Fitting this model into a cryo-EM map generated a pentameric pRNA structure; five helices projecting from the pRNA ring resemble an RNA claw. Biochemical analysis suggested that this shape is important for coordinated motor action required for DNA translocation. PMID:22879380

  11. [Capping strategies in RNA viruses].

    PubMed

    Bouvet, Mickaël; Ferron, François; Imbert, Isabelle; Gluais, Laure; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; Decroly, Etienne

    2012-04-01

    Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential. PMID:22549871

  12. Improving NMR Structures of RNA.

    PubMed

    Bermejo, Guillermo A; Clore, G Marius; Schwieters, Charles D

    2016-05-01

    Here, we show that modern solution nuclear magnetic resonance (NMR) structures of RNA exhibit more steric clashes and conformational ambiguities than their crystallographic X-ray counterparts. To tackle these issues, we developed RNA-ff1, a new force field for structure calculation with Xplor-NIH. Using seven published NMR datasets, RNA-ff1 improves covalent geometry and MolProbity validation criteria for clashes and backbone conformation in most cases, relative to both the previous Xplor-NIH force field and the original structures associated with the experimental data. In addition, with smaller base-pair step rises in helical stems, RNA-ff1 structures enjoy more favorable base stacking. Finally, structural accuracy improves in the majority of cases, as supported by complete residual dipolar coupling cross-validation. Thus, the reported advances show great promise in bridging the quality gap that separates NMR and X-ray structures of RNA. PMID:27066747

  13. RNA Protein Interaction in Neurons

    PubMed Central

    Darnell, Robert B.

    2013-01-01

    Neurons have their own systems for regulating RNA. Several multigene families encode RNA binding proteins (RNABPs) that are uniquely expressed in neurons, including the well-known neuron-specific markers ELAV and NeuN, and the disease antigen NOVA. New technologies have emerged in recent years to assess the function of these proteins in vivo, and the answers are yielding insights into how and why neurons may regulate RNA in special ways—to increase cellular complexity, to spatially localize mRNA, and to regulate their expression in response to synaptic stimuli. The functions of such restricted neuronal proteins is likely to be complimented by more widely expressed RNABPs that may themselves have developed specialized functions in neurons, including Argonaute/miRNAs. Here we review what is known about such RNABPs, and explore the potential biologic and neurologic significance of neuronal RNA regulatory systems. PMID:23701460

  14. Phenotypic MicroRNA Microarrays

    PubMed Central

    Kwon, Yong-Jun; Heo, Jin Yeong; Kim, Hi Chul; Kim, Jin Yeop; Liuzzi, Michel; Soloveva, Veronica

    2013-01-01

    Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.

  15. Statistical Analysis of RNA Backbone

    PubMed Central

    Hershkovitz, Eli; Sapiro, Guillermo; Tannenbaum, Allen; Williams, Loren Dean

    2009-01-01

    Local conformation is an important determinant of RNA catalysis and binding. The analysis of RNA conformation is particularly difficult due to the large number of degrees of freedom (torsion angles) per residue. Proteins, by comparison, have many fewer degrees of freedom per residue. In this work, we use and extend classical tools from statistics and signal processing to search for clusters in RNA conformational space. Results are reported both for scalar analysis, where each torsion angle is separately studied, and for vectorial analysis, where several angles are simultaneously clustered. Adapting techniques from vector quantization and clustering to the RNA structure, we find torsion angle clusters and RNA conformational motifs. We validate the technique using well-known conformational motifs, showing that the simultaneous study of the total torsion angle space leads to results consistent with known motifs reported in the literature and also to the finding of new ones. PMID:17048391

  16. Messenger RNA (mRNA) Nanoparticle Tumour Vaccination

    PubMed Central

    Phua, Kyle K.L.; Nair, Smita K.; Leong, Kam W.

    2014-01-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA’s biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research. PMID:24904987

  17. The RNA WikiProject: community annotation of RNA families.

    PubMed

    Daub, Jennifer; Gardner, Paul P; Tate, John; Ramsköld, Daniel; Manske, Magnus; Scott, William G; Weinberg, Zasha; Griffiths-Jones, Sam; Bateman, Alex

    2008-12-01

    The online encyclopedia Wikipedia has become one of the most important online references in the world and has a substantial and growing scientific content. A search of Google with many RNA-related keywords identifies a Wikipedia article as the top hit. We believe that the RNA community has an important and timely opportunity to maximize the content and quality of RNA information in Wikipedia. To this end, we have formed the RNA WikiProject (http://en.wikipedia.org/wiki/Wikipedia:WikiProject_RNA) as part of the larger Molecular and Cellular Biology WikiProject. We have created over 600 new Wikipedia articles describing families of noncoding RNAs based on the Rfam database, and invite the community to update, edit, and correct these articles. The Rfam database now redistributes this Wikipedia content as the primary textual annotation of its RNA families. Users can, therefore, for the first time, directly edit the content of one of the major RNA databases. We believe that this Wikipedia/Rfam link acts as a functioning model for incorporating community annotation into molecular biology databases. PMID:18945806

  18. The RNA synthesis machinery of negative-stranded RNA viruses

    SciTech Connect

    Ortín, Juan; Martín-Benito, Jaime

    2015-05-15

    The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

  19. mRNA Translocation Occurs During the Second Step of Ribosomal Intersubunit Rotation

    PubMed Central

    Ermolenko, Dmitri N.; Noller, Harry F.

    2010-01-01

    During protein synthesis, mRNA and tRNA undergo coupled translocation through the ribosome in a process that is catalyzed by elongation factor EF-G. Based on cryo-EM reconstructions, counterclockwise and clockwise rotational movements between the large and small ribosomal subunits have been implicated in a proposed ratcheting mechanism to drive the unidirectional movement of translocation. We have used a combination of two fluorescence-based approaches to study the timing of these events: Intersubunit FRET measurements to observe relative rotational movement of the subunits and a fluorescence quenching assay to monitor translocation of mRNA. Binding of EF-G·GTP first induces rapid counterclockwise intersubunit rotation, followed by a slower, clockwise reversal of the rotational movement. Comparison of the rates of these movements reveals that mRNA translocation occurs during the second, clockwise rotation event, corresponding to the transition from the hybrid state to the classical state. PMID:21399643

  20. RNA Viruses: RNA Roles in Pathogenesis, Coreplication and Viral Load

    PubMed Central

    Poltronieri, Palmiro; Sun, Binlian; Mallardo, Massimo

    2015-01-01

    The review intends to present and recapitulate the current knowledge on the roles and importance of regulatory RNAs, such as microRNAs and small interfering RNAs, RNA binding proteins and enzymes processing RNAs or activated by RNAs, in cells infected by RNA viruses. The review focuses on how non-coding RNAs are involved in RNA virus replication, pathogenesis and host response, especially in retroviruses HIV, with examples of the mechanisms of action, transcriptional regulation, and promotion of increased stability of their targets or their degradation. PMID:27047253

  1. RNA template-directed RNA synthesis by T7 RNA polymerase.

    PubMed Central

    Cazenave, C; Uhlenbeck, O C

    1994-01-01

    In an attempt to synthesize an oligoribonucleotide by run-off transcription by bacteriophage T7 RNA polymerase, a major transcript was produced that was much longer than expected. Analysis of the reaction indicated that the product resulted from initial DNA-directed run-off transcription followed by RNA template-directed RNA synthesis. This reaction occurred because the RNA made from the DNA template displayed self-complementarity at its 3' end and therefore could form an intra- or intermolecular primed template. In reactions containing only an RNA template, the rate of incorporation of NTPs was quite comparable to DNA-dependent transcription. RNA template-directed RNA synthesis has been found to occur with a great number of oligoribonucleotides, even with primed templates that are only marginally stable. In one instance, we observed a multistep extension reaction converting the oligonucleotide into a final product longer than twice its original length. Presumably, such a process could have generated some of the RNAs found to be efficiently replicated by T7 RNA polymerase. Images PMID:7518923

  2. The EM Earthquake Precursor

    NASA Astrophysics Data System (ADS)

    Jones, K. B., II; Saxton, P. T.

    2013-12-01

    Many attempts have been made to determine a sound forecasting method regarding earthquakes and warn the public in turn. Presently, the animal kingdom leads the precursor list alluding to a transmission related source. By applying the animal-based model to an electromagnetic (EM) wave model, various hypotheses were formed, but the most interesting one required the use of a magnetometer with a differing design and geometry. To date, numerous, high-end magnetometers have been in use in close proximity to fault zones for potential earthquake forecasting; however, something is still amiss. The problem still resides with what exactly is forecastable and the investigating direction of EM. After the 1989 Loma Prieta Earthquake, American earthquake investigators predetermined magnetometer use and a minimum earthquake magnitude necessary for EM detection. This action was set in motion, due to the extensive damage incurred and public outrage concerning earthquake forecasting; however, the magnetometers employed, grounded or buried, are completely subject to static and electric fields and have yet to correlate to an identifiable precursor. Secondly, there is neither a networked array for finding any epicentral locations, nor have there been any attempts to find even one. This methodology needs dismissal, because it is overly complicated, subject to continuous change, and provides no response time. As for the minimum magnitude threshold, which was set at M5, this is simply higher than what modern technological advances have gained. Detection can now be achieved at approximately M1, which greatly improves forecasting chances. A propagating precursor has now been detected in both the field and laboratory. Field antenna testing conducted outside the NE Texas town of Timpson in February, 2013, detected three strong EM sources along with numerous weaker signals. The antenna had mobility, and observations were noted for recurrence, duration, and frequency response. Next, two

  3. Canine procalcitonin messenger RNA expression.

    PubMed

    Kuzi, Sharon; Aroch, Itamar; Peleg, Keren; Karnieli, Ohad; Klement, Eyal; Dank, Gillian

    2008-09-01

    Procalcitonin is considered an acute phase protein used as both a marker of infection and prognosis in human medicine. Canine procalcitonin has been previously sequenced; however, its use as a diagnostic or prognostic tool in dogs has never been assessed. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay for canine procalcitonin messenger RNA (mRNA) was developed. Whole blood samples were collected from ill and healthy dogs. RNA was extracted and the real-time PCR was assessed. The patients' diagnoses, complete blood cell count, and differential leukocyte count results were recorded. Based on the diagnosis, dogs were divided into 5 groups: inflammatory, infectious, neoplastic, other diseases, and healthy controls. Procalcitonin mRNA expression and the hematological measures were compared between groups, and their correlations were assessed. Procalcitonin mRNA expression was assessed in 70 dogs, including infectious (17), noninfectious inflammatory (17), neoplastic (18), other diseases (7), and healthy controls (11), and was significantly (P < 0.001) higher in all ill dogs versus controls. Procalcitonin may therefore be considered an acutephase protein in dogs. However, there were no significant differences in procalcitonin mRNA expression between ill dog groups and no correlations between its expression levels and hematological measures. In 5 dogs of all disease categories, procalcitonin mRNA expression was measured twice during the course of disease. The changes in its levels were in agreement with the clinical evaluation of improvement or deterioration, suggesting a possible prognostic value. PMID:18776098

  4. Evolution in an RNA World

    PubMed Central

    Joyce, Gerald F.

    2009-01-01

    A longstanding research goal has been to develop a self-sustained chemical system that is capable of undergoing Darwinian evolution. The notion of primitive RNA-based life suggests this goal might be achieved by constructing an RNA enzyme that catalyzes the replication of RNA molecules, including the RNA enzyme itself. This reaction recently was demonstrated in a cross-catalytic system involving two RNA enzymes that catalyze each other’s synthesis from a total of four component substrates. The cross-replicating RNA enzymes undergo self-sustained exponential amplification at a constant temperature in the absence of proteins or other biological materials. Amplification occurs with a doubling time of 30–60 min, and can be continued indefinitely. Small populations of cross-replicating RNA enzymes can be made to compete for limited resources within a common environment. The molecules reproduce with high fidelity, but occasionally give rise to recombinants that also can replicate. Over the course of many “generations” of selective amplification, novel variants arise and grow to dominate the population based on their relative fitness under the chosen reaction conditions. This is the first example, outside of biology, of evolutionary adaptation in a molecular genetic system. PMID:19667013

  5. Cofactors in the RNA World

    NASA Technical Reports Server (NTRS)

    Ditzler, Mark A.

    2014-01-01

    RNA world theories figure prominently in many scenarios for the origin and early evolution of life. These theories posit that RNA molecules played a much larger role in ancient biology than they do now, acting both as the dominant biocatalysts and as the repository of genetic information. Many features of modern RNA biology are potential examples of molecular fossils from an RNA world, such as the pervasive involvement of nucleotides in coenzymes, the existence of natural aptamers that bind these coenzymes, the existence of natural ribozymes, a biosynthetic pathway in which deoxynucleotides are produced from ribonucleotides, and the central role of ribosomal RNA in protein synthesis in the peptidyl transferase center of the ribosome. Here, we uses both a top-down approach that evaluates RNA function in modern biology and a bottom-up approach that examines the capacities of RNA independent of modern biology. These complementary approaches exploit multiple in vitro evolution techniques coupled with high-throughput sequencing and bioinformatics analysis. Together these complementary approaches advance our understanding of the most primitive organisms, their early evolution, and their eventual transition to modern biochemistry.

  6. Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.

    PubMed Central

    Trifillis, P; Day, N; Kiledjian, M

    1999-01-01

    Defects in RNA-binding proteins have been implicated in human genetic disorders. However, efforts in understanding the functions of these proteins have been hampered by the inability to obtain their mRNA substrates. To identify cognate cellular mRNAs associated with an RNA-binding protein, we devised a strategy termed isolation of specific nucleic acids associated with proteins (SNAAP). The SNAAP technique allows isolation and subsequent identification of these mRNAs. To assess the validity of this approach, we utilized cellular mRNA and protein from K562 cells and alphaCP1, a protein implicated in a-globin mRNA stability, as a model system. Immobilization of an RNA-binding protein with the glutathione-S-transferase (GST) domain enables isolation of mRNA within an mRNP context and the identity of the bound mRNAs is determined by the differential display assay. The specificity of protein-RNA interactions was considerably enhanced when the interactions were carried out in the presence of cellular extract rather than purified components. Two of the mRNAs specifically bound by alphaCP1 were mRNAs encoding the transmembrane receptor protein, TAPA-1, and the mitochondrial cytochrome c oxidase subunit II enzyme, coxII. A specific poly(C)-sensitive complex formed on the TAPA-1 and coxII 3' UTRs consistent with the binding of aCP1. Furthermore, direct binding of purified alphaCP proteins to these 3' UTRs was demonstrated and the binding sites determined. These results support the feasibility of the SNAAP technique and suggest a broad applicability for the approach in identifying mRNA targets for clinically relevant RNA-binding proteins that will provide insights into their possible functions. PMID:10445881

  7. tRNA synthetase: tRNA Aminoacylation and beyond

    PubMed Central

    Pang, Yan Ling Joy; Poruri, Kiranmai; Martinis, Susan A.

    2014-01-01

    The aminoacyl-tRNA synthetases are prominently known for their classic function in the first step of protein synthesis, where they bear the responsibility of setting the genetic code. Each enzyme is exquisitely adapted to covalently link a single standard amino acid to its cognate set of tRNA isoacceptors. These ancient enzymes have evolved idiosyncratically to host alternate activities that go far beyond their aminoacylation role and impact a wide range of other metabolic pathways and cell signaling processes. The family of aminoacyl-tRNA synthetases have also been suggested as a remarkable scaffold to incorporate new domains that would drive evolution and the emergence of new organisms with more complex function. Because they are essential, the tRNA synthetases have served as pharmaceutical targets for drug and antibiotic development. The recent unfolding of novel important functions for this family of proteins offers new and promising pathways for therapeutic development to treat diverse human diseases. PMID:24706556

  8. RNA Sociology: Group Behavioral Motifs of RNA Consortia

    PubMed Central

    Witzany, Guenther

    2014-01-01

    RNA sociology investigates the behavioral motifs of RNA consortia from the social science perspective. Besides the self-folding of RNAs into single stem loop structures, group building of such stem loops results in a variety of essential agents that are highly active in regulatory processes in cellular and non-cellular life. RNA stem loop self-folding and group building do not depend solely on sequence syntax; more important are their contextual (functional) needs. Also, evolutionary processes seem to occur through RNA stem loop consortia that may act as a complement. This means the whole entity functions only if all participating parts are coordinated, although the complementary building parts originally evolved for different functions. If complementary groups, such as rRNAs and tRNAs, are placed together in selective pressure contexts, new evolutionary features may emerge. Evolution initiated by competent agents in natural genome editing clearly contrasts with statistical error replication narratives. PMID:25426799

  9. An Integrative Analysis of microRNA and mRNA Profiling in CML Stem Cells.

    PubMed

    Nassar, Farah J; El Eit, Rabab; Nasr, Rihab

    2016-01-01

    Integrative analysis of microRNA (miRNA) and messenger RNA (mRNA) in Chronic Myeloid leukemia (CML) stem cells is an important technique to study the involvement of miRNA and their targets in CML stem cells self-renewal, maintenance, and therapeutic resistance. Here, we describe a simplified integrative analysis using Ingenuity Pathway Analysis software after performing proper RNA extraction, miRNA and mRNA microarray and data analysis. PMID:27581151

  10. Alternative RNA splicing and cancer

    PubMed Central

    Liu, Sali; Cheng, Chonghui

    2015-01-01

    Alternative splicing of pre-messenger RNA (mRNA) is a fundamental mechanism by which a gene can give rise to multiple distinct mRNA transcripts, yielding protein isoforms with different, even opposing, functions. With the recognition that alternative splicing occurs in nearly all human genes, its relationship with cancer-associated pathways has emerged as a rapidly growing field. In this review, we summarize recent findings that have implicated the critical role of alternative splicing in cancer and discuss current understandings of the mechanisms underlying dysregulated alternative splicing in cancer cells. PMID:23765697

  11. The ViennaRNA web services.

    PubMed

    Gruber, Andreas R; Bernhart, Stephan H; Lorenz, Ronny

    2015-01-01

    The ViennaRNA package is a widely used collection of programs for thermodynamic RNA secondary structure prediction. Over the years, many additional tools have been developed building on the core programs of the package to also address issues related to noncoding RNA detection, RNA folding kinetics, or efficient sequence design considering RNA-RNA hybridizations. The ViennaRNA web services provide easy and user-friendly web access to these tools. This chapter describes how to use this online platform to perform tasks such as prediction of minimum free energy structures, prediction of RNA-RNA hybrids, or noncoding RNA detection. The ViennaRNA web services can be used free of charge and can be accessed via http://rna.tbi.univie.ac.at. PMID:25577387

  12. Bioterrorism awareness for EMS.

    PubMed

    Patrick, Richard W

    2004-04-01

    It is important to understand that the issues surrounding bioterrorism and all weapons of mass destruction are complex. In an effort to enhance response to such events, EMS should handle all incidents from the perspective of an all-hazards approach. Prevention, preparation, response and recovery are essential to the safe mitigation of all incidents. Organizations must be prepared. Plan now for a safer tomorrow. Your personnel and communities depend on you. PMID:15131906

  13. Cotranslational microRNA mediated messenger RNA destabilization

    PubMed Central

    Tat, Trinh To; Maroney, Patricia A; Chamnongpol, Sangpen; Coller, Jeff; Nilsen, Timothy W

    2016-01-01

    MicroRNAs are small (22 nucleotide) regulatory molecules that play important roles in a wide variety of biological processes. These RNAs, which bind to targeted mRNAs via limited base pairing interactions, act to reduce protein production from those mRNAs. Considerable evidence indicates that miRNAs destabilize targeted mRNAs by recruiting enzymes that function in normal mRNA decay and mRNA degradation is widely thought to occur when mRNAs are in a ribosome free state. Nevertheless, when examined, miRNA targeted mRNAs are invariably found to be polysome associated; observations that appear to be at face value incompatible with a simple decay model. Here, we provide evidence that turnover of miRNA-targeted mRNAs occurs while they are being translated. Cotranslational mRNA degradation is initiated by decapping and proceeds 5’ to 3’ behind the last translating ribosome. These results provide an explanation for a long standing mystery in the miRNA field. DOI: http://dx.doi.org/10.7554/eLife.12880.001 PMID:27058298

  14. The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli.

    PubMed

    Julián, Patricia; Milon, Pohl; Agirrezabala, Xabier; Lasso, Gorka; Gil, David; Rodnina, Marina V; Valle, Mikel

    2011-07-01

    Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNA(fMet) requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNA(fMet). Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNA(fMet), IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNA(fMet), which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNA(fMet) induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation. PMID:21750663

  15. Deciphering the RNA landscape by RNAome sequencing

    PubMed Central

    Derks, Kasper WJ; Misovic, Branislav; van den Hout, Mirjam CGN; Kockx, Christel EM; Payan Gomez, Cesar; Brouwer, Rutger WW; Vrieling, Harry; Hoeijmakers, Jan HJ; van IJcken, Wilfred FJ; Pothof, Joris

    2015-01-01

    Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods. PMID:25826412

  16. Deciphering the RNA landscape by RNAome sequencing.

    PubMed

    Derks, Kasper W J; Misovic, Branislav; van den Hout, Mirjam C G N; Kockx, Christel E M; Gomez, Cesar Payan; Brouwer, Rutger W W; Vrieling, Harry; Hoeijmakers, Jan H J; van IJcken, Wilfred F J; Pothof, Joris

    2015-01-01

    Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods. PMID:25826412

  17. Nematode endogenous small RNA pathways

    PubMed Central

    Hoogstrate, Suzanne W; Volkers, Rita JM; Sterken, Mark G; Kammenga, Jan E; Snoek, L Basten

    2014-01-01

    The discovery of small RNA silencing pathways has greatly extended our knowledge of gene regulation. Small RNAs have been presumed to play a role in every field of biology because they affect many biological processes via regulation of gene expression and chromatin remodeling. Most well-known examples of affected processes are development, fertility, and maintenance of genome stability. Here we review the role of the three main endogenous small RNA silencing pathways in Caenorhabditis elegans: microRNAs, endogenous small interfering RNAs, and PIWI-interacting RNAs. After providing an entry-level overview on how these pathways function, we discuss research on other nematode species providing insight into the evolution of these small RNA pathways. In understanding the differences between the endogenous small RNA pathways and their evolution, a more comprehensive picture is formed of the functions and effects of small RNAs. PMID:25340013

  18. RNA silencing: an antiviral mechanism.

    PubMed

    Csorba, T; Pantaleo, V; Burgyán, J

    2009-01-01

    RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins which can counteract the host silencing-based antiviral process. After the discovery of virus-encoded silencing suppressors, it was shown that these viral proteins can target one or more key points in the silencing machinery. Here we review recent progress in our understanding of the mechanism and function of antiviral RNA silencing in plants, and on the virus's counterattack by expression of silencing-suppressor proteins. We also discuss emerging evidence that RNA silencing and expression of viral silencing-suppressor proteins are tools forged as a consequence of virus-host coevolution for fine-tuning host-pathogen coexistence. PMID:20109663

  19. Isothermal titration calorimetry of RNA.

    PubMed

    Salim, Nilshad N; Feig, Andrew L

    2009-03-01

    Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. A single well-designed experiment can provide complete thermodynamic characterization of a binding reaction, including K(a), DeltaG, DeltaH, DeltaS and reaction stoichiometry (n). Repeating the experiment at different temperatures allows determination of the heat capacity change (DeltaC(P)) of the interaction. Modern calorimeters are sensitive enough to probe even weak biological interactions making ITC a very popular method among biochemists. Although ITC has been applied to protein studies for many years, it is becoming widely applicable in RNA biochemistry as well, especially in studies which involve RNA folding and RNA interactions with small molecules, proteins and with other RNAs. This review focuses on best practices for planning, designing and executing effective ITC experiments when one or more of the reactants is an RNA. PMID:18835447

  20. The Annotation of RNA Motifs

    PubMed Central

    2002-01-01

    The recent deluge of new RNA structures, including complete atomic-resolution views of both subunits of the ribosome, has on the one hand literally overwhelmed our individual abilities to comprehend the diversity of RNA structure, and on the other hand presented us with new opportunities for comprehensive use of RNA sequences for comparative genetic, evolutionary and phylogenetic studies. Two concepts are key to understanding RNA structure: hierarchical organization of global structure and isostericity of local interactions. Global structure changes extremely slowly, as it relies on conserved long-range tertiary interactions. Tertiary RNA–RNA and quaternary RNA–protein interactions are mediated by RNA motifs, defined as recurrent and ordered arrays of non-Watson–Crick base-pairs. A single RNA motif comprises a family of sequences, all of which can fold into the same three-dimensional structure and can mediate the same interaction(s). The chemistry and geometry of base pairing constrain the evolution of motifs in such a way that random mutations that occur within motifs are accepted or rejected insofar as they can mediate a similar ordered array of interactions. The steps involved in the analysis and annotation of RNA motifs in 3D structures are: (a) decomposition of each motif into non-Watson–Crick base-pairs; (b) geometric classification of each basepair; (c) identification of isosteric substitutions for each basepair by comparison to isostericity matrices; (d) alignment of homologous sequences using the isostericity matrices to identify corresponding positions in the crystal structure; (e) acceptance or rejection of the null hypothesis that the motif is conserved. PMID:18629252

  1. Localized control of oxidized RNA.

    PubMed

    Zhan, Yu; Dhaliwal, James S; Adjibade, Pauline; Uniacke, James; Mazroui, Rachid; Zerges, William

    2015-11-15

    The oxidation of biological molecules by reactive oxygen species (ROS) can render them inactive or toxic. This includes the oxidation of RNA, which appears to underlie the detrimental effects of oxidative stress, aging and certain neurodegenerative diseases. Here, we investigate the management of oxidized RNA in the chloroplast of the green alga Chlamydomonas reinhardtii. Our immunofluorescence microscopy results reveal that oxidized RNA (with 8-hydroxyguanine) is localized in the pyrenoid, a chloroplast microcompartment where CO2 is assimilated by the Calvin cycle enzyme Rubisco. Results of genetic analyses support a requirement for the Rubisco large subunit (RBCL), but not Rubisco, in the management of oxidized RNA. An RBCL pool that can carry out such a 'moonlighting' function is revealed by results of biochemical fractionation experiments. We also show that human (HeLa) cells localize oxidized RNA to cytoplasmic foci that are distinct from stress granules, processing bodies and mitochondria. Our results suggest that the compartmentalization of oxidized RNA management is a general phenomenon and therefore has some fundamental significance. PMID:26449969

  2. Predicting and Modeling RNA Architecture

    PubMed Central

    Westhof, Eric; Masquida, Benoît; Jossinet, Fabrice

    2011-01-01

    SUMMARY A general approach for modeling the architecture of large and structured RNA molecules is described. The method exploits the modularity and the hierarchical folding of RNA architecture that is viewed as the assembly of preformed double-stranded helices defined by Watson-Crick base pairs and RNA modules maintained by non-Watson-Crick base pairs. Despite the extensive molecular neutrality observed in RNA structures, specificity in RNA folding is achieved through global constraints like lengths of helices, coaxiality of helical stacks, and structures adopted at the junctions of helices. The Assemble integrated suite of computer tools allows for sequence and structure analysis as well as interactive modeling by homology or ab initio assembly with possibilities for fitting within electronic density maps. The local key role of non-Watson-Crick pairs guides RNA architecture formation and offers metrics for assessing the accuracy of three-dimensional models in a more useful way than usual root mean square deviation (RMSD) values. PMID:20504963

  3. Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice

    PubMed Central

    Wang, Joseph Che-Yen; Nickens, David G.; Lentz, Thomas B.; Loeb, Daniel D.; Zlotnick, Adam

    2014-01-01

    Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse transcriptase. To generate the circular dsDNA genome of HBV, reverse transcription requires multiple template switches within the confines of the capsid. To date, most anti-HBV therapeutics target this reverse transcription process. The detailed molecular mechanisms of this crucial process are poorly understood because of the lack of structural information. We hypothesized that capsid, RNA, and viral reverse transcriptase would need a precise geometric organization to accomplish reverse transcription. Here we present the asymmetric structure of authentic RNA-filled cores, determined to 14.5-Å resolution from cryo-EM data. Capsid and RNA are concentric. On the interior of the RNA, we see a distinct donut-like density, assigned to viral reverse transcriptase, which pins the viral pregenomic RNA to the capsid inner surface. The observation of a unique ordered structure inside the core suggests that assembly and the first steps of reverse transcription follow a single, determinate pathway and strongly suggests that all subsequent steps in DNA synthesis do as well. PMID:25034253

  4. Summary: the modified nucleosides of RNA.

    PubMed Central

    Limbach, P A; Crain, P F; McCloskey, J A

    1994-01-01

    A comprehensive listing is made of posttranscriptionally modified nucleosides from RNA reported in the literature through mid-1994. Included are chemical structures, common names, symbols, Chemical Abstracts registry numbers (for ribonucleoside and corresponding base), Chemical Abstracts Index Name, phylogenetic sources, and initial literature citations for structural characterization or occurrence, and for chemical synthesis. The listing is categorized by type of RNA: tRNA, rRNA, mRNA, snRNA, and other RNAs. A total of 93 different modified nucleosides have been reported in RNA, with the largest number and greatest structural diversity in tRNA, 79; and 28 in rRNA, 12 in mRNA, 11 in snRNA and 3 in other small RNAs. PMID:7518580

  5. RNA polymerase I–Rrn3 complex at 4.8 Å resolution

    PubMed Central

    Engel, Christoph; Plitzko, Jürgen; Cramer, Patrick

    2016-01-01

    Transcription of ribosomal DNA by RNA polymerase I (Pol I) requires the initiation factor Rrn3. Here we report the cryo-EM structure of the Pol I–Rrn3 complex at 4.8 Å resolution. The structure reveals how Rrn3 binding converts an inactive Pol I dimer into an initiation-competent monomeric complex and provides insights into the mechanisms of Pol I-specific initiation and regulation. PMID:27418309

  6. RNA polymerase I-Rrn3 complex at 4.8 Å resolution.

    PubMed

    Engel, Christoph; Plitzko, Jürgen; Cramer, Patrick

    2016-01-01

    Transcription of ribosomal DNA by RNA polymerase I (Pol I) requires the initiation factor Rrn3. Here we report the cryo-EM structure of the Pol I-Rrn3 complex at 4.8 Å resolution. The structure reveals how Rrn3 binding converts an inactive Pol I dimer into an initiation-competent monomeric complex and provides insights into the mechanisms of Pol I-specific initiation and regulation. PMID:27418309

  7. RNA polymerase I-Rrn3 complex at 4.8 Å resolution

    NASA Astrophysics Data System (ADS)

    Engel, Christoph; Plitzko, Jürgen; Cramer, Patrick

    2016-07-01

    Transcription of ribosomal DNA by RNA polymerase I (Pol I) requires the initiation factor Rrn3. Here we report the cryo-EM structure of the Pol I-Rrn3 complex at 4.8 Å resolution. The structure reveals how Rrn3 binding converts an inactive Pol I dimer into an initiation-competent monomeric complex and provides insights into the mechanisms of Pol I-specific initiation and regulation.

  8. RNA mediated assembly of nanostructures

    NASA Astrophysics Data System (ADS)

    Rouge, Jessica Lynn

    The first chapter of this work presents a comprehensive look at RNA mediated nanoparticle formation. The overall goal of this research is to gain a deeper understanding of the RNA-particle formation mechanism and the basic properties of the materials selected by modified RNA molecules. Understanding such RNA-substrate interactions and how they translate into the physical and chemical characteristics of the nanoparticles they create are important fundamental concepts when considering these biotemplated materials as potential chemical catalysts. The RNA sequences discussed in the first chapter (referred to as Pdases) were discovered using RNA in vitro selection techniques. These Pdases were found to be capable of forming inorganic palladium (Pd) containing nanoparticles with impressive control over an individual particle's size and shape, despite incubation with the same organometallic precursor. This discovery held exciting implications for inorganic nanoparticle design while also generating numerous questions regarding the mechanism of RNA mediated particle growth. The central question that arose after this initial discovery was how could a biomolecule be used to tailor the physical size and shape of inorganic materials? Starting with a chemical proof designed to uncover the composition of the nanoparticles formed by RNA mediation, this chapter investigates the basic material properties of the nanoparticles while also introducing surprising results regarding the effect of multiple sequences on nanoparticle growth outcomes. In the second chapter, the experiments shift to developing methods to investigate nanoparticle growth mechanisms by fluorescence spectroscopy. A fluorescence polarization anisotropy (FPA) assay is presented in which the strengths of the technique are adapted for studying the formation of RNA mediated Pd nanoparticles in real time. This is a unique application of FPA, as it has been adapted to encompass both the biochemical and materials analysis

  9. Surface for Catalysis by Poliovirus RNA-Dependent RNA Polymerase

    PubMed Central

    Wang, Jing; Lyle, John M.; Bullitt, Esther

    2013-01-01

    The poliovirus RNA-dependent RNA polymerase, 3Dpol, replicates the viral genomic RNA on the surface of virus-induced intracellular membranes. Macromolecular assemblies of 3Dpol form linear array of subunits that propagate along a strong protein-protein interaction called interface-I, as was observed in the crystal structure of wild-type poliovirus polymerase. These “filaments” recur with slight modifications in planar sheets and, with additional modifications that accommodate curvature, in helical tubes of the polymerase, by packing filaments together via a second set of interactions. Periodic variations of subunit orientations within 3Dpol tubes give rise to “ghost reflections” in diffraction patterns computed from electron cryomicrographs of helical arrays. The ghost reflections reveal that polymerase tubes are formed by bundles of 4–6 interface-I filaments, which are then connected to the next bundle of filaments with a perturbation of interface interactions between bundles. While enzymatically inactive polymerase is also capable of oligomerization, much thinner tubes are formed that lack interface-I interactions between adjacent subunits, suggesting that long-range allostery produces conformational changes that extend from the active site to the protein-protein interface. Macromolecular assemblies of poliovirus polymerase show repeated use of flexible interface interactions for polymerase lattice formation, suggesting that adaptability of polymerase-polymerase interactions facilitates RNA replication. In addition, the presence of a positively charged groove identified in polymerase arrays may help position and stabilize the RNA template during replication. PMID:23583774

  10. RNA catalyzes nuclear pre-mRNA splicing

    PubMed Central

    Fica, Sebastian M.; Tuttle, Nicole; Novak, Thaddeus; Li, Nan-Sheng; Lu, Jun; Koodathingal, Prakash; Dai, Qing; Staley, Jonathan P.; Piccirilli, Joseph A.

    2014-01-01

    SUMMARY In nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a multi-megadalton machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, following the discovery of self-splicing group II intron RNAs, the snRNAs were hypothesized to catalyze splicing. However, no definitive evidence for a role of either RNA or protein in catalysis by the spliceosome has been reported to date. By using metal rescue strategies, here we show that the U6 snRNA catalyzes both splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction. Strikingly, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA. These findings indicate that group II introns and the spliceosome share common catalytic mechanisms, and likely common evolutionary origins. Our results demonstrate that RNA mediates catalysis within the spliceosome. PMID:24196718

  11. miSolRNA: A tomato micro RNA relational database

    PubMed Central

    2010-01-01

    Background The economic importance of Solanaceae plant species is well documented and tomato has become a model for functional genomics studies. In plants, important processes are regulated by microRNAs (miRNA). Description We describe here a data base integrating genetic map positions of miRNA-targeted genes, their expression profiles and their relations with quantitative fruit metabolic loci and yield associated traits. miSolRNA provides a metadata source to facilitate the construction of hypothesis aimed at defining physiological modes of action of regulatory process underlying the metabolism of the tomato fruit. Conclusions The MiSolRNA database allows the simple extraction of metadata for the proposal of new hypothesis concerning possible roles of miRNAs in the regulation of tomato fruit metabolism. It permits i) to map miRNAs and their predicted target sites both on expressed (SGN-UNIGENES) and newly annotated sequences (BAC sequences released), ii) to co-locate any predicted miRNA-target interaction with metabolic QTL found in tomato fruits, iii) to retrieve expression data of target genes in tomato fruit along their developmental period and iv) to design further experiments for unresolved questions in complex trait biology based on the use of genetic materials that have been proven to be a useful tools for map-based cloning experiments in Solanaceae plant species. PMID:21059227

  12. Kinetic oscillations in the expression of messenger RNA, regulatory protein, and nonprotein coding RNA

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2008-06-01

    The interplay of messenger RNA (mRNA), protein, produced via translation of this RNA, and nonprotein coding RNA (ncRNA) may include regulation of the ncRNA production by protein and (i) ncRNA-mRNA association or (ii) ncRNA-protein association resulting in degradation of the corresponding complex. The kinetic models, describing these two scenarios and taking into account that the association of ncRNA with a target occurs after ncRNA conversion from the initial form to the final form (e.g., from a long RNA to microRNA), are found to predict oscillations provided that the rate of ncRNA formation increases with increasing protein population.

  13. Chaperoning 5S RNA assembly

    PubMed Central

    Madru, Clément; Lebaron, Simon; Blaud, Magali; Delbos, Lila; Pipoli, Juliana; Pasmant, Eric; Réty, Stéphane; Leulliot, Nicolas

    2015-01-01

    In eukaryotes, three of the four ribosomal RNAs (rRNAs)—the 5.8S, 18S, and 25S/28S rRNAs—are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2–Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2–Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2–Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit. PMID:26159998

  14. A 22-nt artificial microRNA mediates widespread RNA silencing in Arabidopsis

    PubMed Central

    McHale, Marcus; Eamens, Andrew L; Finnegan, E Jean; Waterhouse, Peter M

    2013-01-01

    It is known that 22-nucleotide (nt) microRNAs (miRNAs) derived from asymmetric duplexes trigger phased small-interfering RNA (phasiRNA) production from complementary targets. Here we investigate the efficacy of 22-nt artificial miRNA (amiRNA)-mediated RNA silencing relative to conventional hairpin RNA (hpRNA) and 21-nt amiRNA-mediated RNA silencing. CHALCONE SYNTHASE (CHS) was selected as a target in Arabidopsis thaliana due to the obvious and non-lethal loss of anthocyanin accumulation upon widespread RNA silencing. Over-expression of CHS in the pap1-D background facilitated visual detection of both local and systemic RNA silencing. RNA silencing was initiated in leaf tissues from hpRNA and amiRNA plant expression vectors under the control of an Arabidopsis RuBisCo small subunit 1A promoter (SSU). In this system, hpRNA expression triggered CHS silencing in most leaf tissues but not in roots or seed coats. Similarly, 21-nt amiRNA expression from symmetric miRNA/miRNA* duplexes triggered CHS silencing in all leaf tissues but not in roots or seed coats. However, 22-nt amiRNA expression from an asymmetric duplex triggered CHS silencing in all tissues, including roots and seed coats, in the majority of plant lines. This widespread CHS silencing required RNA-DEPENDENT RNA POLYMERASE6-mediated accumulation of phasiRNAs from the endogenous CHS transcript. These results demonstrate the efficacy of asymmetric 22-nt amiRNA-directed RNA silencing and associated phasiRNA production and activity, in mediating widespread RNA silencing of an endogenous target gene. Asymmetric 22-nt amiRNA-directed RNA silencing requires little modification of existing amiRNA technology and is expected to be effective in suppressing other genes and/or members of gene families. PMID:23937661

  15. RNA Matchmaking: Finding Cellular Pairing Partners.

    PubMed

    Graveley, Brenton R

    2016-07-21

    RNA structure is intimately related to function, yet methods to identify base-paired RNA strands in a transcriptome-wide manner in cells have remained elusive. One recent paper in Cell and two in Molecular Cell describe related methods to identify RNA sequences that interact in living cells, setting the stage for breakthroughs in our understanding of RNA structure and function. PMID:27447984

  16. RSRE: RNA structural robustness evaluator.

    PubMed

    Shu, Wenjie; Bo, Xiaochen; Zheng, Zhiqiang; Wang, Shengqi

    2007-07-01

    Biological robustness, defined as the ability to maintain stable functioning in the face of various perturbations, is an important and fundamental topic in current biology, and has become a focus of numerous studies in recent years. Although structural robustness has been explored in several types of RNA molecules, the origins of robustness are still controversial. Computational analysis results are needed to make up for the lack of evidence of robustness in natural biological systems. The RNA structural robustness evaluator (RSRE) web server presented here provides a freely available online tool to quantitatively evaluate the structural robustness of RNA based on the widely accepted definition of neutrality. Several classical structure comparison methods are employed; five randomization methods are implemented to generate control sequences; sub-optimal predicted structures can be optionally utilized to mitigate the uncertainty of secondary structure prediction. With a user-friendly interface, the web application is easy to use. Intuitive illustrations are provided along with the original computational results to facilitate analysis. The RSRE will be helpful in the wide exploration of RNA structural robustness and will catalyze our understanding of RNA evolution. The RSRE web server is freely available at http://biosrv1.bmi.ac.cn/RSRE/ or http://biotech.bmi.ac.cn/RSRE/. PMID:17567615

  17. Viral escape from antisense RNA.

    PubMed

    Bull, J J; Jacobson, A; Badgett, M R; Molineux, I J

    1998-05-01

    RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31-270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3-4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson-Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition. PMID:9643550

  18. Biogenesis, delivery, and function of extracellular RNA.

    PubMed

    Patton, James G; Franklin, Jeffrey L; Weaver, Alissa M; Vickers, Kasey; Zhang, Bing; Coffey, Robert J; Ansel, K Mark; Blelloch, Robert; Goga, Andrei; Huang, Bo; L'Etoille, Noelle; Raffai, Robert L; Lai, Charles P; Krichevsky, Anna M; Mateescu, Bogdan; Greiner, Vanille J; Hunter, Craig; Voinnet, Olivier; McManus, Michael T

    2015-01-01

    The Extracellular RNA (exRNA) Communication Consortium was launched by the National Institutes of Health to focus on the extent to which RNA might function in a non-cell-autonomous manner. With the availability of increasingly sensitive tools, small amounts of RNA can be detected in serum, plasma, and other bodily fluids. The exact mechanism(s) by which RNA can be secreted from cells and the mechanisms for the delivery and uptake by recipient cells remain to be determined. This review will summarize current knowledge about the biogenesis and delivery of exRNA and outline projects seeking to understand the functional impact of exRNA. PMID:26320939

  19. Polysome Preparation, RNA Isolation and Analysis

    PubMed Central

    Zhang, Hailong; Zhou, Muxiang

    2016-01-01

    During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosome can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.

  20. RNA-Based Vaccines in Cancer Immunotherapy

    PubMed Central

    McNamara, Megan A.; Nair, Smita K.; Holl, Eda K.

    2015-01-01

    RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s) of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy. PMID:26665011

  1. RNAome sequencing delineates the complete RNA landscape.

    PubMed

    Derks, Kasper W J; Pothof, Joris

    2015-09-01

    Standard RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species. For example, small and large RNAs from the same sample cannot be sequenced in a single sequence run. We designed RNAome sequencing, which is a strand-specific method to determine the expression of small and large RNAs from ribosomal RNA-depleted total RNA in a single sequence run. RNAome sequencing quantitatively preserves all RNA classes. This characteristic allows comparisons between RNA classes, thereby facilitating relationships between different RNA classes. Here, we describe in detail the experimental procedure associated with RNAome sequencing published by Derks and colleagues in RNA Biology (2015) [1]. We also provide the R code for the developed Total Rna Analysis Pipeline (TRAP), an algorithm to analyze RNAome sequencing datasets (deposited at the Gene Expression Omnibus data repository, accession number GSE48084). PMID:26484291

  2. Detecção inesperada de efeitos de lentes fracas em grupos de galáxias pouco luminosos em raios-X

    NASA Astrophysics Data System (ADS)

    Carrasco, R.; Mendes de Oliveira, C.; Sodrã©, L., Jr.; Lima Neto, G. B.; Cypriano, E. S.; Lengruber, L. L.; Cuevas, H.; Ramirez, A.

    2003-08-01

    Obtivemos, como parte do programa de verificação científica do GMOS Sul, imagens profundas de três grupos de galáxias: G97 e G102 (z~0,4) e G124 (z = 0,17). Esses alvos foram selecionados a partir do catálogo de fontes extensas de Vikhlinin (1998), por terem luminosidades em raios X menores que 3´1043 ergs s-1, valor cerca de uma ou duas ordens de grandeza inferior ao de aglomerados de galáxias. O objetivo primário dessas observações é o estudo da evolução de galáxias em grupos. Grupos são ambientes menos densos que aglomerados, contêm a grande maioria das galáxias do Universo mas que, até o momento, foram estudados detalhadamente apenas no Universo local (z~0). Com esses dados efetuamos uma análise estatística da distorção na forma das galáxias de fundo (lentes gravitacionais fracas) como forma de inferir o conteúdo e a distribuição de massa nesses grupos apesar de que, em princípio, esse efeito não deveria ser detectado uma vez que os critérios de seleção adotados previlegiam sistemas de baixa massa. De fato, para G124 obtivemos apenas um limite superior para sua massa que é compatível com sua luminosidade em raios X. De modo contrário e surpreendente, os objetos G102 e G097, aparentam ter massas que resultariam em dispersões de velocidade maiores que 1000 km s-1, muito maiores do que se espera para grupos de galáxias. Com efeito, para G097 obtivemos, a partir de dados do satélite XMM, uma estimativa para a temperatura do gás intragrupo de kT = 2,6 keV, que é tipica de sistemas com dispersões de velocidade de ~ 600 km s-1, bem característica de grupos. Essas contradições aparentes entre lentes fracas e raios X podem ser explicadas de dois modos: i) a massa obtida por lentes estaria sobreestimada devido à superposição de estruturas massivas ao longo da linha de visada ou ii) a temperatura do gás do meio intra-grupo reflete o potencial gravitacional de estruturas menores que estariam se fundindo para formar uma

  3. CryoEM structures of two spliceosomal complexes: starter and dessert at the spliceosome feast

    PubMed Central

    Nguyen, Thi Hoang Duong; Galej, Wojciech P; Fica, Sebastian M; Lin, Pei-Chun; Newman, Andrew J; Nagai, Kiyoshi

    2016-01-01

    The spliceosome is formed on pre-mRNA substrates from five small nuclear ribonucleoprotein particles (U1, U2, U4/U6 and U5 snRNPs), and numerous non-snRNP factors. Saccharomyces cerevisiae U4/U6.U5 tri-snRNP comprises U5 snRNA, U4/U6 snRNA duplex and approximately 30 proteins and represents a substantial part of the spliceosome before activation. Schizosaccharomyces pombe U2.U6.U5 spliceosomal complex is a post-catalytic intron lariat spliceosome containing U2 and U5 snRNPs, NTC (nineteen complex), NTC-related proteins (NTR), U6 snRNA, and an RNA intron lariat. Two recent papers describe near-complete atomic structures of these complexes based on cryoEM single-particle analysis. The U4/U6.U5 tri-snRNP structure provides crucial insight into the activation mechanism of the spliceosome. The U2.U6.U5 complex reveals the striking architecture of NTC and NTR and important features of the group II intron-like catalytic RNA core remaining after spliced mRNA is released. These two structures greatly advance our understanding of the mechanism of pre-mRNA splicing. PMID:26803803

  4. CryoEM structures of two spliceosomal complexes: starter and dessert at the spliceosome feast.

    PubMed

    Nguyen, Thi Hoang Duong; Galej, Wojciech P; Fica, Sebastian M; Lin, Pei-Chun; Newman, Andrew J; Nagai, Kiyoshi

    2016-02-01

    The spliceosome is formed on pre-mRNA substrates from five small nuclear ribonucleoprotein particles (U1, U2, U4/U6 and U5 snRNPs), and numerous non-snRNP factors. Saccharomyces cerevisiae U4/U6.U5 tri-snRNP comprises U5 snRNA, U4/U6 snRNA duplex and approximately 30 proteins and represents a substantial part of the spliceosome before activation. Schizosaccharomyces pombe U2.U6.U5 spliceosomal complex is a post-catalytic intron lariat spliceosome containing U2 and U5 snRNPs, NTC (nineteen complex), NTC-related proteins (NTR), U6 snRNA, and an RNA intron lariat. Two recent papers describe near-complete atomic structures of these complexes based on cryoEM single-particle analysis. The U4/U6.U5 tri-snRNP structure provides crucial insight into the activation mechanism of the spliceosome. The U2.U6.U5 complex reveals the striking architecture of NTC and NTR and important features of the group II intron-like catalytic RNA core remaining after spliced mRNA is released. These two structures greatly advance our understanding of the mechanism of pre-mRNA splicing. PMID:26803803

  5. Hairpins under tension: RNA versus DNA

    PubMed Central

    Bercy, Mathilde; Bockelmann, Ulrich

    2015-01-01

    We use optical tweezers to control the folding and unfolding of individual DNA and RNA hairpins by force. Four hairpin molecules are studied in comparison: two DNA and two RNA ones. We observe that the conformational dynamics is slower for the RNA hairpins than for their DNA counterparts. Our results indicate that structures made of RNA are dynamically more stable. This difference might contribute to the fact that DNA and RNA play fundamentally different biological roles in spite of chemical similarity. PMID:26323319

  6. Unexpected functions of tRNA and tRNA processing enzymes.

    PubMed

    Hurto, Rebecca L

    2011-01-01

    tRNA and tRNA processing enzymes impact more than protein production. Studies have uncovered roles for tRNA in the regulation of transcription, translation and protein turnover. Induced by stress or as a programmed part of development, nonrandom tRNA fragments can guide mRNA cleavage, inhibit translation and promote morphological changes. Similarly, tRNA processing enzymes, such as RNaseP and tRNA aminoacyl-synthetases participate in tasks affecting more than tRNA function (i.e., mRNA function and cellular signaling). Unraveling the complexities of their functions will increase our understanding of how mutations associated with disease impact these functions and the downstream consequences. This chapter focuses on how tRNA and tRNA processing enzymes influence cellular function and RNA-infrastructure via pathways beyond the decoding activities that tRNA are known for. PMID:21915787

  7. Identified EM Earthquake Precursors

    NASA Astrophysics Data System (ADS)

    Jones, Kenneth, II; Saxton, Patrick

    2014-05-01

    Many attempts have been made to determine a sound forecasting method regarding earthquakes and warn the public in turn. Presently, the animal kingdom leads the precursor list alluding to a transmission related source. By applying the animal-based model to an electromagnetic (EM) wave model, various hypotheses were formed, but the most interesting one required the use of a magnetometer with a differing design and geometry. To date, numerous, high-end magnetometers have been in use in close proximity to fault zones for potential earthquake forecasting; however, something is still amiss. The problem still resides with what exactly is forecastable and the investigating direction of EM. After a number of custom rock experiments, two hypotheses were formed which could answer the EM wave model. The first hypothesis concerned a sufficient and continuous electron movement either by surface or penetrative flow, and the second regarded a novel approach to radio transmission. Electron flow along fracture surfaces was determined to be inadequate in creating strong EM fields, because rock has a very high electrical resistance making it a high quality insulator. Penetrative flow could not be corroborated as well, because it was discovered that rock was absorbing and confining electrons to a very thin skin depth. Radio wave transmission and detection worked with every single test administered. This hypothesis was reviewed for propagating, long-wave generation with sufficient amplitude, and the capability of penetrating solid rock. Additionally, fracture spaces, either air or ion-filled, can facilitate this concept from great depths and allow for surficial detection. A few propagating precursor signals have been detected in the field occurring with associated phases using custom-built loop antennae. Field testing was conducted in Southern California from 2006-2011, and outside the NE Texas town of Timpson in February, 2013. The antennae have mobility and observations were noted for

  8. Shapes of RNA pseudoknot structures.

    PubMed

    Reidys, Christian M; Wang, Rita R

    2010-11-01

    In this article, we study abstract shapes of k-noncrossing, σ-canonical RNA pseudoknot structures. We consider lv1k- and lv5k-shapes, which represent a generalization of the abstract π'- and π-shapes of RNA secondary structures introduced by Giegerich et al. Using a novel approach, we compute the generating functions of lv1k- and lv5k-shapes as well as the generating functions of all lv1k- and lv5k-shapes induced by all k-noncrossing, σ-canonical RNA structures for fixed n. By means of singularity analysis of the generating functions, we derive explicit asymptotic expressions For online Supplementary Material, see www.liebertonline.com. PMID:20868269

  9. Rapid compaction during RNA folding

    NASA Astrophysics Data System (ADS)

    Russell, Rick; Millett, Ian S.; Tate, Mark W.; Kwok, Lisa W.; Nakatani, Bradley; Gruner, Sol M.; Mochrie, Simon G. J.; Pande, Vijay; Doniach, Sebastian; Herschlag, Daniel; Pollack, Lois

    2002-04-01

    We have used small angle x-ray scattering and computer simulations with a coarse-grained model to provide a time-resolved picture of the global folding process of the Tetrahymena group I RNA over a time window of more than five orders of magnitude. A substantial phase of compaction is observed on the low millisecond timescale, and the overall compaction and global shape changes are largely complete within one second, earlier than any known tertiary contacts are formed. This finding indicates that the RNA forms a nonspecifically collapsed intermediate and then searches for its tertiary contacts within a highly restricted subset of conformational space. The collapsed intermediate early in folding of this RNA is grossly akin to molten globule intermediates in protein folding.

  10. Genomic analysis of RNA localization

    PubMed Central

    Taliaferro, J Matthew; Wang, Eric T; Burge, Christopher B

    2014-01-01

    The localization of mRNAs to specific subcellular sites is widespread, allowing cells to spatially restrict and regulate protein production, and playing important roles in development and cellular physiology. This process has been studied in mechanistic detail for several RNAs. However, the generality or specificity of RNA localization systems and mechanisms that impact the many thousands of localized mRNAs has been difficult to assess. In this review, we discuss the current state of the field in determining which RNAs localize, which RNA sequences mediate localization, the protein factors involved, and the biological implications of localization. For each question, we examine prominent systems and techniques that are used to study individual messages, highlight recent genome-wide studies of RNA localization, and discuss the potential for adapting other high-throughput approaches to the study of localization. PMID:25483039

  11. miRNA Isolation from FFPET Specimen: A Technical Comparison of miRNA and Total RNA Isolation Methods.

    PubMed

    Nagy, Zsófia Brigitta; Wichmann, Barnabás; Kalmár, Alexandra; Barták, Barbara Kinga; Tulassay, Zsolt; Molnár, Béla

    2016-07-01

    MiRNA remain stable for detection and PCR-based amplification in FFPE tissue samples. Several miRNA extraction kits are available, however miRNA fraction, as part of total RNA can be isolated using total RNA purification methods, as well. Our primary aim was to compare four different miRNA and total RNA isolation methods from FFPE tissues. Further purposes were to evaluate quantitatively and qualitatively the yield of the isolated miRNA. MiRNAs were isolated from normal colorectal cancer FFPE specimens from the same patients. Two miRNA isolation kits (High Pure miRNA Isolation Kit, miRCURY™ RNA Isolation Kit) and two total RNA isolation kits were compared (High Pure RNA Paraffin Kit, MagNA Pure 96 Cellular RNA LV Kit). Quantity and quality were determined, expression analysis was performed by real-time PCR using qPCR Human Panel I + II (Exiqon) method detecting 742 human miRNAs in parallel. The yield of total RNA was found to be higher than miRNA purification protocols (in CRC: Ex: 0203 ± 0021 μg; HPm: 1,45 ± 0,8 μg; HPp: 21,36 ± 4,98 μg; MP: 8,6 ± 5,1 μg). MiRNAs were detected in lower relative quantity of total RNA compared to the miRNA kits. Higher number of miRNAs could be detected by the miRNA isolation kits in comparison to the total RNA isolation methods. (Ex: 497 ± 16; HPm: 542 ± 11; HPp: 332 ± 36; MP: 295 ± 74). Colon specific miRNAs (miR-21-5p;-34-5p) give satisfying results by miRNA isolation kits. Although miRNA can be detected also after total RNA isolation methods, for reliable and reproducible miRNA expression profiling the use of miRNA isolation kits are more suitable. PMID:26678076

  12. RNA Study Using DNA Nanotechnology.

    PubMed

    Tadakuma, Hisashi; Masubuchi, Takeya; Ueda, Takuya

    2016-01-01

    Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit. PMID:26970193

  13. The rise of regulatory RNA

    PubMed Central

    Morris, K.V.; Mattick, J.S.

    2015-01-01

    Discoveries over the last decade portend a paradigm shift in molecular biology. Evidence suggests that RNA is not only functional as a messenger between DNA and protein but also in the regulation of genome organization and gene expression, which is increasingly elaborated in complex organisms. Regulatory RNAs appear to operate at many levels, but in particular to play an important role in the epigenetic processes that control differentiation and development. These discoveries suggest a central role for RNA in human evolution and ontogeny. Here we survey the emergence of the previously unsuspected world of regulatory RNAs from an historical perspective. PMID:24776770

  14. Prevention and Immunotherapy of Secondary Murine Alveolar Echinococcosis Employing Recombinant EmP29 Antigen

    PubMed Central

    Boubaker, Ghalia; Hemphill, Andrew; Huber, Cristina Olivia; Spiliotis, Markus; Babba, Hamouda; Gottstein, Bruno

    2015-01-01

    Alveolar echinococcosis (AE) is caused by infection with the larval stage of the tapeworm Echinococcus multilocularis. An increasing understanding of immunological events that account for the metacestode survival in human and murine AE infection prompted us to undertake explorative experiments tackling the potential of novel preventive and/or immunotherapeutic measures. In this study, the immunoprotective and immunotherapeutic ability of recombinant EmP29 antigen (rEmP29) was assessed in mice that were intraperitoneally infected with E. multilocularis metacestodes. For vaccination, three intraperitoneal injections with 20μg rEmP29 emulsified in saponin adjuvants were applied over 6 weeks. 2 weeks after the last boost, mice were infected, and at 90 days post-infection, rEmP29-vaccinated mice exhibited a median parasite weight that was reduced by 75% and 59% when compared to NaCl- or saponin–treated control mice, respectively. For immunotherapeutical application, the rEmP29 (20μg) vaccine was administered to experimentally infected mice, starting at 1 month post-infection, three times with 2 weeks intervals. Mice undergoing rEmP29 immunotherapy exhibited a median parasite load that was reduced by 53% and 49% when compared to NaCl- and saponin–treated control mice, respectively. Upon analysis of spleen cells, both, vaccination and treatment with rEmP29, resulted in low ratios of Th2/Th1 (IL-4/IFN-γ) cytokine mRNA and low levels of mRNA coding for IL-10 and IL-2. These results suggest that reduction of the immunosuppressive environment takes place in vaccinated as well as immunotreated mice, and a shift towards a Th1 type of immune response may be responsible for the observed increased restriction of parasite growth. The present study provides the first evidence that active immunotherapy may present a sustainable route for the control of AE. PMID:26053794

  15. miRNA-223 inhibits epithelial-mesenchymal transition in gastric carcinoma cells via Sp1.

    PubMed

    Hu, Jing; Shan, Zhiyan; Hu, Kewei; Ren, Fengyun; Zhang, Wei; Han, Meiling; Li, Yuezhen; Feng, Kejian; Lei, Lei; Feng, Yukuan

    2016-07-01

    Sp1 plays critical roles in epithelial-mesenchymal transition (EMT) of certain cancer. However, few studies have indicated whether Sp1 is involved in the EMT of gastric cancer, and whether abnormal expression of Sp1 in gastric cancer EMT is regulated in a post-transcriptional manner, and the involvement of miRNAs in this regulation. In this study, we selected 20 cases of gastric cancers, their liver metastases and para-carcinoma tissues to examine the levels of Sp1 protein and mRNA by immunohistochemistry and fluorescent PCR, which showed that Sp1 was increased in gastric cancers and their metastases compared with adjacent tissues, but there was no difference in Sp1 mRNA between these three groups, suggesting changes in Sp1 may be attributed to inactivation of post-transcriptional regulation. We verified by a luciferase reporter system that miRNA-223 binds to 3'-UTR of Sp1 gene and inhibits its translation, in agreement with negative correlation between miRNA-223 and Sp1 protein levels in gastric cancer cells. By employing TGF-β1 to induce MGC-803, BGC-823 and SGC-7901, we successfully built cellular EMT model. Then, we overexpressed miRNA-223 in the model by using a lentiviral system, which diminished EMT indicators and suppressed proliferation and invasion ability, and induced apoptosis. Finally, we verified the specificity of the regulation pathway miRNA-223/Sp1/EMT. These findings suggest that low expression of miRNA-223 in gastric cancer cells is an important cause for EMT. miRNA-223 specifically regulates the EMT process of gastric cancer cells through its target gene Sp1. Overexpression of miRNA-223 in these cells inhibits EMT via the miRNA-223/Sp1/EMT pathway. PMID:27212195

  16. Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways* #

    PubMed Central

    Dai, Zhen; Wu, Rong; Zhao, Yi-cheng; Wang, Kan-kan; Huang, Yong-ye; Yang, Xin; Xie, Zi-cong; Tu, Chang-chun; Ouyang, Hong-sheng; Wang, Tie-dong; Pang, Da-xin

    2014-01-01

    RNA interference (RNAi) is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus (CSFV) replication by short hairpin RNA (shRNA) in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon (IFN)-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs (miRNA), and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNA/shRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer (SCNT) embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA-induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation. PMID:24793764

  17. Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation

    PubMed Central

    Sun, Chaomin; Querol-Audí, Jordi; Mortimer, Stefanie A.; Arias-Palomo, Ernesto; Doudna, Jennifer A.; Nogales, Eva; Cate, Jamie H. D.

    2013-01-01

    The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome. PMID:23766293

  18. Translational arrest by a prokaryotic signal recognition particle is mediated by RNA interactions.

    PubMed

    Beckert, Bertrand; Kedrov, Alexej; Sohmen, Daniel; Kempf, Georg; Wild, Klemens; Sinning, Irmgard; Stahlberg, Henning; Wilson, Daniel N; Beckmann, Roland

    2015-10-01

    The signal recognition particle (SRP) recognizes signal sequences of nascent polypeptides and targets ribosome-nascent chain complexes to membrane translocation sites. In eukaryotes, translating ribosomes are slowed down by the Alu domain of SRP to allow efficient targeting. In prokaryotes, however, little is known about the structure and function of Alu domain-containing SRPs. Here, we report a complete molecular model of SRP from the Gram-positive bacterium Bacillus subtilis, based on cryo-EM. The SRP comprises two subunits, 6S RNA and SRP54 or Ffh, and it facilitates elongation slowdown similarly to its eukaryotic counterpart. However, protein contacts with the small ribosomal subunit observed for the mammalian Alu domain are substituted in bacteria by RNA-RNA interactions of 6S RNA with the α-sarcin-ricin loop and helices H43 and H44 of 23S rRNA. Our findings provide a structural basis for cotranslational targeting and RNA-driven elongation arrest in prokaryotes. PMID:26344568

  19. RNA isolation and fractionation with compaction agents

    NASA Technical Reports Server (NTRS)

    Murphy, J. C.; Fox, G. E.; Willson, R. C.

    2001-01-01

    A new approach to the isolation of RNA from bacterial lysates employs selective precipitation by compaction agents, such as hexammine cobalt and spermidine. Using 3.5 mM hexammine cobalt, total RNA can be selectively precipitated from a cell lysate. At a concentration of 2 mM hexammine cobalt, rRNA can be fractionated from low molecular weight RNA. The resulting RNA mixture is readily resolved to pure 5S and mixed 16S/23S rRNA by nondenaturing anion-exchange chromatography. Using a second stage of precipitation at 8 mM hexammine cobalt, the low molecular weight RNA fraction can be isolated by precipitation. Compaction precipitation was also applied to the purification of an artificial stable RNA derived from Escherichia coli 5S rRNA and to the isolation of an Escherichia coli-expressed ribozyme. Copyright 2001 Academic Press.

  20. RNA helicases in infection and disease.

    PubMed

    Steimer, Lenz; Klostermeier, Dagmar

    2012-06-01

    RNA helicases unwind their RNA substrates in an ATP-dependent reaction, and are central to all cellular processes involving RNA. They have important roles in viral life cycles, where RNA helicases are either virus-encoded or recruited from the host. Vertebrate RNA helicases sense viral infections, and trigger the innate antiviral immune response. RNA helicases have been implicated in protozoic, bacterial and fungal infections. They are also linked to neurological disorders, cancer, and aging processes.   Genome-wide studies continue to identify helicase genes that change their expression patterns after infection or disease outbreak, but the mechanism of RNA helicase action has been defined for only a few diseases. RNA helicases are prognostic and diagnostic markers and suitable drug targets, predominantly for antiviral and anti-cancer therapies. This review summarizes the current knowledge on RNA helicases in infection and disease, and their growing potential as drug targets. PMID:22699555

  1. Conformational readout of RNA by small ligands

    PubMed Central

    Kligun, Efrat; Mandel-Gutfreund, Yael

    2013-01-01

    RNA molecules have highly versatile structures that can fold into myriad conformations, providing many potential pockets for binding small molecules. The increasing number of available RNA structures, in complex with proteins, small ligands and in free form, enables the design of new therapeutically useful RNA-binding ligands. Here we studied RNA ligand complexes from 10 RNA groups extracted from the protein data bank (PDB), including adaptive and non-adaptive complexes. We analyzed the chemical, physical, structural and conformational properties of binding pockets around the ligand. Comparing the properties of ligand-binding pockets to the properties of computed pockets extracted from all available RNA structures and RNA-protein interfaces, revealed that ligand-binding pockets, mainly the adaptive pockets, are characterized by unique properties, specifically enriched in rare conformations of the nucleobase and the sugar pucker. Further, we demonstrate that nucleotides possessing the rare conformations are preferentially involved in direct interactions with the ligand. Overall, based on our comprehensive analysis of RNA-ligand complexes, we suggest that the unique conformations adopted by RNA nucleotides play an important role in RNA recognition by small ligands. We term the recognition of a binding site by a ligand via the unique RNA conformations “RNA conformational readout.” We propose that “conformational readout” is a general way by which RNA binding pockets are recognized and selected from an ensemble of different RNA states. PMID:23618839

  2. Relicts and models of the RNA world

    NASA Astrophysics Data System (ADS)

    Lehto, Kirsi; Karetnikov, Alexey

    2005-01-01

    It is widely believed that the current DNA-RNA-protein-based life forms have evolved from preceding RNA-protein-based life forms, and these again, from mere RNA replicons. By rationale, it can be assumed that the early RNA replicons were fully heterotrophic in terms of obtaining all their building blocks from their environment. In the absence of protein catalysts, their essential life functions had to be mediated by simple functional structures and mechanisms, such as RNA secondary structures, RNA-RNA interactions and RNA-mediated catalysis, and possibly by catalytic minerals or clays. The central role of RNA catalysts in early life forms is supported by the fact that several catalytic RNAs still perform central biological functions in current life forms, and at least some of these may be derived as molecular relicts from the early RNA-based life. The RNA-catalysed metabolic reactions and molecular fossils are more conserved in the eukaryotic life forms than in the prokaryotes, suggesting that the linear eukaryote genomes may more closely resemble the structure and function of the early RNA replicons, than what do the circular prokaryote genomes. Present-day RNA viruses and viroids utilize ultimately simple life strategies, which may be similar to those used by the early RNA replicons. Thus, molecular and functional properties of viruses and viroids may be considered as examples or models of the structures and replication mechanisms, which might have been used for the replication of the early biopolymers.

  3. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking.

    PubMed

    Dolgosheina, Elena V; Jeng, Sunny C Y; Panchapakesan, Shanker Shyam S; Cojocaru, Razvan; Chen, Patrick S K; Wilson, Peter D; Hawkins, Nancy; Wiggins, Paul A; Unrau, Peter J

    2014-10-17

    Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes. PMID:25101481

  4. Avian influenza virus RNA extraction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The efficient extraction and purification of viral RNA is critical for down-stream molecular applications whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of avian influenza (AI) virus by molecular methods from expe...

  5. Mitomycin C Inhibits Ribosomal RNA

    PubMed Central

    Snodgrass, Ryan G.; Collier, Abby C.; Coon, Amy E.; Pritsos, Chris A.

    2010-01-01

    Mitomycin C (MMC) is a commonly used and extensively studied chemotherapeutic agent requiring biological reduction for activity. Damage to nuclear DNA is thought to be its primary mechanism of cell death. Due to a lack of evidence for significant MMC activation in the nucleus and for in vivo studies demonstrating the formation of MMC-DNA adducts, we chose to investigate alternative nucleic acid targets. Real-time reverse transcription-PCR was used to determine changes in mitochondrial gene expression induced by MMC treatment. Although no consistent effects on mitochondrial mRNA expression were observed, complementary results from reverse transcription-PCR experiments and gel-shift and binding assays demonstrated that MMC rapidly decreased the transcript levels of 18S ribosomal RNA in a concentration-dependent manner. Under hypoxic conditions, transcript levels of 18S rRNA decreased by 1.5-fold compared with untreated controls within 30 min. Recovery to base line required several hours, indicating that de novo synthesis of 18S was necessary. Addition of MMC to an in vitro translation reaction significantly decreased protein production in the cell-free system. Functional assays performed using a luciferase reporter construct in vivo determined that protein translation was inhibited, further confirming this mechanism of toxicity. The interaction of MMC with ribosomal RNA and subsequent inhibition of protein translation is consistent with mechanisms proposed for other natural compounds. PMID:20418373

  6. Circular RNA expands its territory.

    PubMed

    Bao, Chunyang; Lyu, Dongbin; Huang, Shenglin

    2016-03-01

    Circular RNAs (circRNAs) represent a novel class of widespread non-coding RNAs in eukaryotes. They are unusually stable RNA molecules with cell type-specific expression patterns, and are predominantly present in the cytoplasm. We recently demonstrated the existence of abundant circRNAs in exosomes and suggest a potential application of exosomal circRNAs for cancer detection. PMID:27308606

  7. Predicting RNA pseudoknot folding thermodynamics.

    PubMed

    Cao, Song; Chen, Shi-Jie

    2006-01-01

    Based on the experimentally determined atomic coordinates for RNA helices and the self-avoiding walks of the P (phosphate) and C4 (carbon) atoms in the diamond lattice for the polynucleotide loop conformations, we derive a set of conformational entropy parameters for RNA pseudoknots. Based on the entropy parameters, we develop a folding thermodynamics model that enables us to compute the sequence-specific RNA pseudoknot folding free energy landscape and thermodynamics. The model is validated through extensive experimental tests both for the native structures and for the folding thermodynamics. The model predicts strong sequence-dependent helix-loop competitions in the pseudoknot stability and the resultant conformational switches between different hairpin and pseudoknot structures. For instance, for the pseudoknot domain of human telomerase RNA, a native-like and a misfolded hairpin intermediates are found to coexist on the (equilibrium) folding pathways, and the interplay between the stabilities of these intermediates causes the conformational switch that may underlie a human telomerase disease. PMID:16709732

  8. RNA location and modeling of a WD40 repeat domain within the vault.

    PubMed Central

    Kong, L B; Siva, A C; Kickhoefer, V A; Rome, L H; Stewart, P L

    2000-01-01

    The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap. PMID:10864046

  9. RNA location and modeling of a WD40 repeat domain within the vault.

    PubMed

    Kong, L B; Siva, A C; Kickhoefer, V A; Rome, L H; Stewart, P L

    2000-06-01

    The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-A-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined "barrel only" images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap. PMID:10864046

  10. RNA cleavage and chain elongation by Escherichia coli DNA-dependent RNA polymerase in a binary enzyme.RNA complex.

    PubMed Central

    Altmann, C R; Solow-Cordero, D E; Chamberlin, M J

    1994-01-01

    In the absence of DNA, Escherichia coli RNA polymerase (EC 2.7.7.6) can bind RNA to form an equimolar binary complex with the concomitant release of the sigma factor. We show now that E. coli RNA polymerase binds at a region near the 3' terminus of the RNA and that an RNA in such RNA.RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA. These include GreA/GreB-dependent cleavage of the RNA and elongation by 3'-terminal addition of NMP from NTP. Both of these reactions are inhibited by rifampicin. Hence, by several criteria, the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes. These findings can be explained by a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein binding sites located up to 10 nt apart. In this model, the stability of RNA binding to the polymerase in the ternary complex is due primarily to its interaction with the protein. Images PMID:7513426

  11. Roles of the Coding and Noncoding Regions of Rift Valley Fever Virus RNA Genome Segments in Viral RNA Packaging

    PubMed Central

    Murakami, Shin; Terasaki, Kaori; Narayanan, Krishna

    2012-01-01

    We characterized the RNA elements involved in the packaging of Rift Valley fever virus RNA genome segments, L, M, and S. The 5′-terminal 25 nucleotides of each RNA segment were equally competent for RNA packaging and carried an RNA packaging signal, which overlapped with the RNA replication signal. Only the deletion mutants of L RNA, but not full-length L RNA, were efficiently packaged, implying the possible requirement of RNA compaction for L RNA packaging. PMID:22278239

  12. Multiple RNA interaction: beyond two.

    PubMed

    Mneimneh, Saad; Ahmed, Syed Ali

    2015-03-01

    The interaction of two RNA molecules involves a complex interplay between folding and binding that warranted recent developments in RNA-RNA interaction algorithms. However, biological mechanisms in which more than two RNAs take part in an interaction also exist. It is reasonable to believe that interactions involving multiple RNAs are generally more complex to be treated pairwise. In addition, given a pool of RNAs, it is not trivial to predict which RNAs interact without sufficient biological knowledge. Therefore, structures resulting from multiple RNA interactions often cannot be predicted by the existing algorithms that handle RNAs pairwise and may simply favor the best interacting pair. We propose a system for multiple RNA interaction that overcomes the difficulties mentioned above by formulating a combinatorial optimization problem called Pegs and Rubber Bands. A solution to this problem encodes a structure of interacting RNAs. The problem, not surprisingly, is NP-hard. However, our experiments with approximation algorithms and heuristics for the problem suggest that this formulation is adequate to predict known interaction patterns of multiple RNAs. In general, however, the optimal solution obtained does not necessarily correspond to the actual structure observed in biological experiments. Moreover, a structure produced by interacting RNAs may not be unique. We extend our approach to generate multiple suboptimal solutions. By clustering these solutions, we are able to reveal representatives that correspond to realistic structures. Specifically, our results on the U2-U6 complex with introns in the spliceosome of human/yeast and the CopA-CopT complex in E. coli are consistent with published biological structures. PMID:25680214

  13. Template-free generation of RNA species that replicate with bacteriophage T7 RNA polymerase.

    PubMed Central

    Biebricher, C K; Luce, R

    1996-01-01

    A large variety of different RNA species that are replicated by DNA-dependent RNA polymerase from bacteriophage T7 have been generated by incubating high concentrations of this enzyme with substrate for extended time periods. The products differed from sample to sample in molecular weight and sequence, their chain lengths ranging from 60 to 120. The mechanism of autocatalytic amplification of RNA by T7 RNA polymerase proved to be analogous to that observed with viral RNA-dependent RNA polymerases (replicases): only single-stranded templates are accepted and complementary replica strands are synthesized. With enzyme in excess, exponential growth was observed; linear growth resulted when the enzyme was saturated by RNA template. The plus strands, present at 90% of the replicating RNA species, were found to have GG residues at both termini. Consensus sequences were not found among the sequences of the replicating RNA species. The secondary structures of all species sequenced turned out to be hairpins. The RNA species were specifically replicated by T7 RNA polymerase; they were not accepted as templates by the RNA polymerases from Escherichia coli or bacteriophage SP6 or by Qbeta replicase; T3 RNA polymerase was partially active. Template-free production of RNA was completely suppressed by addition of DNA to the incubation mixture. When both DNA and RNA templates were present, transcription and replication competed, but T7 RNA polymerase preferred DNA as a template. No replicating RNA species were detected in vivo in cells expressing T7 RNA polymerase. Images PMID:8670848

  14. Spontaneous Formation of RNA Strands, Peptidyl RNA, and Cofactors

    PubMed Central

    Jauker, Mario; Griesser, Helmut; Richert, Clemens

    2015-01-01

    How the biochemical machinery evolved from simple precursors is an open question. Here we show that ribonucleotides and amino acids condense to peptidyl RNAs in the absence of enzymes under conditions established for genetic copying. Untemplated formation of RNA strands that can encode genetic information, formation of peptidyl chains linked to RNA, and formation of the cofactors NAD+, FAD, and ATP all occur under the same conditions. In the peptidyl RNAs, the peptide chains are phosphoramidate-linked to a ribonucleotide. Peptidyl RNAs with long peptide chains were selected from an initial pool when a lipophilic phase simulating the interior of membranes was offered, and free peptides were released upon acidification. Our results show that key molecules of genetics, catalysis, and metabolism can emerge under the same conditions, without a mineral surface, without an enzyme, and without the need for chemical pre-activation. PMID:26435376

  15. EPA LABORATORIES IMPLEMENT EMS PROGRAM

    EPA Science Inventory

    This paper highlights the breadth and magnitude of carrying out an effective Environmental Management System (EMS) program at the U.S. EPA's research and development laboratories. Federal research laboratories have unique operating challenges compared to more centralized industr...

  16. Role of RNA polymerase IV in plant small RNA metabolism.

    PubMed

    Zhang, Xiaoyu; Henderson, Ian R; Lu, Cheng; Green, Pamela J; Jacobsen, Steven E

    2007-03-13

    In addition to the three RNA polymerases (RNAP I-III) shared by all eukaryotic organisms, plant genomes encode a fourth RNAP (RNAP IV) that appears to be specialized in the production of siRNAs. Available data support a model in which dsRNAs are generated by RNAP IV and RNA-dependent RNAP 2 (RDR2) and processed by DICER (DCL) enzymes into 21- to 24-nt siRNAs, which are associated with different ARGONAUTE (AGO) proteins for transcriptional or posttranscriptional gene silencing. However, it is not yet clear what fraction of genomic siRNA production is RNAP IV-dependent, and to what extent these siRNAs are preferentially processed by certain DCL(s) or associated with specific AGOs for distinct downstream functions. To address these questions on a genome-wide scale, we sequenced approximately 335,000 siRNAs from wild-type and RNAP IV mutant Arabidopsis plants by using 454 technology. The results show that RNAP IV is required for the production of >90% of all siRNAs, which are faithfully produced from a discrete set of genomic loci. Comparisons of these siRNAs with those accumulated in rdr2 and dcl2 dcl3 dcl4 and those associated with AGO1 and AGO4 provide important information regarding the processing, channeling, and functions of plant siRNAs. We also describe a class of RNAP IV-independent siRNAs produced from endogenous single-stranded hairpin RNA precursors. PMID:17360559

  17. siRNA and RNAi optimization.

    PubMed

    Alagia, Adele; Eritja, Ramon

    2016-05-01

    The discovery and examination of the posttranscriptional gene regulatory mechanism known as RNA interference (RNAi) contributed to the identification of small interfering RNA (siRNA) and the comprehension of its enormous potential for clinical purposes. Theoretically, the ability of specific target gene downregulation makes the RNAi pathway an appealing solution for several diseases. Despite numerous hurdles resulting from the inherent properties of siRNA molecule and proper delivery to the target tissue, more than 50 RNA-based drugs are currently under clinical testing. In this work, we analyze the recent literature in the optimization of siRNA molecules. In detail, we focused on describing the most recent advances of siRNA field aimed at optimize siRNA pharmacokinetic properties. Special attention has been given in describing the impact of RNA modifications in the potential off-target effects (OTEs) such as saturation of the RNAi machinery, passenger strand-mediated silencing, immunostimulation, and miRNA-like OTEs as well as to recent developments on the delivery issue. The novel delivery systems and modified siRNA provide significant steps toward the development of reliable siRNA molecules for therapeutic use. WIREs RNA 2016, 7:316-329. doi: 10.1002/wrna.1337 For further resources related to this article, please visit the WIREs website. PMID:26840434

  18. Perda de massa em ventos empoeirados de estrelas supergigantes

    NASA Astrophysics Data System (ADS)

    Vidotto, A. A.; Jatenco-Pereira, V.

    2003-08-01

    Em praticamente todas as regiões do diagrama HR, as estrelas apresentam evidências observacionais de perda de massa. Na literatura, pode-se encontrar trabalhos que tratam tanto do diagnóstico da perda de massa como da construção de modelos que visam explicá-la. O amortecimento de ondas Alfvén tem sido utilizado como mecanismo de aceleração de ventos homogêneos. Entretanto, sabe-se que os envelopes de estrelas frias contêm grãos sólidos e moléculas. Com o intuito de estudar a interação entre as ondas Alfvén e a poeira e a sua conseqüência na aceleração do vento estelar, Falceta-Gonçalves & Jatenco-Pereira (2002) desenvolveram um modelo de perda de massa para estrelas supergigantes. Neste trabalho, apresentamos um estudo do modelo acima proposto para avaliar a dependência da taxa de perda de massa com alguns parâmetros iniciais como, por exemplo, a densidade r0, o campo magnético B0, o comprimento de amortecimento da onda L0, seu fluxo f0, entre outros. Sendo assim, aumentando f0 de 10% a partir de valores de referência, vimos que aumenta consideravelmente, enquanto que um aumento de mesmo valor em r0, B0 e L0 acarreta uma diminuição em .

  19. Linking RNA biology to lncRNAs

    PubMed Central

    Goff, Loyal A.; Rinn, John L.

    2015-01-01

    The regulatory potential of RNA has never ceased to amaze: from RNA catalysis, to RNA-mediated splicing, to RNA-based silencing of an entire chromosome during dosage compensation. More recently, thousands of long noncoding RNA (lncRNA) transcripts have been identified, the majority with unknown function. Thus, it is tempting to think that these lncRNAs represent a cadre of new factors that function through ribonucleic mechanisms. Some evidence points to several lncRNAs with tantalizing physiological contributions and thought-provoking molecular modalities. However, dissecting the RNA biology of lncRNAs has been difficult, and distinguishing the independent contributions of functional RNAs from underlying DNA elements, or the local act of transcription, is challenging. Here, we aim to survey the existing literature and highlight future approaches that will be needed to link the RNA-based biology and mechanisms of lncRNAs in vitro and in vivo. PMID:26430155

  20. Total Cellular RNA Modulates Protein Activity.

    PubMed

    Majumder, Subhabrata; DeMott, Christopher M; Reverdatto, Sergey; Burz, David S; Shekhtman, Alexander

    2016-08-16

    RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states. PMID:27456029

  1. Unmasking Upstream Gene Expression Regulators with miRNA-corrected mRNA Data

    PubMed Central

    Bollmann, Stephanie; Bu, Dengpan; Wang, Jiaqi; Bionaz, Massimo

    2015-01-01

    Expressed micro-RNA (miRNA) affects messenger RNA (mRNA) abundance, hindering the accuracy of upstream regulator analysis. Our objective was to provide an algorithm to correct such bias. Large mRNA and miRNA analyses were performed on RNA extracted from bovine liver and mammary tissue. Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%). Using four levels of target scores from TargetScan (all miRNA:mRNA target gene pairs or only the top 25%, 50%, or 75%) and four levels of the magnitude of miRNA effect (ME) on mRNA expression (30%, 50%, 75%, and 83% mRNA reduction), we generated 17 different datasets (including the original dataset). For each dataset, we performed upstream regulator analysis using two bioinformatics tools. We detected an increased effect on the upstream regulator analysis with larger miRNA:mRNA pair bins and higher ME. The miRNA correction allowed identification of several upstream regulators not present in the analysis of the original dataset. Thus, the proposed algorithm improved the prediction of upstream regulators. PMID:27279737

  2. RNAseq by Total RNA Library Identifies Additional RNAs Compared to Poly(A) RNA Library

    PubMed Central

    Guo, Yan; Zhao, Shilin; Sheng, Quanhu; Guo, Mingsheng; Lehmann, Brian; Pietenpol, Jennifer; Samuels, David C.; Shyr, Yu

    2015-01-01

    The most popular RNA library used for RNA sequencing is the poly(A) captured RNA library. This library captures RNA based on the presence of poly(A) tails at the 3′ end. Another type of RNA library for RNA sequencing is the total RNA library which differs from the poly(A) library by capture method and price. The total RNA library costs more and its capture of RNA is not dependent on the presence of poly(A) tails. In practice, only ribosomal RNAs and small RNAs are washed out in the total RNA library preparation. To evaluate the ability of detecting RNA for both RNA libraries we designed a study using RNA sequencing data of the same two breast cancer cell lines from both RNA libraries. We found that the RNA expression values captured by both RNA libraries were highly correlated. However, the number of RNAs captured was significantly higher for the total RNA library. Furthermore, we identify several subsets of protein coding RNAs that were not captured efficiently by the poly(A) library. One of the most noticeable is the histone-encode genes, which lack the poly(A) tail. PMID:26543871

  3. A Three-Helix Junction Is the Interface between Two Functional Domains of Prohead RNA in ϕ29 DNA Packaging

    PubMed Central

    Zhao, Wei; Saha, Mitul; Ke, Ailong; Morais, Marc C.; Jardine, Paul J.

    2012-01-01

    The double-stranded-DNA bacteriophages employ powerful molecular motors to translocate genomic DNA into preformed capsids during the packaging step in phage assembly. Bacillus subtilis bacteriophage ϕ29 has an oligomeric prohead RNA (pRNA) that is an essential component of its packaging motor. The crystal structure of the pRNA-prohead binding domain suggested that a three-helix junction constitutes both a flexible region and part of a rigid RNA superhelix. Here we define the functional role of the three-helix junction in motor assembly and DNA packaging. Deletion mutagenesis showed that a U-rich region comprising two sides of the junction plays a role in the stable assembly of pRNA to the prohead. The retention of at least two bulged residues in this region was essential for pRNA binding and thereby subsequent DNA packaging. Additional deletions resulted in the loss of the ability of pRNA to multimerize in solution, consistent with the hypothesis that this region provides the flexibility required for pRNA oligomerization and prohead binding. The third side of the junction is part of a large RNA superhelix that spans the motor. The insertion of bases into this feature resulted in a loss of DNA packaging and an impairment of initiation complex assembly. Additionally, cryo-electron microscopy (cryoEM) analysis of third-side insertion mutants showed an increased flexibility of the helix that binds the ATPase, suggesting that the rigidity of the RNA superhelix is necessary for efficient motor assembly and function. These results highlight the critical role of the three-way junction in bridging the prohead binding and ATPase assembly functions of pRNA. PMID:22896620

  4. Dynamic RNA modifications in disease.

    PubMed

    Klungland, Arne; Dahl, John Arne

    2014-06-01

    While the presence of 6-methyladenosine (m6A) modifications in mRNA was noted several decades ago, the first enzyme reversing this modification was identified very recently. Today we know of two methyltransferases introducing m6A in mRNA--METTL3 and METTL14--and two demethylases that remove it have been identified-FTO (ALKBH9) and ALKBH5. The conserved role of m6A seems to relate to meiosis, and mice lacking ALKBH5 are infertile. While loss-of-function mutation in FTO causes a recessive lethal syndrome, sequence variants in introns of the FTO gene are associated with obesity and type 2 diabetes. PMID:25005745

  5. Current techniques for visualizing RNA in cells

    PubMed Central

    Mannack, Lilith V.J.C.; Eising, Sebastian; Rentmeister, Andrea

    2016-01-01

    Labeling RNA is of utmost interest, particularly in living cells, and thus RNA imaging is an emerging field. There are numerous methods relying on different concepts ranging from hybridization-based probes, over RNA-binding proteins to chemo-enzymatic modification of RNA. These methods have different benefits and limitations. This review aims to outline the current state-of-the-art techniques and point out their benefits and limitations. PMID:27158473

  6. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. PMID:25611733

  7. Estudo comparativo entre estrelas centrais de nebulosas planetárias deficientes em hidrogênio

    NASA Astrophysics Data System (ADS)

    Marcolino, W. L. F.; de Araújo, F. X.

    2003-08-01

    Apresentamos neste trabalho o resultado de um estudo das principais características espectrais das estrelas centrais de nebulosas planetárias (ECNP) deficientes em hidrogênio. A origem e a evolução dessas estrelas ainda constitui um problema em aberto na evolução estelar. Geralmente esses objetos são divididos em [WCE], [WCL] e [WELS]. Os tipos [WCE] e [WCL] apresentam um espectro típico de uma estrela Wolf-Rayet carbonada de população I e as [WELS] apresentam linhas fracas de carbono e oxigênio em emissão. Existem evidências que apontam a seguinte sequência evolutiva : [WCL] = > [WCE] = > [WELS] = > PG 1159 (pré anã-branca). No entanto, tal cenário apresenta falhas como por exemplo a falta de ECNP entre os tipos [WCL] e [WCE]. Baseados em uma amostra de 24 objetos obtida no telescópio de 1.52m em La Silla, Chile (acordo ESO/ON), ao longo do ano 2000, apresentamos os resultados da comparação das larguras equivalentes de diversas linhas relevantes entre os tipos [WCL], [WCE] e [WELS]. Verificamos que nossos dados estão de acordo com a sequência evolutiva. Baseado nas linhas de C IV, conseguimos dividir pela primeira vez as [WELS] em dois grupos principais. Além disso, os dados reforçam a afirmação de que as [WCE] são as estrelas que possuem a maior temperatura entre as ECNP deficientes em hidrogênio. Discutimos ainda, a escassez de dados disponíveis na literatura e a necessidade da obtenção de parametros físicos para estes objetos.

  8. RNA decay machines: the exosome.

    PubMed

    Chlebowski, Aleksander; Lubas, Michał; Jensen, Torben Heick; Dziembowski, Andrzej

    2013-01-01

    The multisubunit RNA exosome complex is a major ribonuclease of eukaryotic cells that participates in the processing, quality control and degradation of virtually all classes of RNA in Eukaryota. All this is achieved by about a dozen proteins with only three ribonuclease activities between them. At first glance, the versatility of the pathways involving the exosome and the sheer multitude of its substrates are astounding. However, after fifteen years of research we have some understanding of how exosome activity is controlled and applied inside the cell. The catalytic properties of the eukaryotic exosome are fairly well described and attention is now drawn to how the interplay between these activities impacts cell physiology. Also, it has become evident that exosome function relies on many auxiliary factors, which are intensely studied themselves. In this way, the focus of exosome research is slowly leaving the test tube and moving back into the cell. The exosome also has an interesting evolutionary history, which is evident within the eukaryotic lineage but only fully appreciated when considering similar protein complexes found in Bacteria and Archaea. Thus, while we keep this review focused on the most comprehensively described yeast and human exosomes, we shall point out similarities or dissimilarities to prokaryotic complexes and proteins where appropriate. The article is divided into three parts. In Part One we describe how the exosome is built and how it manifests in cells of different organisms. In Part Two we detail the enzymatic properties of the exosome, especially recent data obtained for holocomplexes. Finally, Part Three presents an overview of the RNA metabolism pathways that involve the exosome. This article is part of a Special Issue entitled: RNA Decay mechanisms. PMID:23352926

  9. Discos de acresção em sistemas Be-X

    NASA Astrophysics Data System (ADS)

    Lopes de Oliveira, R.; Janot-Pacheco, E.

    2003-08-01

    Alguns fenômenos de outbursts em Be-X sugerem a existência, mesmo que temporária, de um disco de acresção quando da passagem do objeto compacto pelo periastro orbital. Neste trabalho avaliamos a possibilidade de formação do disco de acresção em sistemas Be+estrela de neutrons e Be+anã branca, e a influência da excentricidade orbital na ocorrência deste fenômeno. Utilizamos a expressão analítica para o momento angular específico da matéria constituinte de um meio em expansão lenta, como é o caso do disco circunstelar das estrelas Be, proposta por Wang(1981), sob a condição básica de que o raio de circularização deva ser maior do que o raio de Alfvén. Concluímos que existe um limite para o período orbital do sistema acima do qual não é possível a formação do disco de acresção, e que este valor aumenta para sistemas com excentricidade orbital maior.

  10. Discovery of Nuclear DNA-like RNA (dRNA, hnRNA) and Ribonucleoproteins Particles Containing hnRNA.

    PubMed

    Georgiev, G P

    2016-01-01

    On August 9-11, 2014, Cold Spring Harbor (USA) hosted a special symposium dedicated to the discovery of messenger or informational RNA and the main events in the subsequent studies of its synthesis, regulation of synthesis, maturation, and transport. The existence of mRNA in bacteria was first suggested in 1961 by Jacob and Monod, based on genetic studies [1]. The same year, Brenner et al. confirmed the hypothesis [2]. Our laboratory played a key role in the discovery of messenger RNA in eukaryotes, as well as in the discovery of the nuclear ribonucleoproteins that contain it and in the elucidation of their structural organization. Therefore, I was invited to represent Russia at the Symposium and deliver a speech on these topics. However, my visa had only been issued after the end of the Symposium, and, therefore, the presentation was delivered by my former colleague G.N. Yenikolopov, who works at Cold Spring Harbor Laboratory. The transcript of the lecture is presented below. PMID:27099780