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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. 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

  2. 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

  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. 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

  17. 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)

  18. 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

  19. 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

  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. 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

  15. 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

  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. 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

  5. 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

  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

    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.

  12. 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

  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. 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

  19. 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

  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. 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.

  14. 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

  15. 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

  16. 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

  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. 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

  3. 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

  4. 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.

  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. 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.

  8. 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

  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

    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 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

  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. 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

  20. 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

  1. 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

  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. 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.

  12. 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

  13. 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

  14. 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.

  15. 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

  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. 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.

  4. 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

  5. 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...

  6. 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.

  7. 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.

  8. 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.

  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. 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

  12. 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

  13. 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.

  14. [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

  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. 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

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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

  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. 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

  8. 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

  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. 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

  11. 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

  12. 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

  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

    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

  16. 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

  17. 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

  18. 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

  19. 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

  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. 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

  5. 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

  6. 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.

  7. 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

  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. 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

  10. 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

  11. 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

  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. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  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. 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

  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. 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

  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. 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.

  2. 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

  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. 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

  5. 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

  6. 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...

  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. 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...

  14. 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

  15. 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

  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. 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

  2. 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

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

  10. Almost lost in translation. Cryo-EM of a dynamic macromolecular complex: the ribosome.

    PubMed

    Valle, Mikel

    2011-05-01

    Ribosomes are dynamic biological machines that perform numerous tasks during translation, the biosynthesis of proteins. Translocation, the movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) to progress in the reading frame of codons in the mRNA, takes place after the addition of each amino acid. This process involves large ribosome conformational changes, where tRNAs proceed through intermediate states. The structural characterization of these translocation intermediates has remained elusive. Cryo-electron microscopy (cryo-EM) produces three-dimensional averages, and translocating ribosomes poise distinct conformational states, and hence, structurally heterogeneous populations. During the last decade, the quest for visualization of translocation intermediates has progressed together with the development of classification tools in cryo-EM. Some of these new tools have recently been tested in ribosomal translocation, uncovering a clearer picture of the process. This success goes along with the latest advances in cryo-EM and illustrates how the technique offers multiple possibilities for studying macromolecular complexes engaged in dynamic reactions. PMID:21336521

  11. Directly Reconstructing Principal Components of Heterogeneous Particles from Cryo-EM Images

    PubMed Central

    Tagare, Hemant D.; Kucukelbir, Alp; Sigworth, Fred J.; Wang, Hongwei; Rao, Murali

    2015-01-01

    Structural heterogeneity of particles can be investigated by their three-dimensional principal components. This paper addresses the question of whether, and with what algorithm, the three-dimensional principal components can be directly recovered from cryo-EM images. The first part of the paper extends the Fourier slice theorem to covariance functions showing that the three-dimensional covariance, and hence the principal components, of a heterogeneous particle can indeed be recovered from two-dimensional cryo-EM images. The second part of the paper proposes a practical algorithm for reconstructing the principal components directly from cryo-EM images without the intermediate step of calculating covariances. This algorithm is based on maximizing the (posterior) likelihood using the Expectation-Maximization algorithm. The last part of the paper applies this algorithm to simulated data and to two real cryo-EM data sets: a data set of the 70S ribosome with and without Elongation Factor-G (EF-G), and a data set of the inluenza virus RNA dependent RNA Polymerase (RdRP). The first principal component of the 70S ribosome data set reveals the expected conformational changes of the ribosome as the EF-G binds and unbinds. The first principal component of the RdRP data set reveals a conformational change in the two dimers of the RdRP. PMID:26049077

  12. Análise da medição do raio solar em ultravioleta

    NASA Astrophysics Data System (ADS)

    Saraiva, A. C. V.; Giménez de Castro, C. G.; Costa, J. E. R.; Selhorst, C. L.; Simões, P. J. A.

    2003-08-01

    A medição acurada do raio solar em qualquer banda do espectro eletromagnético é de relevância na formulação e calibração de modelos da estrutura e atmosfera solar. Esses modelos atribuem emissão do contínuo do Sol calmo em microondas à mesma região da linha Ha do Hell. Apresentamos a medição do raio solar em UV com imagens do EIT (Extreme Ultraviolet Image Telescope) entre 1996 e 2002, no comprimento de onda 30,9 nm (Ha do Hell), que se forma na região de transição/cromosfera solar. A técnica utilizada para o cálculo do raio UV foi baseada na transformada Wavelet B3spline. Fizemos um banco de dados com 1 imagem por dia durante o período citado. Obtivemos como resultado o raio médio da ordem de 975.61" e uma diminuição do mesmo para o período citado variando em média -0,45" /ano. Comparamos estes dados com os valores obtidos pelo ROI (Radio Observatório de Itapetinga) em 22/48 GHz e Nobeyama Radio Heliograph em 17 GHz mostrando que os raios médios são muito próximos o que indica que a região de formação nessas freqüências é a mesma conforme os modelos. Comparamos os resultados também com outros índices de atividade solar.

  13. Riboswitches and the RNA World

    PubMed Central

    Breaker, Ronald R.

    2012-01-01

    Summary Riboswitches are structured noncoding RNA domains that selectively bind metabolites and control gene expression (Mandal and Breaker 2004a; Coppins et al. 2007; Roth and Breaker 2009). Nearly all examples of the known riboswitches reside in noncoding regions of messenger RNAs where they control transcription or translation. Newfound classes of riboswitches are being reported at a rate of about three per year (Ames and Breaker 2009), and these have been shown to selectively respond to fundamental metabolites including coenzymes, nucleobases or their derivatives, amino acids, and other small molecule ligands. The characteristics of some riboswitches suggest they could be modern descendents of an ancient sensory and regulatory system that likely functioned before the emergence of enzymes and genetic factors made of protein (Nahvi et al. 2002; Vitreschak et al. 2004; Breaker 2006). If true, then some of the riboswitch structures and functions that serve modern cells so well may accurately reflect the capabilities of RNA sensors and switches that existed in the RNA World. This article will address some of the characteristics of modern riboswitches that may be relevant to ancient versions of these metabolite-sensing RNAs. PMID:21106649

  14. 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

  15. Salt RNA protection against thermodegradation

    NASA Astrophysics Data System (ADS)

    Vergne, J.; Maurel, M.-C.

    2003-04-01

    We shown the structural integrity of tRNA at high temperature, 82^oC for 30h, in high salt concentrations (Tehei et al, 2002). Stability were also performed by measuring the residual specific tRNA charge capacity after heat treatment for 30 h at 82^oC. We have undertaken in vitro selection of RNA molecules at high temperature in presence of an ancient halite (NaCl) sample (reference : EZ08-K6-C9). This sample, collected in a borehole at 720.15 m depth, belongs to the Rupelian Upper Salt Formation of the Bresse salt basin (France). Its age is estimated to about 31±3 millions years. These studies provide support for the importance of salt to protect macromolecules against thermal degradation allowing activity to be recovered. These could be useful for searching traces of life in ancient sediments and in planetary exploration. Reference: Tehei Moeva, Franzetti Bruno, Maurel Marie-Christine, Vergne Jacques, Hountondji Codjo and Zaccai Giuseppe, Extremophiles, (2002), 6: 427-430.

  16. Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design.

    PubMed

    Krol, Jacek; Sobczak, Krzysztof; Wilczynska, Urszula; Drath, Maria; Jasinska, Anna; Kaczynska, Danuta; Krzyzosiak, Wlodzimierz J

    2004-10-01

    We have established the structures of 10 human microRNA (miRNA) precursors using biochemical methods. Eight of these structures turned out to be different from those that were computer-predicted. The differences localized in the terminal loop region and at the opposite side of the precursor hairpin stem. We have analyzed the features of these structures from the perspectives of miRNA biogenesis and active strand selection. We demonstrated the different thermodynamic stability profiles for pre-miRNA hairpins harboring miRNAs at their 5'- and 3'-sides and discussed their functional implications. Our results showed that miRNA prediction based on predicted precursor structures may give ambiguous results, and the success rate is significantly higher for the experimentally determined structures. On the other hand, the differences between the predicted and experimentally determined structures did not affect the stability of termini produced through "conceptual dicing." This result confirms the value of thermodynamic analysis based on mfold as a predictor of strand section by RNAi-induced silencing complex (RISC). PMID:15292246

  17. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    PubMed Central

    Dedduwa-Mudalige, Gayani N. P.; Chow, Christine S.

    2015-01-01

    Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA) intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA) including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human. PMID:26370969

  18. Small catalytic RNA: Structure, function and application

    SciTech Connect

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the paperclip'' and hammerhead'' RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a hammerhead,'' to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  19. Endogenous polyamine function—the RNA perspective

    PubMed Central

    Lightfoot, Helen L.; Hall, Jonathan

    2014-01-01

    Recent progress with techniques for monitoring RNA structure in cells such as ‘DMS-Seq’ and ‘Structure-Seq’ suggests that a new era of RNA structure-function exploration is on the horizon. This will also include systematic investigation of the factors required for the structural integrity of RNA. In this context, much evidence accumulated over 50 years suggests that polyamines play important roles as modulators of RNA structure. Here, we summarize and discuss recent literature relating to the roles of these small endogenous molecules in RNA function. We have included studies directed at understanding the binding interactions of polyamines with polynucleotides, tRNA, rRNA, mRNA and ribozymes using chemical, biochemical and spectroscopic tools. In brief, polyamines bind RNA in a sequence-selective fashion and induce changes in RNA structure in context-dependent manners. In some cases the functional consequences of these interactions have been observed in cells. Most notably, polyamine-mediated effects on RNA are frequently distinct from those of divalent cations (i.e. Mg2+) confirming their roles as independent molecular entities which help drive RNA-mediated processes. PMID:25232095

  20. Implementação de um algoritmo para a limpeza de mapas da RCFM

    NASA Astrophysics Data System (ADS)

    Souza, C. L.; Wuensche, C. A.

    2003-08-01

    A Radiação Cósmica de Fundo em Microondas (RCFM), descoberta por Penzias e Wilson em 1965, é uma das ferramentas mais poderosas para o estudo da cosmologia. Com a descoberta de flutuações de temperatura na RCFM, da ordem de uma parte em 105, pelo COBE (1992), uma nova era teve início. Nos últimos onze anos, diversos instrumentos fizeram novas medidas de alta precisão, refinando os resultados apresentados pelo COBE, culminando com os resultados recentes do satélite WMAP. A análise de dados da RCFM, especialmente no caso de experimentos com pequena cobertura do céu, apresenta uma série de dificuldades devido a emissões de contaminantes externos, tais como a emissão da Galáxia e de fontes pontuais, e de ruídos intrínsecos tanto ao sistema de detecção quanto à estratégia de observação do céu. Uma das soluções típicas para a filtragem de dados brutos de um experimento para medir flutuações de temperatura é aplicar um gabarito (template) e um filtro passa alta ao produzir mapas simplificados (sem considerar matrizes de correlação ou covariância). No caso de experimentos que utilizam detectores HEMT, essa combinação de filtros remove, satisfatoriamente, ruídos do tipo 1/f gerados pela instabilidade no ganho do detector acoplado ao movimento do instrumento, definido pela estratégia de observação. Entretanto, o sinal resultante medido, tanto em simulações quanto em séries temporais reais, sugere que parte do sinal cosmológico pode estar sendo removido junto com o ruído dos detectores. Este trabalho descreve as etapas para a produção de um mapa típico (simulado) e os testes preliminares de um algoritmo para remover ruídos do tipo 1/f introduzidos pela estratégia de observação sem prejudicar a qualidade do sinal cosmológico presente no mapa.

  1. The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing

    PubMed Central

    Vourekas, Anastassios; Fu, Qi; Maragkakis, Manolis; Alexiou, Panagiotis; Ma, Jing; Pillai, Ramesh S.

    2015-01-01

    Piwi–piRNA (Piwi-interacting RNA) ribonucleoproteins (piRNPs) enforce retrotransposon silencing, a function critical for preserving the genome integrity of germ cells. The molecular functions of most of the factors that have been genetically implicated in primary piRNA biogenesis are still elusive. Here we show that MOV10L1 exhibits 5′-to-3′ directional RNA-unwinding activity in vitro and that a point mutation that abolishes this activity causes a failure in primary piRNA biogenesis in vivo. We demonstrate that MOV10L1 selectively binds piRNA precursor transcripts and is essential for the generation of intermediate piRNA processing fragments that are subsequently loaded to Piwi proteins. Multiple analyses suggest an intimate coupling of piRNA precursor processing with elements of local secondary structures such as G quadruplexes. Our results support a model in which MOV10L1 RNA helicase activity promotes unwinding and funneling of the single-stranded piRNA precursor transcripts to the endonuclease that catalyzes the first cleavage step of piRNA processing. PMID:25762440

  2. PredcircRNA: computational classification of circular RNA from other long non-coding RNA using hybrid features.

    PubMed

    Pan, Xiaoyong; Xiong, Kai

    2015-08-01

    Recently circular RNA (circularRNA) has been discovered as an increasingly important type of long non-coding RNA (lncRNA), playing an important role in gene regulation, such as functioning as miRNA sponges. So it is very promising to identify circularRNA transcripts from de novo assembled transcripts obtained by high-throughput sequencing, such as RNA-seq data. In this study, we presented a machine learning approach, named as PredcircRNA, focused on distinguishing circularRNA from other lncRNAs using multiple kernel learning. Firstly we extracted different sources of discriminative features, including graph features, conservation information and sequence compositions, ALU and tandem repeats, SNP densities and open reading frames (ORFs) from transcripts. Secondly, to better integrate features from different sources, we proposed a computational approach based on a multiple kernel learning framework to fuse those heterogeneous features. Our preliminary 5-fold cross-validation result showed that our proposed method can classify circularRNA from other types of lncRNAs with an accuracy of 0.778, sensitivity of 0.781, specificity of 0.770, precision of 0.784 and MCC of 0.554 in our constructed gold-standard dataset, respectively. Our feature importance analysis based on Random Forest illustrated some discriminative features, such as conservation features and a GTAG sequence motif. Our PredcircRNA tool is available for download at . PMID:26028480

  3. Identification and characterization of two novel genomic RNA segments RNA5 and RNA6 in rose rosette virus infecting roses.

    PubMed

    Babu, B; Washburn, B K; Poduch, K; Knox, G W; Paret, M L

    2016-06-01

    Rose rosette virus (RRV), a negative-strand RNA virus belonging to the genus Emaravirus, has recently been characterized to be the causal agent of rose rosette disease. Roses showing typical symptoms of RRV collected from a rose nursery in Florida were subjected to reverse transcription-PCR (RT-PCR) assay using primers corresponding to the conserved inverted 13 nucleotide long stretches found at the termini of the RRV genomic RNA segments. RT-PCR analysis yielded two novel genomic RNA segments, RNA5 and RNA6, in addition to the previously identified four RNA segments. The RNA5 is 1650 bp long and encodes for a polypeptide of 465 amino acids (54.3 K), while RNA6 is 1400 bp long and encodes for a polypeptide of 233 amino acids (27.05 K). RACE analysis showed that, both the RNA segments posses at their 5' and 3' termini, stretches of conserved inverted complementary13 nucleotides long sequence with two nucleotide mismatches as previously identified in other genomic RNA segments. Northern blot analysis as well as RT-PCR using specific primers showed the presence of the novel genomic RNA segments in infected plants, but absent in the non-infected plants. The GenBank Acc. Nos. for the sequences reported in this paper are KT007556 and KT007557. PMID:27265465

  4. Initiation of minus-strand RNA synthesis by the brome mosaicvirus RNA-dependent RNA polymerase: use of oligoribonucleotide primers.

    PubMed Central

    Kao, C C; Sun, J H

    1996-01-01

    Various DNA- and RNA-dependent RNA polymerases have been reported to use oligoribonucleotide primers to initiate nucleic acid synthesis. For the brome mosaic virus RNA-dependent RNA polymerase (RdRp), we determined that in reactions performed with limited GTP concentrations, minus-strand RNA synthesis can be stimulated by the inclusion of guanosine monophosphate or specific oligoribonucleotides. Furthermore, guanylyl-3',5'-guanosine (GpG) was incorporated into minus-strand RNA and increased the rate of minus-strand RNA synthesis. In the presence of GpG, RdRp's Km for GTP decreased from 50 microM to approximately 3 microM while the Kms for other nucleotides were unaffected. These results have implications for the mechanism of initiation by RdRp. PMID:8794323

  5. Effect of the "RNA control" locus in Escherichia coli on RNA bacteriophage R23 replication.

    PubMed Central

    Ernberg, J; Sköld, O

    1976-01-01

    The effect of the rel gene of Escherichia coli on the RNA synthesis induced by phage R23 was studied. This RNA phage has the property of inhibiting ribosomal RNA formation and completely dominating the RNA synthesis of the host. Phage-specific RNA formation was found to be dependent on the allelic state of the rel gene. Determinations of RNA synthesis were made by both cumulative and short-term incorporations of uracil and adenine. Variations in labeling of nucleotide pools were compensated for by determining specific activities of ATP and UTP and using these values to obtain true, relative rates of RNA synthesis. PMID:768516

  6. Multisubunit RNA Polymerases IV and V: Purveyors of Non-Coding RNA for Plant Gene Silencing

    SciTech Connect

    Haag, Jeremy R.; Pikaard, Craig S.

    2011-08-01

    In all eukaryotes, nuclear DNA-dependent RNA polymerases I, II and III synthesize the myriad RNAs that are essential for life. Remarkably, plants have evolved two additional multisubunit RNA polymerases, RNA polymerases IV and V, which orchestrate non-coding RNA-mediated gene silencing processes affecting development, transposon taming, antiviral defence and allelic crosstalk. Biochemical details concerning the templates and products of RNA polymerases IV and V are lacking. However, their subunit compositions reveal that they evolved as specialized forms of RNA polymerase II, which provides the unique opportunity to study the functional diversification of a eukaryotic RNA polymerase family.

  7. Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development.

    PubMed

    Song, Xianwei; Wang, Dekai; Ma, Lijia; Chen, Zhiyu; Li, Pingchuan; Cui, Xia; Liu, Chunyan; Cao, Shouyun; Chu, Chengcai; Tao, Yuezhi; Cao, Xiaofeng

    2012-08-01

    Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development. PMID:22443269

  8. A conserved RNA polymerase III promoter required for gammaherpesvirus TMER transcription and microRNA processing

    PubMed Central

    Diebel, Kevin W.; Claypool, David J.; van Dyk, Linda F.

    2014-01-01

    Canonical RNA polymerase III (pol III) type 2 promoters contain a single A and B box and are well documented for their role in tRNA and SINE transcription in eukaryotic cells. The genome of Murid herpesvirus 4 (MuHV-4) contains eight polycistronic tRNA-microRNA encoded RNA (TMER) genes that are transcribed from a RNA pol III type 2-like promoter containing triplicated A box elements. Here, we demonstrate that the triplicated A box sequences are required in their entirety to produce functional MuHV-4 miRNAs. We also identify that these RNA pol III type 2-like promoters are conserved in eukaryotic genomes. Human and mouse predicted tRNA genes containing these promoters also show enrichment of alternative RNA pol III transcription termination sequences and are predicted to give rise to longer tRNA primary transcripts. PMID:24747015

  9. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    SciTech Connect

    Rumi, Mohammad; Ishihara, Shunji . E-mail: si360405@med.shimane-u.ac.jp; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-13

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor {alpha}-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.

  10. Structure of the initiation-competent RNA polymerase I and its implication for transcription

    NASA Astrophysics Data System (ADS)

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-07-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation.

  11. Structure of the initiation-competent RNA polymerase I and its implication for transcription

    PubMed Central

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-01-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation. PMID:27418187

  12. Structure of the initiation-competent RNA polymerase I and its implication for transcription.

    PubMed

    Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

    2016-01-01

    Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation. PMID:27418187

  13. RNA catalysis and the origins of life

    NASA Technical Reports Server (NTRS)

    Orgel, Leslie E.

    1986-01-01

    The role of RNA catalysis in the origins of life is considered in connection with the discovery of riboszymes, which are RNA molecules that catalyze sequence-specific hydrolysis and transesterification reactions of RNA substrates. Due to this discovery, theories positing protein-free replication as preceding the appearance of the genetic code are more plausible. The scope of RNA catalysis in biology and chemistry is discussed, and it is noted that the development of methods to select (or predict) RNA sequences with preassigned catalytic functions would be a major contribution to the study of life's origins.

  14. Double-Stranded RNA Resists Condensation

    NASA Astrophysics Data System (ADS)

    Li, Li; Pabit, Suzette A.; Meisburger, Steve P.; Pollack, Lois

    2011-03-01

    Much attention has been focused on DNA condensation because of its fundamental biological importance. The recent discovery of new roles for RNA duplexes demands efficient packaging of double-stranded RNA for therapeutics. Here we report measurements of short DNA and RNA duplexes in the presence of trivalent ions. Under conditions where UV spectroscopy indicates condensation of DNA duplexes into (insoluble) precipitates, RNA duplexes remain soluble. Small angle x-ray scattering results suggest that the differing surface topologies of RNA and DNA may be crucial in generating the attractive forces that result in precipitation.

  15. Engineering biological systems with synthetic RNA molecules

    PubMed Central

    Liang, Joe C.; Bloom, Ryan J.; Smolke, Christina D.

    2011-01-01

    RNA molecules play diverse functional roles in natural biological systems. There has been growing interest in designing synthetic RNA counterparts for programming biological function. The design of synthetic RNA molecules that exhibit diverse activities, including sensing, regulatory, information processing, and scaffolding activities, has highlighted the advantages of RNA as a programmable design substrate. Recent advances in implementing these engineered RNA molecules as key control elements in synthetic genetic networks are highlighting the functional relevance of this class of synthetic elements in programming cellular behaviors. PMID:21925380

  16. TruSeq Stranded mRNA and Total RNA Sample Preparation Kits

    Cancer.gov

    Total RNA-Seq enabled by ribosomal RNA (rRNA) reduction is compatible with formalin-fixed paraffin embedded (FFPE) samples, which contain potentially critical biological information. The family of TruSeq Stranded Total RNA sample preparation kits provides a unique combination of unmatched data quality for both mRNA and whole-transcriptome analyses, robust interrogation of both standard and low-quality samples and workflows compatible with a wide range of study designs.

  17. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  18. Characteristics and Prediction of RNA Structure

    PubMed Central

    Zhu, Daming; Zhang, Caiming; Han, Huijian; Crandall, Keith A.

    2014-01-01

    RNA secondary structures with pseudoknots are often predicted by minimizing free energy, which is NP-hard. Most RNAs fold during transcription from DNA into RNA through a hierarchical pathway wherein secondary structures form prior to tertiary structures. Real RNA secondary structures often have local instead of global optimization because of kinetic reasons. The performance of RNA structure prediction may be improved by considering dynamic and hierarchical folding mechanisms. This study is a novel report on RNA folding that accords with the golden mean characteristic based on the statistical analysis of the real RNA secondary structures of all 480 sequences from RNA STRAND, which are validated by NMR or X-ray. The length ratios of domains in these sequences are approximately 0.382L, 0.5L, 0.618L, and L, where L is the sequence length. These points are just the important golden sections of sequence. With this characteristic, an algorithm is designed to predict RNA hierarchical structures and simulate RNA folding by dynamically folding RNA structures according to the above golden section points. The sensitivity and number of predicted pseudoknots of our algorithm are better than those of the Mfold, HotKnots, McQfold, ProbKnot, and Lhw-Zhu algorithms. Experimental results reflect the folding rules of RNA from a new angle that is close to natural folding. PMID:25110687

  19. Emerging roles of RNA modifications in bacteria.

    PubMed

    Marbaniang, Carmelita Nora; Vogel, Jörg

    2016-04-01

    RNA modifications are known to abound in stable tRNA and rRNA, where they cluster around functionally important regions. However, RNA-seq based techniques profiling entire transcriptomes are now uncovering an abundance of modified ribonucleotides in mRNAs and noncoding RNAs, too. While most of the recent progress in understanding the regulatory influence of these new RNA modifications stems from eukaryotes, there is growing evidence in bacteria for modified nucleotides beyond the stable RNA species, including modifications of small regulatory RNAs. Given their small genome size, good genetic tractability, and ample knowledge of modification enzymes, bacteria offer excellent model systems to decipher cellular functions of RNA modifications in many diverse physiological contexts. This review highlights how new global approaches combining classic analysis with new sequencing techniques may usher in an era of bacterial epitranscriptomics. PMID:26803287

  20. Developing mRNA-vaccine technologies

    PubMed Central

    Schlake, Thomas; Thess, Andreas; Fotin-Mleczek, Mariola; Kallen, Karl-Josef

    2012-01-01

    mRNA vaccines combine desirable immunological properties with an outstanding safety profile and the unmet flexibility of genetic vaccines. Based on in situ protein expression, mRNA vaccines are capable of inducing a balanced immune response comprising both cellular and humoral immunity while not subject to MHC haplotype restriction. In addition, mRNA is an intrinsically safe vector as it is a minimal and only transient carrier of information that does not interact with the genome. Because any protein can be expressed from mRNA without the need to adjust the production process, mRNA vaccines also offer maximum flexibility with respect to development. Taken together, mRNA presents a promising vector that may well become the basis of a game-changing vaccine technology platform. Here, we outline the current knowledge regarding different aspects that should be considered when developing an mRNA-based vaccine technology. PMID:23064118

  1. Optimal alphabets for an RNA world.

    PubMed Central

    Gardner, Paul P; Holland, Barbara R; Moulton, Vincent; Hendy, Mike; Penny, David

    2003-01-01

    Experiments have shown that the canonical AUCG genetic alphabet is not the only possible nucleotide alphabet. In this work we address the question 'is the canonical alphabet optimal?' We make the assumption that the genetic alphabet was determined in the RNA world. Computational tools are used to infer the RNA secondary structure (shape) from a given RNA sequence, and statistics from RNA shapes are gathered with respect to alphabet size. Then, simulations based upon the replication and selection of fixed-sized RNA populations are used to investigate the effect of alternative alphabets upon RNA's ability to step through a fitness landscape. These results show that for a low copy fidelity the canonical alphabet is fitter than two-, six- and eight-letter alphabets. In higher copy-fidelity experiments, six-letter alphabets outperform the four-letter alphabets, suggesting that the canonical alphabet is indeed a relic of the RNA world. PMID:12816657

  2. Use of Deoxyribozymes in RNA Research

    PubMed Central

    Baum, Dana A.

    2010-01-01

    Since their first identification by in vitro selection in 1994, deoxyribozymes have been developed to catalyze a variety of chemical reactions. The first DNA-catalyzed reaction was cleavage of a ribonucleotide linkage within an oligonucleotide substrate. In subsequent years, growing collections of deoxyribozymes have been developed for several reactions that have practical utility for RNA research. These deoxyribozymes are useful for site-specific RNA cleavage as well as ligation to form linear, branched, and lariat RNA products. An application related to RNA ligation is deoxyribozyme-catalyzed labeling of RNA (DECAL), which is used to attach a biophysical tag to a desired RNA sequence at a specific position. With current achievements and likely future developments, deoxyribozymes are a useful contributor to the toolbox of RNA research methods. PMID:20946786

  3. Understanding the transcriptome through RNA structure

    PubMed Central

    Wan, Yue; Kertesz, Michael; Spitale, Robert C.; Segal, Eran; Chang, Howard

    2013-01-01

    RNA structure is critical for gene regulation and function. In the past, transcriptomes have been largely parsed by primary sequences and expression levels, but it is now becoming feasible to annotate and compare transcriptomes based on RNA structure. In addition to computational prediction methods, the recent advent of experimental techniques to probe RNA structure by deep sequencing has enabled genome-wide measurements of RNA structure, and provided the first picture of the structural organization of an eukaryotic transcriptome—the “RNA structurome”. With additional advances in method refinement and interpretation, structural views of the transcriptome should help to identify and validate regulatory RNA motifs that are involved in diverse cellular processes, and thereby increase understanding of RNA function. PMID:21850044

  4. Polyadenylation of stable RNA precursors in vivo

    PubMed Central

    Li, Zhongwei; Pandit, Shilpa; Deutscher, Murray P.

    1998-01-01

    Polyadenylation at the 3′ terminus has long been considered a specific feature of mRNA and a few other unstable RNA species. Here we show that stable RNAs in Escherichia coli can be polyadenylated as well. RNA molecules with poly(A) tails are the major products that accumulate for essentially all stable RNA precursors when RNA maturation is slowed because of the absence of processing exoribonucleases; poly(A) tails vary from one to seven residues in length. The polyadenylation process depends on the presence of poly(A) polymerase I. A stochastic competition between the exoribonucleases and poly(A) polymerase is proposed to explain the accumulation of polyadenylated RNAs. These data indicate that polyadenylation is not unique to mRNA, and its widespread occurrence suggests that it serves a more general function in RNA metabolism. PMID:9770456

  5. RNA sequence analysis using covariance models.

    PubMed Central

    Eddy, S R; Durbin, R

    1994-01-01

    We describe a general approach to several RNA sequence analysis problems using probabilistic models that flexibly describe the secondary structure and primary sequence consensus of an RNA sequence family. We call these models 'covariance models'. A covariance model of tRNA sequences is an extremely sensitive and discriminative tool for searching for additional tRNAs and tRNA-related sequences in sequence databases. A model can be built automatically from an existing sequence alignment. We also describe an algorithm for learning a model and hence a consensus secondary structure from initially unaligned example sequences and no prior structural information. Models trained on unaligned tRNA examples correctly predict tRNA secondary structure and produce high-quality multiple alignments. The approach may be applied to any family of small RNA sequences. Images PMID:8029015

  6. Crystal structure of a plectonemic RNA supercoil

    SciTech Connect

    Stagno, Jason R.; Ma, Buyong; Li, Jess; Altieri, Amanda S.; Byrd, R. Andrew; Ji, Xinhua

    2012-12-14

    Genome packaging is an essential housekeeping process in virtually all organisms for proper storage and maintenance of genetic information. Although the extent and mechanisms of packaging vary, the process involves the formation of nucleic-acid superstructures. Crystal structures of DNA coiled coils indicate that their geometries can vary according to sequence and/or the presence of stabilizers such as proteins or small molecules. However, such superstructures have not been revealed for RNA. Here we report the crystal structure of an RNA supercoil, which displays one level higher molecular organization than previously reported structures of DNA coiled coils. In the presence of an RNA-binding protein, two interlocking RNA coiled coils of double-stranded RNA, a 'coil of coiled coils', form a plectonemic supercoil. Molecular dynamics simulations suggest that protein-RNA interaction is required for the stability of the supercoiled RNA. This study provides structural insight into higher order packaging mechanisms of nucleic acids.

  7. PNPASE Regulates RNA Import into Mitochondria

    PubMed Central

    Wang, Geng; Chen, Hsiao-Wen; Oktay, Yavuz; Zhang, Jin; Allen, Eric L.; Smith, Geoffrey M.; Fan, Kelly C.; Hong, Jason S.; French, Samuel W.; McCaffery, J. Michael; Lightowlers, Robert N.; Morse, Herbert C.; Koehler, Carla M.; Teitell, Michael A.

    2010-01-01

    SUMMARY RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, we localized polynucleotide phosphorylase (PNPASE), a 3′ → 5′ exoribonuclease and poly-A polymerase, in the mitochondrial intermembrane space, a location lacking resident RNAs. Here, we show a new role for PNPASE in regulating the import of nuclear-encoded RNAs into the mitochondrial matrix. PNPASE reduction impaired mitochondrial RNA processing and polycistronic transcripts accumulated. Augmented import of RNase P, 5S rRNA, and MRP RNAs depended on PNPASE expression and PNPASE–imported RNA interactions were identified. PNPASE RNA processing and import activities were separable and a mitochondrial RNA targeting signal was isolated that enabled RNA import in a PNPASE-dependent manner. Combined, these data strongly support an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria. PMID:20691904

  8. Delivery materials for siRNA therapeutics

    NASA Astrophysics Data System (ADS)

    Kanasty, Rosemary; Dorkin, Joseph Robert; Vegas, Arturo; Anderson, Daniel

    2013-11-01

    RNA interference (RNAi) has broad potential as a therapeutic to reversibly silence any gene. To achieve the clinical potential of RNAi, delivery materials are required to transport short interfering RNA (siRNA) to the site of action in the cells of target tissues. This Review provides an introduction to the biological challenges that siRNA delivery materials aim to overcome, as well as a discussion of the way that the most effective and clinically advanced classes of siRNA delivery systems, including lipid nanoparticles and siRNA conjugates, are designed to surmount these challenges. The systems that we discuss are diverse in their approaches to the delivery problem, and provide valuable insight to guide the design of future siRNA delivery materials.

  9. Regulation of Flavivirus RNA synthesis and replication.

    PubMed

    Selisko, Barbara; Wang, Chunling; Harris, Eva; Canard, Bruno

    2014-12-01

    RNA synthesis and replication of the members of the Flavivirus genus (including dengue, West Nile and Japanese encephalitis viruses) is regulated by a wide variety of mechanisms and actors. These include the sequestration of the RNA-dependent RNA polymerase (RdRp) for functions other than RNA synthesis, regulatory interactions with other viral and host proteins within the replication complex (RC), and regulatory elements within the RNA genome itself. In this review, we discuss our current knowledge of the multiple levels at which Flavivirus RNA synthesis is controlled. We aim to bring together two active research fields: the structural and functional biology of individual proteins of the RC and the impressive wealth of knowledge acquired regarding the viral genomic RNA. PMID:25462437

  10. Regulation of Flavivirus RNA synthesis and replication

    PubMed Central

    Selisko, Barbara; Wang, Chunling; Harris, Eva; Canard, Bruno

    2014-01-01

    RNA synthesis and replication of the members of the Flavivirus genus (including dengue, West Nile and Japanese encephalitis viruses) is regulated by a wide variety of mechanisms and actors. These include the sequestration of the RNA-dependent RNA polymerase (RdRp) for functions other than RNA synthesis, regulatory interactions with other viral and host proteins within the replication complex (RC), and regulatory elements within the RNA genome itself. In this review, we discuss our current knowledge of the multiple levels at which Flavivirus RNA synthesis is controlled. We aim to bring together two active research fields: the structural and functional biology of individual proteins of the RC and the impressive wealth of knowledge acquired regarding the viral genomic RNA. PMID:25462437

  11. Estudo em microondas do aprisionamento e precipitação de elétrons em explosões solares

    NASA Astrophysics Data System (ADS)

    Rosal, A. C.; Costa, J. E. R.

    2003-08-01

    Uma explosão solar é uma variação rápida e intensa do brilho que ocorre nas chamadas regiões ativas da atmosfera, constituídas por um plasma magnetizado com intensa indução magnética. Os modelos de explosões solares atuais, discutidos na literatura, apresentam características de aprisionamento e precipitação de elétrons em ambientes magnéticos simplificados. Neste trabalho, nos propusemos a separar a emissão dos elétrons aprisionados da emissão dos elétrons em precipitação apenas a partir da emissão em microondas, melhorando portanto o controle sobre o conjunto de parâmetros inferidos. A emissão em microondas da população em precipitação é bastante fraca e portanto da nossa base de dados de 130 explosões observadas pelo Rádio Polarímetro de Nobeyama, em sete freqüências, apenas para 32 foi possível separar as duas componentes de emissão com uma boa razão sinal/ruído. A partir de estudos das escalas de tempo das emissões devidas à variação gradual da emissão no aprisionamento e da variação rápida da emissão dos elétrons em precipitação foi possível obter a separação utilizando um filtro temporal nas emissões resultantes. Em nossa análise destas explosões estudamos os espectros girossincrotrônicos da emissão gradual, a qual associamos provir do topo dos arcos magnéticos e da emissão de variação rápida associada aos elétrons em precipitação. Estes espectros foram calculados e dos quais inferimos que a indução magnética efetiva do topo e dos pés foi em média, Btopo = 236 G e Bpés = 577 G, inferidas das freqüências de pico dos espectros em ntopo = 11,8 GHz e npés = 14,6 GHz com leve anisotropia (pequeno alargamento espectral). O índice espectral da distribuição não-térmica de elétrons d, inferido do índice espectral de fótons da emissão em regime opticamente fino, foi de dtopo = 3,3 e dpés = 3,9. Estes parâmetros são típicos da maioria das análises realizadas em ambiente único de

  12. Is EMS communicating with the FCC?

    PubMed

    Johnson, M S; VanCott, C; Glass, C; Anderson, P B

    1989-07-01

    Radio communication problems in EMS run the spectrum from annoying to deadly. Dedicated radio frequencies for EMS, much like those exclusive to police and fire departments, are long overdue. PMID:10293680

  13. MicroRNA-mediated target mRNA cleavage and 3'-uridylation in human cells.

    PubMed

    Xu, Kai; Lin, Jing; Zandi, Roza; Roth, Jack A; Ji, Lin

    2016-01-01

    MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5'-fragments were 3'-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5'-3' degradation or other molecular fates. Some 3'-5' decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation. PMID:27440378

  14. Self-assembled RNA interference microsponges for efficient siRNA delivery

    NASA Astrophysics Data System (ADS)

    Lee, Jong Bum; Hong, Jinkee; Bonner, Daniel K.; Poon, Zhiyong; Hammond, Paula T.

    2012-04-01

    The encapsulation and delivery of short interfering RNA (siRNA) has been realized using lipid nanoparticles, cationic complexes, inorganic nanoparticles, RNA nanoparticles and dendrimers. Still, the instability of RNA and the relatively ineffectual encapsulation process of siRNA remain critical issues towards the clinical translation of RNA as a therapeutic. Here we report the synthesis of a delivery vehicle that combines carrier and cargo: RNA interference (RNAi) polymers that self-assemble into nanoscale pleated sheets of hairpin RNA, which in turn form sponge-like microspheres. The RNAi-microsponges consist entirely of cleavable RNA strands, and are processed by the cell’s RNA machinery to convert the stable hairpin RNA to siRNA only after cellular uptake, thus inherently providing protection for siRNA during delivery and transport to the cytoplasm. More than half a million copies of siRNA can be delivered to a cell with the uptake of a single RNAi-microsponge. The approach could lead to novel therapeutic routes for siRNA delivery.

  15. Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins.

    PubMed

    de Beauchene, Isaure Chauvot; de Vries, Sjoerd J; Zacharias, Martin

    2016-06-01

    Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein-RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA-RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins. PMID:27131381

  16. The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing.

    PubMed

    Stalder, Lukas; Heusermann, Wolf; Sokol, Lena; Trojer, Dominic; Wirz, Joel; Hean, Justin; Fritzsche, Anja; Aeschimann, Florian; Pfanzagl, Vera; Basselet, Pascal; Weiler, Jan; Hintersteiner, Martin; Morrissey, David V; Meisner-Kober, Nicole C

    2013-04-17

    Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing. PMID:23511973

  17. Sequence Dependence of Viral RNA Encapsidation.

    PubMed

    Kelly, Joshua; Grosberg, Alexander Y; Bruinsma, Robijn

    2016-07-01

    We develop a Flory mean-field theory for viral RNA (vRNA) molecules that extends the current RNA folding algorithms to include interactions between different sections of the secondary structure. The theory is applied to sequence-selective vRNA encapsidation. The dependence on sequence enters through a single parameter: the largest eigenvalue of the Kramers matrix of the branched polymer obtained by coarse graining the secondary structure. Differences between the work of encapsidation of vRNA molecules and of randomized isomers are found to be in the range of 20 kBT, more than sufficient to provide a strong bias in favor of vRNA encapsidation. The method is applied to a packaging competition experiment where large vRNA molecules compete for encapsidation with two smaller RNA species that together have the same nucleotide sequence as the large molecule. We encounter a substantial, generic free energy bias, that also is of the order of 20 kBT, in favor of encapsidating the single large RNA molecule. The bias is mainly the consequence of the fact that dividing up a large vRNA molecule involves the release of stored elastic energy. This provides an important, nonspecific mechanism for preferential encapsidation of single larger vRNA molecules over multiple smaller mRNA molecules with the same total number of nucleotides. The result is also consistent with recent RNA packaging competition experiments by Comas-Garcia et al.1 Finally, the Flory method leads to the result that when two RNA molecules are copackaged, they are expected to remain segregated inside the capsid. PMID:27116641

  18. Improving miRNA-mRNA interaction predictions

    PubMed Central

    2014-01-01

    Background MicroRNAs are short RNA molecules that post-transcriptionally regulate gene expression. Today, microRNA target prediction remains challenging since very few have been experimentally validated and sequence-based predictions have large numbers of false positives. Furthermore, due to the different measuring rules used in each database of predicted interactions, the selection of the most reliable ones requires extensive knowledge about each algorithm. Results Here we propose two methods to measure the confidence of predicted interactions based on experimentally validated information. The output of the methods is a combined database where new scores and statistical confidences are re-assigned to each predicted interaction. The new scores allow the robust combination of several databases without the effect of low-performing algorithms dragging down good-performing ones. The combined databases obtained using both algorithms described in this paper outperform each of the existing predictive algorithms that were considered for the combination. Conclusions Our approaches are a useful way to integrate predicted interactions from different databases. They reduce the selection of interactions to a unique database based on an intuitive score and allow comparing databases between them. PMID:25559987

  19. Evaluation of Commercially Available RNA Amplification Kits for RNA Sequencing Using Very Low Input Amounts of Total RNA

    PubMed Central

    Shanker, Savita; Paulson, Ariel; Edenberg, Howard J.; Peak, Allison; Perera, Anoja; Alekseyev, Yuriy O.; Beckloff, Nicholas; Bivens, Nathan J.; Donnelly, Robert; Gillaspy, Allison F.; Grove, Deborah; Gu, Weikuan; Jafari, Nadereh; Kerley-Hamilton, Joanna S.; Lyons, Robert H.; Tepper, Clifford

    2015-01-01

    This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.Multiple recent publications on RNA sequencing (RNA-seq) have demonstrated the power of next-generation sequencing technologies in whole-transcriptome analysis. Vendor-specific protocols used for RNA library construction often require at least 100 ng total RNA. However, under certain conditions, much less RNA is available for library construction. In these cases, effective transcriptome profiling requires amplification of subnanogram amounts of RNA. Several commercial RNA amplification kits are available for amplification prior to library construction for next-generation sequencing, but these kits have not been comprehensively field evaluated for accuracy and performance of RNA-seq for picogram amounts of RNA. To address this, 4 types of amplification kits were tested with 3 different concentrations, from 5 ng to 50 pg, of a commercially available RNA. Kits were tested at multiple sites to assess reproducibility and ease of use. The human total reference RNA used was spiked with a control pool of RNA molecules in order to further evaluate quantitative recovery of input material. Additional control data sets were generated from libraries constructed following polyA selection or ribosomal depletion using established kits and protocols. cDNA was collected from the different sites, and libraries were synthesized at a single site using established protocols. Sequencing runs were carried out on the Illumina platform. Numerous metrics were compared among the kits and dilutions used. Overall, no single kit appeared to meet all the challenges of small input material. However, it is encouraging that excellent data can be recovered with even the 50 pg input total RNA. PMID:25649271

  20. Observações no infravermelho médio de objetos estelares jovens em NGC 3576

    NASA Astrophysics Data System (ADS)

    Barbosa, C.; Damineli, A.; Blum, R.; Conti, P.

    2003-08-01

    Apresentamos os resultados de observações no infravermelho médio de candidatos a objetos estelares jovens e massivos em NGC 3576. As imagens de alta resolução foram obtidas no observatório Gemini Sul com o uso dos filtros em 10,8, 7,9, 9,8, 12,5 e 18,2 mm. Nossas imagens mostram a fonte IRS 1 resolvida em 4 objetos pela primeira vez em 10 mm. Para cada objeto obtivemos a distribuição espectral de energia de 1.2 até 18 mm, bem como a temperatura de cor, a distribuição espacial e a profundidade óptica em 9,8 mm da poeira circunstelar. Apresentamos uma estimativa das massas dos objetos estudados, baseados na luminosidade emitida no infravermelho médio, bem como um modelo para explicar as diferentes características observadas de cada objeto. Finalmente discutimos a possível localização da(s) fonte(s) de ionização de NGC 3576.

  1. Pseudoknots in RNA folding landscapes

    PubMed Central

    Kucharík, Marcel; Hofacker, Ivo L.; Stadler, Peter F.; Qin, Jing

    2016-01-01

    Motivation: The function of an RNA molecule is not only linked to its native structure, which is usually taken to be the ground state of its folding landscape, but also in many cases crucially depends on the details of the folding pathways such as stable folding intermediates or the timing of the folding process itself. To model and understand these processes, it is necessary to go beyond ground state structures. The study of rugged RNA folding landscapes holds the key to answer these questions. Efficient coarse-graining methods are required to reduce the intractably vast energy landscapes into condensed representations such as barrier trees or basin hopping graphs (BHG) that convey an approximate but comprehensive picture of the folding kinetics. So far, exact and heuristic coarse-graining methods have been mostly restricted to the pseudoknot-free secondary structures. Pseudoknots, which are common motifs and have been repeatedly hypothesized to play an important role in guiding folding trajectories, were usually excluded. Results: We generalize the BHG framework to include pseudoknotted RNA structures and systematically study the differences in predicted folding behavior depending on whether pseudoknotted structures are allowed to occur as folding intermediates or not. We observe that RNAs with pseudoknotted ground state structures tend to have more pseudoknotted folding intermediates than RNAs with pseudoknot-free ground state structures. The occurrence and influence of pseudoknotted intermediates on the folding pathway, however, appear to depend very strongly on the individual RNAs so that no general rule can be inferred. Availability and implementation: The algorithms described here are implemented in C++ as standalone programs. Its source code and Supplemental material can be freely downloaded from http://www.tbi.univie.ac.at/bhg.html. Contact: qin@bioinf.uni-leipzig.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID

  2. An all RNA hypercycle network

    NASA Astrophysics Data System (ADS)

    Vaidya, Nilesh; Lehman, Niles

    The RNA world hypothesis suggests RNA-based catalysis and information storage as the first step in the evolution of life on the Earth. The central process of the RNA world was the replica-tion of RNA, which may have involved the joining of oligonucleotides, perhaps by recombination rather than organization along a linear template. To assist this build-up of information, a hy-percycle may have played a significant role by allowing cooperation between autocatalytic units in a cyclic linkage in such a way that there is a mutual survival and regulated growth of all the units involved (1). Compared to non-coupled self-replicating units, which can only sustain a limited amount of genetic information, the hypercycle allows the maintenance of large amounts of information through cooperation among otherwise competitive units. However, hypercycles have never been empirically demonstrated in the absence of cell-like compartmentalization. In the current work, hypercyclic behavior is demonstrated in the autocatalytic assembly of Azoar-cus group I ribozyme (2). Three different constructs of the Azoarcus ribozyme with different internal guide sequences (IGS) -GUG (canonical), GAG, and GCG -are capable of a min-imal amount of self-assembly when broken into two fragments. Here, self-assembly depends on a mismatch with non-complementary sequences, CGU, CAU and CUU, respectively, to be recognized by IGS via autocatalysis. Yet when all three constructs are present in the same reaction vessel, concomitant assembly of all three is enhanced through an interdependent hy-percyclic reaction network. Analysis of these reactions indicates that each system is capable of guiding its own reproduction weakly, along with providing enhanced catalytic support for the reproduction of one other construct system through matched IGS-tag interactions. Also, when co-incubated with non-interacting (i.e., selfish) yet efficient self-assembly systems, the hypercyclic assembly outcompetes the selfish self

  3. Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis

    PubMed Central

    Zhang, Wei; Chang, Jae-Woong; Lin, Lilong; Minn, Kay; Wu, Baolin; Chien, Jeremy; Yong, Jeongsik; Zheng, Hui; Kuang, Rui

    2015-01-01

    High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/. PMID:26699225

  4. Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis.

    PubMed

    Zhang, Wei; Chang, Jae-Woong; Lin, Lilong; Minn, Kay; Wu, Baolin; Chien, Jeremy; Yong, Jeongsik; Zheng, Hui; Kuang, Rui

    2015-12-01

    High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/. PMID:26699225

  5. Separating para and ortho water.

    PubMed

    Horke, Daniel A; Chang, Yuan-Pin; Długołęcki, Karol; Küpper, Jochen

    2014-10-27

    Water exists as two nuclear-spin isomers, para and ortho, determined by the overall spin of its two hydrogen nuclei. For isolated water molecules, the conversion between these isomers is forbidden and they act as different molecular species. Yet, these species are not readily separated, and no pure para sample has been produced. Accordingly, little is known about their specific physical and chemical properties, conversion mechanisms, or interactions. The production of isolated samples of both spin isomers is demonstrated in pure beams of para and ortho water in their respective absolute ground state. These single-quantum-state samples are ideal targets for unraveling spin-conversion mechanisms, for precision spectroscopy and fundamental symmetry-breaking studies, and for spin-enhanced applications, for example laboratory astrophysics and astrochemistry or hypersensitized NMR experiments. PMID:25196938

  6. Establishment of a continuous culture system for Entamoeba muris and analysis of the small subunit rRNA gene.

    PubMed

    Kobayashi, S; Suzuki, J; Takeuchi, T

    2009-06-01

    We established a culture system for Entamoeba muris (MG-EM-01 strain isolated from a Mongolian gerbil) using a modified Balamuth's egg yolk infusion medium supplemented with 4% adult bovine serum and Bacteroides fragilis cocultured with Escherichia coli. Further, encystation was observed in the culture medium. The morphological characteristics of E. muris are similar to those of Entamoeba coli (E. coli); moreover, the malic isoenzyme electrophoretic band, which shows species-specific electrophoretic mobility, of E. muris had almost the same mobility as that observed with the malic isoenzyme electrophorectic band of E. coli (UZG-EC-01 strain isolated from a gorilla). We determined the small subunit rRNA (SSU-rRNA) gene sequence of the MG-EM-01 strain, and this sequence was observed to show 82.7% homology with that of the UZG-EC-01 strain. Further, the resultant phylogenetic tree for molecular taxonomy based on the SSU-rRNA genes of the 21 strains of the intestinal parasitic amoeba species indicated that the MG-EM-01 strain was most closely related to E. coli. PMID:19585892

  7. Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production.

    PubMed

    Han, Bo W; Wang, Wei; Li, Chengjian; Weng, Zhiping; Zamore, Phillip D

    2015-05-15

    PIWI-interacting RNAs (piRNAs) protect the animal germ line by silencing transposons. Primary piRNAs, generated from transcripts of genomic transposon "junkyards" (piRNA clusters), are amplified by the "ping-pong" pathway, yielding secondary piRNAs. We report that secondary piRNAs, bound to the PIWI protein Ago3, can initiate primary piRNA production from cleaved transposon RNAs. The first ~26 nucleotides (nt) of each cleaved RNA becomes a secondary piRNA, but the subsequent ~26 nt become the first in a series of phased primary piRNAs that bind Piwi, allowing piRNAs to spread beyond the site of RNA cleavage. The ping-pong pathway increases only the abundance of piRNAs, whereas production of phased primary piRNAs from cleaved transposon RNAs adds sequence diversity to the piRNA pool, allowing adaptation to changes in transposon sequence. PMID:25977554

  8. Computational prediction of microRNA genes.

    PubMed

    Hertel, Jana; Langenberger, David; Stadler, Peter F

    2014-01-01

    The computational identification of novel microRNA (miRNA) genes is a challenging task in bioinformatics. Massive amounts of data describing unknown functional RNA transcripts have to be analyzed for putative miRNA candidates with automated computational pipelines. Beyond those miRNAs that meet the classical definition, high-throughput sequencing techniques have revealed additional miRNA-like molecules that are derived by alternative biogenesis pathways. Exhaustive bioinformatics analyses on such data involve statistical issues as well as precise sequence and structure inspection not only of the functional mature part but also of the whole precursor sequence of the putative miRNA. Apart from a considerable amount of species-specific miRNAs, the majority of all those genes are conserved at least among closely related organisms. Some miRNAs, however, can be traced back to very early points in the evolution of eukaryotic species. Thus, the investigation of the conservation of newly found miRNA candidates comprises an important step in the computational annotation of miRNAs.Topics covered in this chapter include a review on the obvious problem of miRNA annotation and family definition, recommended pipelines of computational miRNA annotation or detection, and an overview of current computer tools for the prediction of miRNAs and their limitations. The chapter closes discussing how those bioinformatic approaches address the problem of faithful miRNA prediction and correct annotation. PMID:24639171

  9. Advances in Systemic siRNA Delivery

    PubMed Central

    Leng, Qixin; Woodle, Martin C; Lu, Patrick Y; Mixson, A James

    2009-01-01

    Sequence-specific gene silencing with small interfering RNA (siRNA) has transformed basic science research, and the efficacy of siRNA therapeutics toward a variety of diseases is now being evaluated in pre-clinical and clinical trials. Despite its potential value, the highly negatively charged siRNA has the classic delivery problem of requiring transport across cell membranes to the cytosol. Consequently, carrier development for siRNA delivery is one of the most important problems to solve before siRNA can achieve widespread clinical use. An assortment of non-viral carriers including liposomes, peptides, polymers, and aptamers are being evaluated for their ability to shepherd siRNA to the target tissue and cross the plasma membrane barrier into the cell. Several promising carriers with low toxicity and increased specificity for disease targets have emerged for siRNA-based therapeutics. This review will discuss non-viral approaches for siRNA therapeutics, with particular focus on synthetic carriers for in vivo systemic delivery of siRNA. PMID:20161621

  10. Modulation of RNA function by aminoglycoside antibiotics.

    PubMed

    Schroeder, R; Waldsich, C; Wank, H

    2000-01-01

    One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science. PMID:10619838

  11. MicroRNA control of ovarian function

    PubMed Central

    Christenson, L. K.

    2011-01-01

    Post-transcriptional gene regulation, a regulatory mechanism classically involved in female and male germ cell function has recently been implicated in control of somatic cells of the ovary and testis. Recent advancements in this field may be attributed primarily to the discovery and study of microRNAs (miRNA), small RNA transcripts that can influence mRNA expression via post-transcriptional gene regulatory mechanisms. In the ovary, targeted deletion of Dicer 1, a key enzyme in miRNA biogenesis, provided the first empirical evidence that miRNA/siRNA were critically involved in multiple aspects of ovarian function (folliculogenesis, oocyte maturation, ovulation, and luteal function). Functional studies of miRNA in the ovary have mostly focused on granulosa cells during the critical period of the ovarian cycle surrounding the ovulatory surge of luteinizing hormone (LH). Specific miRNA have been implicated in ovarian responses, due to their transcriptional induction by the LH surge (i.e., miR-21, -132 and -212) or through bioinformatic approaches (miR-224, -17-5p and let-7b). Numerous other miRNA are highly abundant in ovarian somatic tissues, suggesting that we have much to discover with respect to the role of miRNA and regulation of ovarian function. This review will recap the key observations of these early studies and provide insight into future experiments that might further our understanding of ovarian function. PMID:21666774

  12. The evolution of chloroplast RNA editing.

    PubMed

    Tillich, Michael; Lehwark, Pascal; Morton, Brian R; Maier, Uwe G

    2006-10-01

    RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria. PMID:16835291

  13. Evaluation of microRNA alignment techniques.

    PubMed

    Ziemann, Mark; Kaspi, Antony; El-Osta, Assam

    2016-08-01

    Genomic alignment of small RNA (smRNA) sequences such as microRNAs poses considerable challenges due to their short length (∼21 nucleotides [nt]) as well as the large size and complexity of plant and animal genomes. While several tools have been developed for high-throughput mapping of longer mRNA-seq reads (>30 nt), there are few that are specifically designed for mapping of smRNA reads including microRNAs. The accuracy of these mappers has not been systematically determined in the case of smRNA-seq. In addition, it is unknown whether these aligners accurately map smRNA reads containing sequence errors and polymorphisms. By using simulated read sets, we determine the alignment sensitivity and accuracy of 16 short-read mappers and quantify their robustness to mismatches, indels, and nontemplated nucleotide additions. These were explored in the context of a plant genome (Oryza sativa, ∼500 Mbp) and a mammalian genome (Homo sapiens, ∼3.1 Gbp). Analysis of simulated and real smRNA-seq data demonstrates that mapper selection impacts differential expression results and interpretation. These results will inform on best practice for smRNA mapping and enable more accurate smRNA detection and quantification of expression and RNA editing. PMID:27284164

  14. Chicken rRNA Gene Cluster Structure

    PubMed Central

    Dyomin, Alexander G.; Koshel, Elena I.; Kiselev, Artem M.; Saifitdinova, Alsu F.; Galkina, Svetlana A.; Fukagawa, Tatsuo; Kostareva, Anna A.

    2016-01-01

    Ribosomal RNA (rRNA) genes, whose activity results in nucleolus formation, constitute an extremely important part of genome. Despite the extensive exploration into avian genomes, no complete description of avian rRNA gene primary structure has been offered so far. We publish a complete chicken rRNA gene cluster sequence here, including 5’ETS (1836 bp), 18S rRNA gene (1823 bp), ITS1 (2530 bp), 5.8S rRNA gene (157 bp), ITS2 (733 bp), 28S rRNA gene (4441 bp) and 3’ETS (343 bp). The rRNA gene cluster sequence of 11863 bp was assembled from raw reads and deposited to GenBank under KT445934 accession number. The assembly was validated through in situ fluorescent hybridization analysis on chicken metaphase chromosomes using computed and synthesized specific probes, as well as through the reference assembly against de novo assembled rRNA gene cluster sequence using sequenced fragments of BAC-clone containing chicken NOR (nucleolus organizer region). The results have confirmed the chicken rRNA gene cluster validity. PMID:27299357

  15. Regulation of Cell Death by Transfer RNA

    PubMed Central

    2013-01-01

    Abstract Significance: Both transfer RNA (tRNA) and cytochrome c are essential molecules for the survival of cells. tRNA decodes mRNA codons into amino-acid-building blocks in protein in all organisms, whereas cytochrome c functions in the electron transport chain that powers ATP synthesis in mitochondrion-containing eukaryotes. Additionally, in vertebrates, cytochrome c that is released from mitochondria is a potent inducer of apoptosis, activating apoptotic proteins (caspases) in the cytoplasm to dismantle cells. A better understanding of both tRNA and cytochrome c is essential for an insight into the regulation of cell life and death. Recent Advances: A recent study showed that the mitochondrion-released cytochrome c can be removed from the cell-death pathway by tRNA molecules. The direct binding of cytochrome c by tRNA provides a mechanism for tRNA to regulate cell death, beyond its role in gene expression. Critical Issues: The nature of the tRNA–cytochrome c binding interaction remains unknown. The questions of how this interaction affects tRNA function, cellular metabolism, and apoptotic sensitivity are unanswered. Future Directions: Investigations into the critical issues raised above will improve the understanding of tRNA in the fundamental processes of cell death and metabolism. Such knowledge will inform therapies in cell death-related diseases. Antioxid. Redox Signal. 19, 583–594. PMID:23350625

  16. MicroRNA mimicry blocks pulmonary fibrosis

    PubMed Central

    Montgomery, Rusty L; Yu, Guoying; Latimer, Paul A; Stack, Christianna; Robinson, Kathryn; Dalby, Christina M; Kaminski, Naftali; van Rooij, Eva

    2014-01-01

    Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR-29 family has gained a lot of attention for its clear function in tissue fibrosis. This fibroblast-enriched miRNA family is downregulated in fibrotic diseases which induces a coordinate increase of many extracellular matrix genes. Here, we show that intravenous injection of synthetic RNA duplexes can increase miR-29 levels in vivo for several days. Moreover, therapeutic delivery of these miR-29 mimics during bleomycin-induced pulmonary fibrosis restores endogenous miR-29 function whereby decreasing collagen expression and blocking and reversing pulmonary fibrosis. Our data support the feasibility of using miRNA mimics to therapeutically increase miRNAs and indicate miR-29 to be a potent therapeutic miRNA for treating pulmonary fibrosis. PMID:25239947

  17. Movement of regulatory RNA between animal cells

    PubMed Central

    Jose, Antony M.

    2015-01-01

    Summary Recent studies suggest that RNA can move from one cell to another and regulate genes through specific base-pairing. Mechanisms that modify or select RNA for secretion from a cell are unclear. Secreted RNA can be stable enough to be detected in the extracellular environment and can enter the cytosol of distant cells to regulate genes. Mechanisms that import RNA into the cytosol of an animal cell can enable uptake of RNA from many sources including other organisms. This role of RNA is akin to that of steroid hormones, which cross cell membranes to regulate genes. The potential diagnostic use of RNA in human extracellular fluids has ignited interest in understanding mechanisms that enable the movement of RNA between animal cells. Genetic model systems will be essential to gain more confidence in proposed mechanisms of RNA transport and to connect an extracellular RNA with a specific biological function. Studies in the worm C. elegans and in other animals have begun to reveal parts of this novel mechanism of cell-to-cell communication. Here, I summarize the current state of this nascent field, highlight the many unknowns, and suggest future directions. PMID:26138457

  18. Initiator RNA in Discontinuous Polyoma DNA Synthesis*

    PubMed Central

    Reichard, Peter; Eliasson, Rolf; Söderman, Gunilla

    1974-01-01

    During replication of polyoma DNA in isolated nuclei, RNA was found attached to the 5′ ends of growing progeny strands. This RNA starts with either ATP or GTP and can be labeled at its 5′ end with 32P from β-labeled nucleotides. Digestion of progeny strands with pancreatic DNase released 32P-labeled RNA that, on gel electrophoresis, gave a distinct peak in the position expected for a decanucleotide. We believe that this short RNA is involved in the initiation of the discontinuous synthesis of DNA and propose the name “initiator RNA” for it. The covalent linkage of initiator RNA to 5′ ends of growing DNA chains was substantiated by the finding that 32P was transferred to ribonucleotides by alkaline hydrolysis of purified initiator RNA obtained by DNase digestion of polyoma progeny strands synthesized from [α-32P]dTTP. While initiator RNA was quite homogeneous in size, it had no unique base sequence since digestion with pancreatic RNase of initiator RNA labeled at its 5′ end with 32P released a variety of different [32P]oligonucleotides. The switch from RNA to DNA synthesis during strand elongation may thus depend on the size of initiator RNA rather than on a specific base sequence. PMID:4373733

  19. Controlled evolution of an RNA enzyme

    NASA Technical Reports Server (NTRS)

    Joyce, G. F.

    1991-01-01

    It is generally thought that prior to the origin of protein synthesis, life on earth was based on self-replicating RNA molecules. This idea has become especially popular recently due to the discovery of catalytic RNA (ribozymes). RNA has both genotypic and phenotypic properties, suggesting that it is capable of undergoing Darwinian evolution. RNA evolution is likely to have played a critical role in the early history of life on earth, and thus is important in considering the possibility of life elsewhere in the solar system. We have constructed an RNA-based evolving system in the laboratory, combining amplification and mutation of an RNA genotype with selection of a corresponding RNA phenotype. This system serves as a functional model of a primitive organism. It can also be used as a tool to explore the catalytic potential of RNA. By altering the selection constraints, we are attempting to modify the substrate specificity of an existing ribozyme in order to develop ribozymes with novel catalytic function. In this way, we hope to gain a better understanding of RNA's catalytic versatility and to assess its suitability for the role of primordial catalyst. All of the RNA enzymes that are known to exist in contemporary biology carry out cleavage/ligation reactions involving RNA substrates. The Tetrahymena ribozyme, for example, catalyzes phosphoester transfer between a guanosine containing and an oligopyrimidine containing substrate. We tested the ability of mutant forms of the Tetrahymena ribozyme to carry out a comparable reaction using DNA, rather than RNA substrate. An ensemble of structural variants of the ribozyme was prepared and tested for their ability to specifically cleave d(GGCCCTCT-A3TA3TA) at the phosphodiester bond following the sequence CCCTCT. We recovered a mutant form of the enzyme that cleaves DNA more efficiently than does the wild-type. Beginning with this selected mutant we have now scattered random mutations throughout the ribozyme and have begun

  20. Construction of RNA nanocages by re-engineering the packaging RNA of Phi29 bacteriophage

    NASA Astrophysics Data System (ADS)

    Hao, Chenhui; Li, Xiang; Tian, Cheng; Jiang, Wen; Wang, Guansong; Mao, Chengde

    2014-05-01

    RNA nanotechnology promises rational design of RNA nanostructures with wide array of structural diversities and functionalities. Such nanostructures could be used in applications such as small interfering RNA delivery and organization of in vivo chemical reactions. Though having impressive development in recent years, RNA nanotechnology is still quite limited and its programmability and complexity could not rival the degree of its closely related cousin: DNA nanotechnology. Novel strategies are needed for programmed RNA self-assembly. Here, we have assembled RNA nanocages by re-engineering a natural, biological RNA motif: the packaging RNA of phi29 bacteriophage. The resulting RNA nanostructures have been thoroughly characterized by gel electrophoresis, cryogenic electron microscopy imaging and dynamic light scattering.

  1. REDIdb: the RNA editing database

    PubMed Central

    Picardi, Ernesto; Regina, Teresa Maria Rosaria; Brennicke, Axel; Quagliariello, Carla

    2007-01-01

    The RNA Editing Database (REDIdb) is an interactive, web-based database created and designed with the aim to allocate RNA editing events such as substitutions, insertions and deletions occurring in a wide range of organisms. The database contains both fully and partially sequenced DNA molecules for which editing information is available either by experimental inspection (in vitro) or by computational detection (in silico). Each record of REDIdb is organized in a specific flat-file containing a description of the main characteristics of the entry, a feature table with the editing events and related details and a sequence zone with both the genomic sequence and the corresponding edited transcript. REDIdb is a relational database in which the browsing and identification of editing sites has been simplified by means of two facilities to either graphically display genomic or cDNA sequences or to show the corresponding alignment. In both cases, all editing sites are highlighted in colour and their relative positions are detailed by mousing over. New editing positions can be directly submitted to REDIdb after a user-specific registration to obtain authorized secure access. This first version of REDIdb database stores 9964 editing events and can be freely queried at . PMID:17175530

  2. Circulating microbial RNA and health

    PubMed Central

    Leung, Ross Ka-Kit; Wu, Ying-Kit

    2015-01-01

    Measurement of health indicators in the blood is a commonly performed diagnostic procedure. Two blood studies one involving extended observations on the health of an individual by integrative Personal Omics Profiling (iPOP), and the other tracking the impact of Left Ventricular Assist Device (LVAD) placement on nine heart failure patients were examined for the association of change in health status with change in microbial RNA species. Decrease in RNA expression ratios of human to bacteria and viruses accompanying deteriorated conditions was evident in both studies. Despite large between-subject variations in bacterial composition before LVAD implantation among all the patients, on day 180 after the implantation they manifested apparent between-subject bacterial similarity. In the iPOP study three periods, namely, pre-respiratory syncytial virus (RSV) infection with normal blood glucose level, RSV infection with normal blood glucose level, and post-RSV infection with high blood glucose level could be defined. The upsurge of Enterobacteria phage PhiX 174 sensu lato and Escherichia coli gene expression, in which membrane transporters, membrane receptors for environment signalling, carbohydrate catabolic genes and carbohydrate-active enzymes were enriched only throughout the second period, which suggests a potentially overlooked microbial response to or modulation of the host blood glucose level. PMID:26576508

  3. RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA.

    PubMed

    Chapman, Erich G; Moon, Stephanie L; Wilusz, Jeffrey; Kieft, Jeffrey S

    2014-01-01

    Dengue virus is a growing global health threat. Dengue and other flaviviruses commandeer the host cell's RNA degradation machinery to generate the small flaviviral RNA (sfRNA), a noncoding RNA that induces cytopathicity and pathogenesis. Host cell exonuclease Xrn1 likely loads on the 5' end of viral genomic RNA and degrades processively through ∼10 kB of RNA, halting near the 3' end of the viral RNA. The surviving RNA is the sfRNA. We interrogated the architecture of the complete Dengue 2 sfRNA, identifying five independently-folded RNA structures, two of which quantitatively confer Xrn1 resistance. We developed an assay for real-time monitoring of Xrn1 resistance that we used with mutagenesis and RNA folding experiments to show that Xrn1-resistant RNAs adopt a specific fold organized around a three-way junction. Disrupting the junction's fold eliminates the buildup of disease-related sfRNAs in human cells infected with a flavivirus, directly linking RNA structure to sfRNA production. DOI: http://dx.doi.org/10.7554/eLife.01892.001. PMID:24692447

  4. RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA

    PubMed Central

    Chapman, Erich G; Moon, Stephanie L; Wilusz, Jeffrey; Kieft, Jeffrey S

    2014-01-01

    Dengue virus is a growing global health threat. Dengue and other flaviviruses commandeer the host cell’s RNA degradation machinery to generate the small flaviviral RNA (sfRNA), a noncoding RNA that induces cytopathicity and pathogenesis. Host cell exonuclease Xrn1 likely loads on the 5′ end of viral genomic RNA and degrades processively through ∼10 kB of RNA, halting near the 3′ end of the viral RNA. The surviving RNA is the sfRNA. We interrogated the architecture of the complete Dengue 2 sfRNA, identifying five independently-folded RNA structures, two of which quantitatively confer Xrn1 resistance. We developed an assay for real-time monitoring of Xrn1 resistance that we used with mutagenesis and RNA folding experiments to show that Xrn1-resistant RNAs adopt a specific fold organized around a three-way junction. Disrupting the junction’s fold eliminates the buildup of disease-related sfRNAs in human cells infected with a flavivirus, directly linking RNA structure to sfRNA production. DOI: http://dx.doi.org/10.7554/eLife.01892.001 PMID:24692447

  5. FLDS: A Comprehensive dsRNA Sequencing Method for Intracellular RNA Virus Surveillance.

    PubMed

    Urayama, Syun-Ichi; Takaki, Yoshihiro; Nunoura, Takuro

    2016-03-26

    Knowledge of the distribution and diversity of RNA viruses is still limited in spite of their possible environmental and epidemiological impacts because RNA virus-specific metagenomic methods have not yet been developed. We herein constructed an effective metagenomic method for RNA viruses by targeting long double-stranded (ds)RNA in cellular organisms, which is a hallmark of infection, or the replication of dsRNA and single-stranded (ss)RNA viruses, except for retroviruses. This novel dsRNA targeting metagenomic method is characterized by an extremely high recovery rate of viral RNA sequences, the retrieval of terminal sequences, and uniform read coverage, which has not previously been reported in other metagenomic methods targeting RNA viruses. This method revealed a previously unidentified viral RNA diversity of more than 20 complete RNA viral genomes including dsRNA and ssRNA viruses associated with an environmental diatom colony. Our approach will be a powerful tool for cataloging RNA viruses associated with organisms of interest. PMID:26877136

  6. Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA.

    PubMed

    Wei, Zhiyun; Batagov, Arsen O; Carter, David R F; Krichevsky, Anna M

    2016-01-01

    Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA. PMID:27503761

  7. piRNA cluster database: a web resource for piRNA producing loci

    PubMed Central

    Rosenkranz, David

    2016-01-01

    Piwi proteins and their guiding small RNAs, termed Piwi-interacting (pi-) RNAs, are essential for silencing of transposons in the germline of animals. A substantial fraction of piRNAs originates from genomic loci termed piRNA clusters and sequences encoded in these piRNA clusters determine putative targets for the Piwi/piRNA system. In the past decade, studies of piRNA transcriptomes in different species revealed additional roles for piRNAs beyond transposon silencing, reflecting the astonishing plasticity of the Piwi/piRNA system along different phylogenetic branches. Moreover, piRNA transcriptomes can change drastically during development and vary across different tissues. Since piRNA clusters crucially shape piRNA profiles, analysis of these loci is imperative for a thorough understanding of functional and evolutionary aspects of the piRNA pathway. But despite the ever-growing amount of available piRNA sequence data, we know little about the factors that determine differential regulation of piRNA clusters, nor the evolutionary events that cause their gain or loss. In order to facilitate addressing these subjects, we established a user-friendly piRNA cluster database (http://www.smallrnagroup-mainz.de/piRNAclusterDB.html) that provides comprehensive data on piRNA clusters in multiple species, tissues and developmental stages based on small RNA sequence data deposited at NCBI's Sequence Read Archive (SRA). PMID:26582915

  8. Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins

    PubMed Central

    de Beauchene, Isaure Chauvot; de Vries, Sjoerd J.; Zacharias, Martin

    2016-01-01

    Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein–RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA–RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins. PMID:27131381

  9. Prohead RNA: a noncoding viral RNA of novel structure and function.

    PubMed

    Hill, Alyssa C; Bartley, Laura E; Schroeder, Susan J

    2016-07-01

    Prohead RNA (pRNA) is an essential component of the powerful Φ29-like bacteriophage DNA packaging motor. However, the specific role of this unique RNA in the Φ29 packaging motor remains unknown. This review examines pRNA as a noncoding RNA of novel structure and function. In order to highlight the reasons for exploring the structure and function of pRNA, we (1) provide an overview of Φ29-like bacteriophage and the Φ29 DNA packaging motor, including putative motor mechanisms and structures of its component parts; (2) discuss pRNA structure and possible roles for pRNA in the Φ29 packaging motor; (3) summarize pRNA self-assembly; and (4) describe the prospective therapeutic applications of pRNA. Many questions remain to be answered in order to connect what is currently known about pRNA structure to its novel function in the Φ29 packaging motor. The knowledge gained from studying the structure, function, and sequence variation in pRNA will help develop tools to better navigate the conformational landscapes of RNA. WIREs RNA 2016, 7:428-437. doi: 10.1002/wrna.1330 For further resources related to this article, please visit the WIREs website. PMID:26810250

  10. Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision

    PubMed Central

    Denise, Hubert; Moschos, Sterghios A.; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

    2014-01-01

    TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034–encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5′ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC). PMID:24496437

  11. Identification of dengue RNA binding proteins using RNA chromatography and quantitative mass spectrometry.

    PubMed

    Ward, Alex M; Gunaratne, J; Garcia-Blanco, Mariano A

    2014-01-01

    A major challenge in dengue virus (DENV) research has been to understand the interaction of the viral RNA with host cell proteins during infection. Until recently, there were no comprehensive studies identifying host RNA binding proteins that interact with DENV RNA (Ward et al. RNA Biol 8 (6):1173-1186, 2011). Here, we describe a method for identifying proteins that associate with DENV RNA using RNA chromatography and quantitative mass spectrometry. The method utilizes a tobramycin RNA aptamer incorporated into an RNA containing the dengue 5' and 3' untranslated regions (UTRs) in order to reversibly bind RNA to a tobramycin matrix. The RNA-tobramycin matrix is incubated with SILAC-labeled cell lysates, and bound proteins are eluted using an excess of tobramycin. The eluate is analyzed using quantitative mass spectrometry, which allows direct and quantitative comparison of proteins bound to DENV UTRs and a control RNA-tobramycin matrix. This technique has the advantage of allowing one to distinguish between specific and nonspecific binding proteins based on the ratio of protein preferentially bound to the DENV UTRs versus the control RNA. This methodology can also be used for validation of quantitative mass spectrometry results using conventional Western blotting for specific proteins. Furthermore, though it was specifically developed to identify DENV RNA binding proteins, the RNA chromatography method described here can be applied to a broad range of viral and cellular RNAs for identification of interacting proteins. PMID:24696342

  12. FLDS: A Comprehensive dsRNA Sequencing Method for Intracellular RNA Virus Surveillance

    PubMed Central

    Urayama, Syun-ichi; Takaki, Yoshihiro; Nunoura, Takuro

    2016-01-01

    Knowledge of the distribution and diversity of RNA viruses is still limited in spite of their possible environmental and epidemiological impacts because RNA virus-specific metagenomic methods have not yet been developed. We herein constructed an effective metagenomic method for RNA viruses by targeting long double-stranded (ds)RNA in cellular organisms, which is a hallmark of infection, or the replication of dsRNA and single-stranded (ss)RNA viruses, except for retroviruses. This novel dsRNA targeting metagenomic method is characterized by an extremely high recovery rate of viral RNA sequences, the retrieval of terminal sequences, and uniform read coverage, which has not previously been reported in other metagenomic methods targeting RNA viruses. This method revealed a previously unidentified viral RNA diversity of more than 20 complete RNA viral genomes including dsRNA and ssRNA viruses associated with an environmental diatom colony. Our approach will be a powerful tool for cataloging RNA viruses associated with organisms of interest. PMID:26877136

  13. piRNA cluster database: a web resource for piRNA producing loci.

    PubMed

    Rosenkranz, David

    2016-01-01

    Piwi proteins and their guiding small RNAs, termed Piwi-interacting (pi-) RNAs, are essential for silencing of transposons in the germline of animals. A substantial fraction of piRNAs originates from genomic loci termed piRNA clusters and sequences encoded in these piRNA clusters determine putative targets for the Piwi/piRNA system. In the past decade, studies of piRNA transcriptomes in different species revealed additional roles for piRNAs beyond transposon silencing, reflecting the astonishing plasticity of the Piwi/piRNA system along different phylogenetic branches. Moreover, piRNA transcriptomes can change drastically during development and vary across different tissues.Since piRNA clusters crucially shape piRNA profiles, analysis of these loci is imperative for a thorough understanding of functional and evolutionary aspects of the piRNA pathway. But despite the ever-growing amount of available piRNA sequence data, we know little about the factors that determine differential regulation of piRNA clusters, nor the evolutionary events that cause their gain or loss.In order to facilitate addressing these subjects, we established a user-friendly piRNA cluster database (http://www.smallrnagroup-mainz.de/piRNAclusterDB.html) that provides comprehensive data on piRNA clusters in multiple species, tissues and developmental stages based on small RNA sequence data deposited at NCBI's Sequence Read Archive (SRA). PMID:26582915

  14. Fetal Bovine Serum RNA Interferes with the Cell Culture derived Extracellular RNA

    PubMed Central

    Wei, Zhiyun; Batagov, Arsen O.; Carter, David R. F.; Krichevsky, Anna M.

    2016-01-01

    Fetal bovine serum (FBS) has been used in eukaryotic cell cultures for decades. However, little attention has been paid to the biological effects associated with RNA content of FBS on cell cultures. Here, using RNA sequencing, we demonstrate that FBS contains a diverse repertoire of protein-coding and regulatory RNA species, including mRNA, miRNA, rRNA, and snoRNA. The majority of them (>70%) are retained even after extended ultracentrifugation in the preparations of vesicle-depleted FBS (vdFBS) commonly utilized in the studies of extracellular vesicles (EV) and intercellular communication. FBS-associated RNA is co-isolated with cell-culture derived extracellular RNA (exRNA) and interferes with the downstream RNA analysis. Many evolutionally conserved FBS-derived RNA species can be falsely annotated as human or mouse transcripts. Notably, specific miRNAs abundant in FBS, such as miR-122, miR-451a and miR-1246, have been previously reported as enriched in cell-culture derived EVs, possibly due to the confounding effect of the FBS. Analysis of publically available exRNA datasets supports the notion of FBS contamination. Furthermore, FBS transcripts can be taken up by cultured cells and affect the results of highly sensitive gene expression profiling technologies. Therefore, precautions for experimental design are warranted to minimize the interference and misinterpretations caused by FBS-derived RNA. PMID:27503761

  15. Protein mediated miRNA detection and siRNA enrichment using p19.

    PubMed

    Jin, Jingmin; Cid, Melissa; Poole, Catherine B; McReynolds, Larry A

    2010-06-01

    p19 RNA binding protein from the Carnation Italian ringspot virus (CIRV) is an RNA-silencing suppressor that binds small interfering RNA (siRNA) with high affinity. We created a bifunctional p19 fusion protein with an N-terminal maltose binding protein (MBP), for protein purification, and a C-terminal chitin binding domain (CBD) to bind p19 to chitin magnetic beads. The fusion protein binds dsRNAs in the size range of 20-23 nucleotides, but does not bind ssRNA or dsDNA. Relative affinities of the p19 fusion protein for different-length RNA and DNA substrates were determined. Binding specificity of the p19 fusion protein for small dsRNA allows detection of miRNA:RNA probe duplexes. Using radioactive RNA probes, we were able to detect low levels of miRNAs in the sub-femtomole range and in the presence of a million-fold excess of total RNA. Detection is linear over three logs. Unlike most nucleic acid detection methods, p19 selects for RNA hybrids of correct length and structure. Rules for designing optimal RNA probes for p19 detection of miRNAs were determined by in vitro binding of 18 different dsRNA oligos to p19. These studies demonstrate the potential of p19 fusion protein to detect miRNAs and isolate endogenous siRNAs. PMID:20569217

  16. Hairpin DNA-Templated Silver Nanoclusters as Novel Beacons in Strand Displacement Amplification for MicroRNA Detection.

    PubMed

    Zhang, Jingpu; Li, Chao; Zhi, Xiao; Ramón, Gabriel Alfranca; Liu, Yanlei; Zhang, Chunlei; Pan, Fei; Cui, Daxiang

    2016-01-19

    MicroRNA (miRNA) biomarkers display great potential for cancer diagnosis and prognosis. The development of rapid and specific methods for miRNA detection has become a hotspot. Herein, hairpin DNA-templated silver nanoclusters (AgNCs/HpDNA) were prepared and integrated into strand-displacement amplification (SDA) as a novel beacon for miRNA detection. The light-up platform was established based on guanine (G)-rich fluorescence enhancement that essentially converted the excitation/emission pair of AgNCs/HpDNAs from a shorter wavelength to a longer wavelength, and then achieved fluorescent enhancement at longer wavelength. On the basis of the validation of the method, the single and duplex detection were conducted in two plasma biomarkers (miR-16-5p and miR-19b-3p) for the diagnosis of gastric cancer. The probe (AgNCs/RED 16(7s)C) utilized for miR-16-5p detection adopted a better conformation with high specificity to recognize single-base mismatches by producing dramatically opposite signals (increase or decrease at 580 nm ex/640 nm em) while the probe (AgNCs/GRE 19b(5s)C) for miR-19b-3p generated dual signals (increase at 490 nm ex/570 nm em and decrease at 430 nm ex/530 nm em) with bright fluorescence in one reaction during the amplification, but unexpectedly was partially digested. This is for the first time to allow the generation of enhanced fluorescent AgNCs and the target recognition integrated into a single process, which offers great opportunity for specific miRNA detection in an easy and rapid way. PMID:26675240

  17. Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

    PubMed

    Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

    2015-12-01

    Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs

  18. Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity

    PubMed Central

    Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

    2015-01-01

    Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory

  19. Alveolar Echinococcosis: Characterization of Diagnostic Antigen Em18 and Serological Evaluation of Recombinant Em18

    PubMed Central

    Sako, Yasuhito; Nakao, Minoru; Nakaya, Kazuhiro; Yamasaki, Hiroshi; Gottstein, Bruno; Lightowers, Marshall W.; Schantz, Peter M.; Ito, Akira

    2002-01-01

    The Echinococcus multilocularis protein Em18 is one of the most promising antigens for use in serodiagnosis of alveolar echinococcosis in human patients. Here we identify an antigenic relationship between Em18 and a 65-kDa immunodominant E. multilocularis surface protein previously identified as either EM10 or EmII/3. The NH2-terminal sequence of native Em18 was determined, revealing it to be a fragment of EM10. Experiments were undertaken to investigate the effect of proteinase inhibitors on the degradation of EM10 in crude extracts of E. multilocularis protoscoleces. Em18 was found to be the product of degradation of EM10 by cysteine proteinase. A recombinant Em18 (RecEm18, derived from 349K to 508K of EM10) was successfully expressed by using Escherichia coli expression system and then evaluated for use in serodiagnosis of alveolar echinococcosis. RecEm18 was recognized by 27 (87.1%) and 28 (90.3%) of 31 serum samples from clinically and/or pathologically confirmed alveolar echinococcosis patients by enzyme-linked immunosorbent assay and immunoblotting, respectively. Of 33 serum samples from cystic echinococcosis patients, 1 was recorded as having a weak positive reaction to RecEm18; however, none of the serum samples which were tested from neurocysticercosis patients (n = 10) or healthy people (n = 15) showed positive reactions. RecEm18 has the potential for use in the differential serodiagnosis of alveolar echinococcosis. PMID:12149326

  20. Tethering in RNA: An RNA-Binding Fragment Discovery Tool

    PubMed Central

    Tran, Kiet; Arkin, Michelle R.; Beal, Peter A.

    2016-01-01

    Tethering has been extensively used to study small molecule interactions with proteins through reversible disulfide bond forming reactions to cysteine residues. We describe the adaptation of Tethering to the study of small molecule binding to RNA using a thiol-containing adenosine analog (ASH). Among 30 disulfide-containing small molecules screened for efficient Tethering to ASH-bearing RNAs derived from pre-miR21, a benzotriazole-containing compound showed prominent adduct formation and selectivity for one of the RNAs tested. The results of this screen demonstrate the viability of using thiol-modified nucleic acids to discover molecules with binding affinity and specificity for the purpose of therapeutic compound lead discovery. PMID:25749683

  1. Disrupted tRNA Genes and tRNA Fragments: A Perspective on tRNA Gene Evolution.

    PubMed

    Kanai, Akio

    2015-01-01

    Transfer RNAs (tRNAs) are small non-coding RNAs with lengths of approximately 70-100 nt. They are directly involved in protein synthesis by carrying amino acids to the ribosome. In this sense, tRNAs are key molecules that connect the RNA world and the protein world. Thus, study of the evolution of tRNA molecules may reveal the processes that led to the establishment of the central dogma: genetic information flows from DNA to RNA to protein. Thanks to the development of DNA sequencers in this century, we have determined a huge number of nucleotide sequences from complete genomes as well as from transcriptomes in many species. Recent analyses of these large data sets have shown that particular tRNA genes, especially in Archaea, are disrupted in unique ways: some tRNA genes contain multiple introns and some are split genes. Even tRNA molecules themselves are fragmented post-transcriptionally in many species. These fragmented small RNAs are known as tRNA-derived fragments (tRFs). In this review, I summarize the progress of research into the disrupted tRNA genes and the tRFs, and propose a possible model for the molecular evolution of tRNAs based on the concept of the combination of fragmented tRNA halves. PMID:25629271

  2. siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.

    PubMed

    Galka-Marciniak, Paulina; Olejniczak, Marta; Starega-Roslan, Julia; Szczesniak, Michal W; Makalowska, Izabela; Krzyzosiak, Wlodzimierz J

    2016-04-01

    shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology. PMID:26921501

  3. Battle against RNA oxidation: molecular mechanisms for reducing oxidized RNA to protect cells.

    PubMed

    Li, Zhongwei; Malla, Sulochan; Shin, Brian; Li, James M

    2014-01-01

    Oxidation is probably the most common type of damage that occurs in cellular RNA. Oxidized RNA may be dysfunctional and is implicated in the pathogenesis of age-related human diseases. Cellular mechanisms controlling oxidized RNA have begun to be revealed. Currently, a number of ribonucleases and RNA-binding proteins have been shown to reduce oxidized RNA and to protect cells under oxidative stress. Although information about how these factors work is still very limited, we suggest several mechanisms that can be used to minimize oxidized RNA in various organisms. PMID:24375979

  4. BATTLE AGAINST RNA OXIDATION: Molecular mechanisms for reducing oxidized RNA to protect cells

    PubMed Central

    Li, Zhongwei; Malla, Sulochan; Shin, Brian; Li, James M.

    2014-01-01

    Oxidation is probably the most common type of damage that occurs in cellular RNA. Oxidized RNA may be dysfunctional and is implicated in the pathogenesis of age-related human diseases. Cellular mechanisms controlling oxidized RNA have begun to be revealed. Currently, a number of ribonucleases and RNA binding proteins have been shown to reduce oxidized RNA and to protect cells under oxidative stress. Although information about how these factors work is still very limited, we suggest several mechanisms that can be used to minimize oxidized RNA in various organisms. PMID:24375979

  5. Primer-Dependent and Primer-Independent Initiation of Double Stranded RNA Synthesis by Purified Arabidopsis RNA-Dependent RNA Polymerases RDR2 and RDR6

    PubMed Central

    Devert, Anthony; Fabre, Nicolas; Floris, Maïna; Canard, Bruno; Robaglia, Christophe; Crété, Patrice

    2015-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants. PMID:25793874

  6. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified Arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6.

    PubMed

    Devert, Anthony; Fabre, Nicolas; Floris, Maïna; Canard, Bruno; Robaglia, Christophe; Crété, Patrice

    2015-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants. PMID:25793874

  7. Rna catalysis and the origin of life

    NASA Astrophysics Data System (ADS)

    Pace, Norman R.; Marsh, Terry L.

    1985-06-01

    Until the discovery of catalytic RNAs, first the self-splicing intron inTetrahymena and then the bacterial RNAse P, cellular enzymes had always seemed to be protein in nature. The recognition that RNA can catalytically make and break phosphodiester bonds simplifies some of the assumptions required of a rudimentary self-replicating entity. Available information on the chemistry of RNA-catalyzed reactions is reviewed, with particular attention to self-splicing introns and tRNA processing by RNase P. An explicit model for a self-replicating RNA is described. The model postulates a nucleotide binding/polymerization site in the RNA, and takes advantage of intrinsic fluidity in RNA higher order structure to dissociate parent and progeny complementary strands.

  8. Quantitative transcriptome analysis using RNA-seq.

    PubMed

    Külahoglu, Canan; Bräutigam, Andrea

    2014-01-01

    RNA-seq has emerged as the technology of choice to quantify gene expression. This technology is a convenient accurate tool to quantify diurnal changes in gene expression, gene discovery, differential use of promoters, and splice variants for all genes expressed in a single tissue. Thus, RNA-seq experiments provide sequence information and absolute expression values about transcripts in addition to relative quantification available with microarrays or qRT-PCR. The depth of information by sequencing requires careful assessment of RNA intactness and DNA contamination. Although the RNA-seq is comparatively recent, a standard analysis framework has emerged with the packages of Bowtie2, TopHat, and Cufflinks. With rising popularity of RNA-seq tools have become manageable for researchers without much bioinformatical knowledge or programming skills. Here, we present a workflow for a RNA-seq experiment from experimental planning to biological data extraction. PMID:24792045

  9. Argonaute: The executor of small RNA function.

    PubMed

    Azlan, Azali; Dzaki, Najat; Azzam, Ghows

    2016-08-20

    The discovery of small non-coding RNAs - microRNA (miRNA), short interfering RNA (siRNA) and PIWI-interacting RNA (piRNA) - represents one of the most exciting frontiers in biology specifically on the mechanism of gene regulation. In order to execute their functions, these small RNAs require physical interactions with their protein partners, the Argonaute (AGO) family proteins. Over the years, numerous studies have made tremendous progress on understanding the roles of AGO in gene silencing in various organisms. In this review, we summarize recent progress of AGO-mediated gene silencing and other cellular processes in which AGO proteins have been implicated with a particular focus on progress made in flies, humans and other model organisms as compliment. PMID:27569398

  10. The European Mobile System (EMS)

    NASA Technical Reports Server (NTRS)

    Jongejans, A.; Rogard, R.; Mistretta, I.; Ananasso, F.

    1993-01-01

    The European Space Agency is presently procuring an L band payload in order to promote a regional European L band system coping with the specific needs of the European market. The payload, and the two communications systems to be supported, are described below. The potential market for EMS in Europe is discussed.

  11. Mensaje para alumnos y padres

    NASA Video Gallery

    El astronauta de la NASA José Hernández alienta a los estudiantes a que sigan sus sueños. Hernández también habla acerca del papel que juegan los padres para ayudar a que sus hijos hagan realidad s...

  12. Analysis of the miRNA-mRNA-lncRNA networks in ER+ and ER- breast cancer cell lines.

    PubMed

    Wu, Qian; Guo, Li; Jiang, Fei; Li, Lei; Li, Zhong; Chen, Feng

    2015-12-01

    Recently, rapid advances in bioinformatics analysis have expanded our understanding of the transcriptome to a genome-wide level. miRNA-mRNA-lncRNA interactions have been shown to play critical regulatory role in cancer biology. In this study, we discussed the use of an integrated systematic approach to explore new facets of the oestrogen receptor (ER)-regulated transcriptome. The identification of RNAs that are related to the expression status of the ER may be useful in clinical therapy and prognosis. We used a network modelling strategy. First, microarray expression profiling of mRNA, lncRNA and miRNA was performed in MCF-7 (ER-positive) and MDA-MB-231 cells (ER- negative). A co-expression network was then built using co-expression relationships of the differentially expressed mRNAs and lncRNAs. Finally, the selected miRNA-mRNA network was added to the network. The key miRNA-mRNA-lncRNA interaction can be inferred from the network. The mRNA and non-coding RNA expression profiles of the cells with different ER phenotypes were distinct. Among the aberrantly expressed miRNAs, the expression levels of miR-19a-3p, miR-19b-3p and miR-130a-3p were much lower in the MCF-7 cells, whereas that of miR-148b-3p was much higher. In a cluster of miR-17-92, the expression levels of six of seven miRNAs were lower in the MCF-7 cells, in addition to miR-20b in the miR-106a-363 cluster. However, the levels of all the miRNAs in the miR-106a-25 cluster were higher in the MCF-7 cells. In the co-expression networking, CD74 and FMNL2 gene which is involved in the immune response and metastasis, respectively, had a stronger correlation with ER. Among the aberrantly expressed lncRNAs, lncRNA-DLEU1 was highly expressed in the MCF-7 cells. A statistical analysis revealed that there was a co-expression relationship between ESR1 and lncRNA-DLEU1. In addition, miR-19a and lncRNA-DLEU1 are both located on the human chromosome 13q. We speculate that miR-19a might be co-expressed with lncRNA-DLEU1

  13. RNA Virus Reverse Genetics and Vaccine Design

    PubMed Central

    Stobart, Christopher C.; Moore, Martin L.

    2014-01-01

    RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines. PMID:24967693

  14. A Cross-chiral RNA Polymerase Ribozyme

    PubMed Central

    Sczepanski, Jonathan T.; Joyce, Gerald F.

    2014-01-01

    Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer.1 This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition2 or chiral symmetry was broken through chemical processes prior to the origin of RNA-based life.3,4 Once an RNA enzyme arose that could catalyze the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating D-and L-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyze the templated polymerization of activated mononucleotides of the opposite handedness. Such a cross-chiral RNA polymerase has now been developed using in vitro evolution. The D-RNA enzyme, consisting of 83 nucleotides, catalyzes the joining of L-mono- or oligonucleotide substrates on a complementary L-RNA template, and similarly for the L-enzyme with D-substrates and a D-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme's activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides. PMID:25363769

  15. Small catalytic RNA: Structure, function and application

    SciTech Connect

    Monforte, J.A.

    1991-04-01

    We have utilized a combination of photochemical cross-linking techniques and site-directed mutagenesis to obtain secondary and tertiary structure information for the self-cleaving, self-ligating subsequence of RNA from the negative strand of Satellite Tobacco Ringspot Virus. We have found that the helical regions fold about a hinge to promoting four different possible tertiary interactions, creating a molecular of similar shape to a paperclip. A model suggesting that the ``paperclip`` and ``hammerhead`` RNAs share a similar three dimensional structure is proposed. We have used a self-cleaving RNA molecule related to a subsequence of plant viroids, a ``hammerhead,`` to study the length-dependent folding of RNA produced during transcription by RNA polymerase. We have used this method to determine the length of RNA sequestered within elongating E. coli and T7 RNA polymerase complexes. The data show that for E. coli RNA polymerase 12{plus_minus}1 nucleotides are sequestered within the ternary complex, which is consistent with the presence of an RNA-DNA hybrid within the transcription bubble, as proposed by others. The result for T7 RNA polymerase differs from E. coli RNA polymerase, with only 10{plus_minus}1 nucleotides sequestered within the ternary complex, setting a new upper limit for the minimum RNA-DNA required for a stable elongating complex. Comparisons between E. coli and T7 RNA polymerase are made. The relevance of the results to models or transcription termination, abortive initiation, and initiation to elongation mode transitions are discussed.

  16. Circulating Extracellular RNA Markers of Liver Regeneration

    PubMed Central

    Yan, Irene K.; Wang, Xue; Asmann, Yan W.; Haga, Hiroaki; Patel, Tushar

    2016-01-01

    Background and Aims Although a key determinant of hepatic recovery after injury is active liver regeneration, the ability to detect ongoing regeneration is lacking. The restoration of liver mass after hepatectomy involves systemic changes with coordinated changes in gene expression guiding regenerative responses, activation of progenitor cells, and proliferation of quiescent hepatocytes. We postulated that these responses involve intercellular communication involving extracellular RNA and that these could represent biomarkers of active regenerative responses. Methods RNA sequencing was performed to identify temporal changes in serum extracellular non-coding RNA after partial hepatectomy in C57BL/6 male mice. Tissue expression of selected RNA was performed by microarray analysis and validated using qRT-PCR. Digital PCR was used to detect and quantify serum expression of selected RNA. Results A peak increase in extracellular RNA content occurred six hours after hepatectomy. RNA sequencing identified alterations in several small non-coding RNA including known and novel microRNAs, snoRNAs, tRNA, antisense and repeat elements after partial hepatectomy. Combinatorial effects and network analyses identified signal regulation, protein complex assembly, and signal transduction as the most common biological processes targeted by miRNA that altered. miR-1A and miR-181 were most significantly altered microRNA in both serum and in hepatic tissues, and their presence in serum was quantitated using digital PCR. Conclusions Extracellular RNA selectively enriched during acute regeneration can be detected within serum and represent biomarkers of ongoing liver regeneration in mice. The ability to detect ongoing active regeneration would improve the assessment of hepatic recovery from liver injury. PMID:27415797

  17. U1 RNA Induces Innate Immunity Signaling

    PubMed Central

    Hoffman, Robert W.; Gazitt, Tal; Foecking, Mark F.; Ortmann, Robert A.; Misfeldt, Michael; Jorgenson, Rebecca; Young, Steven L.; Greidinger, Eric L.

    2006-01-01

    Objective The U1–70-kd RNP is a prominent target of autoimmunity in connective tissue diseases. In this study, we explored whether its endogenous ligand, U1 RNA, mediates a proimmune signal and may be immunogenic. Methods We assayed the proliferation of control and MyD88-knockout splenocytes in response to in vitro–synthesized U1 RNA, and measured interleukin-6 (IL-6) and IL-8 secretion induced by U1 RNA in a human cell line competent for signaling through Toll-like receptor 3 (TLR-3) and TLR-5. Results Treatment with U1 RNA or with poly(I-C), a known agonist of TLR-3, induced approximately twice as much control splenocyte proliferation as did treatment with RNase-digested U1 RNA. Proliferation in response to either poly(I-C) or U1 RNA by MyD88-knockout splenocytes was similarly attenuated. Similar to poly(I-C), U1 RNA induced significant secretion of both IL-6 and IL-8 from a TLR-3–expressing human cell line; in contrast, the TLR-5 agonist flagellin induced predominantly IL-8 secretion. Pretreatment of U1 RNA with RNase abolished IL-6 and IL-8 secretion. Conclusion U1 RNA is capable of inducing manifestations consistent with TLR-3 activation. The ability of U1 RNA (which has a substantial double-stranded secondary structure) to activate TLR-3 may contribute to the immunogenicity of the U1–70-kd autoantigen. Stimulation of innate immunity by native RNA molecules with a double-stranded secondary structure may help explain the high prevalence of autoimmunity to RNA binding proteins. PMID:15457457

  18. DNA and RNA technology in soil biodiversity

    NASA Astrophysics Data System (ADS)

    Pereg, Lily

    2016-04-01

    DNA technology has come a long way and state of the art techniques are currently used in the analysis of soil biodiversity. Current methods will be presented and their strengths and limitations discussed. RNA technology, for the study of gene expression and potential activity of functional groups in the soil, is lagging behind, mostly due to the difficulties of extracting stable RNA from the soil. The potentials and challenges of adopting RNA technology for soil analysis will be discussed.

  19. A chemical screen for biological small molecule–RNA conjugates reveals CoA-linked RNA

    PubMed Central

    Kowtoniuk, Walter E.; Shen, Yinghua; Heemstra, Jennifer M.; Agarwal, Isha; Liu, David R.

    2009-01-01

    Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3′-aminoacylated tRNAs, nucleobase-modified RNAs, and 5′-capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule–RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNA-linked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule–RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule–RNA conjugates, including 3′-dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5′ terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (≲200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood. PMID:19416889

  20. A chemical screen for biological small molecule-RNA conjugates reveals CoA-linked RNA.

    PubMed

    Kowtoniuk, Walter E; Shen, Yinghua; Heemstra, Jennifer M; Agarwal, Isha; Liu, David R

    2009-05-12

    Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3'-aminoacylated tRNAs, nucleobase-modified RNAs, and 5'-capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule-RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNA-linked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule-RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule-RNA conjugates, including 3'-dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5' terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (approximately < 200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood. PMID:19416889

  1. Complete sequence of RNA1 and subgenomic RNA3 of Atlantic halibut nodavirus (AHNV).

    PubMed

    Sommerset, Ingunn; Nerland, Audun H

    2004-03-10

    The Nodaviridae are divided into the alphanodavirus genus, which infects insects, and the betanodavirus genus, which infects fishes. Betanodaviruses are the causative agent of viral encephalopathy and retinopathy (VER) in a number of cultivated marine fish species. The Nodaviridae are small non-enveloped RNA viruses that contain a genome consisting of 2 single-stranded positivesense RNA segments: RNA1 (3.1 kb), which encodes the viral part of the RNA-dependent RNA polymerase (RdRp); and RNA2 (1.4 kb), which encodes the capsid protein. In addition to RNA1 and RNA2, a subgenomic transcript of RNA1, RNA3, is present in infected cells. We have cloned and sequenced RNA1 from the Atlantic halibut Hippoglossus hippoglossus nodavirus (AHNV), and for the first time, the sequence of a betanodaviral subgenomic RNA3 has been determined. AHNV RNA1 was 3100 nucleotides in length and contained a main open reading frame encoding a polypeptide of 981 amino acids. Conservative motifs for RdRp were found in the deduced amino acid sequence. RNA3 was 371 nucleotides in length, and contained an open reading frame encoding a peptide of 75 amino acids corresponding to a hypothetical B2 protein, although sequence alignments with the alphanodavirus B2 proteins showed only marginal similarities. AHNV RNA replication in the fish cell-line SSN-1 (derived from striped snakehead) was analysed by Northern blot analysis, which indicated that RNA3 was synthesised in large amounts (compared to RNA1) at an early point in time post-infection. PMID:15109133

  2. Identifying miRNA/mRNA negative regulation pairs in colorectal cancer

    PubMed Central

    Zhou, Xile; Xu, Xiangming; Wang, Jinhai; Lin, Jianjiang; Chen, Wenbin

    2015-01-01

    Although considerable progress has been made in the molecular biology of Colorectal cancer (CRC), novel approaches are still required to uncover the detailed molecular mechanism of CRC. We aim to explore the potential negatively regulated miRNA-mRNA pairs and investigate their regulatory roles so as to elaborate the potential roles of the critical proteins in the signaling pathways enriched by the differential target genes of negatively regulated miRNA in CRC. Firstly, the differential miRNA-mRNA pairs were selected, followed by pairs of miRNA and their target genes. The obtained relationships were subjected to do functional enrichment analysis and those enriched in CRC pathways were chose to further construct a protein interaction network. Finally, we analyzed the regulatory roles of these relationships and constructed a regulatory network of negatively regulated miRNA and mRNA relationships. A total of 372 pairs of miRNA-mRNA were found and 108 target genes of miRNA were obtained. Three miRNAs including hsa-mir-23b, hsa-mir-365-1 and hsa-mir-365-2 showed significant influence on prognosis of CRC patients. To conclude, the miRNA/mRNA deregulations pairs identified in this study have high potentials to be further applied in diagnosis and treatment of CRC. PMID:26269151

  3. Structure of an Rrp6-RNA exosome complex bound to poly(A) RNA.

    PubMed

    Wasmuth, Elizabeth V; Januszyk, Kurt; Lima, Christopher D

    2014-07-24

    The eukaryotic RNA exosome processes and degrades RNA by directing substrates to the distributive or processive 3' to 5' exoribonuclease activities of Rrp6 or Rrp44, respectively. The non-catalytic nine-subunit exosome core (Exo9) features a prominent central channel. Although RNA can pass through the channel to engage Rrp44, it is not clear how RNA is directed to Rrp6 or whether Rrp6 uses the central channel. Here we report a 3.3 Å crystal structure of a ten-subunit RNA exosome complex from Saccharomyces cerevisiae composed of the Exo9 core and Rrp6 bound to single-stranded poly(A) RNA. The Rrp6 catalytic domain rests on top of the Exo9 S1/KH ring above the central channel, the RNA 3' end is anchored in the Rrp6 active site, and the remaining RNA traverses the S1/KH ring in an opposite orientation to that observed in a structure of a Rrp44-containing exosome complex. Solution studies with human and yeast RNA exosome complexes suggest that the RNA path to Rrp6 is conserved and dependent on the integrity of the S1/KH ring. Although path selection to Rrp6 or Rrp44 is stochastic in vitro, the fate of a particular RNA may be determined in vivo by the manner in which cofactors present RNA to the RNA exosome. PMID:25043052

  4. Interactions between 23S rRNA and tRNA in the ribosomal E site.

    PubMed Central

    Bocchetta, M; Xiong, L; Shah, S; Mankin, A S

    2001-01-01

    Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification-interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3' end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold. PMID:11214181

  5. Messenger RNAs bearing tRNA-like features exemplified by interferon alfa 5 mRNA.

    PubMed

    Díaz-Toledano, Rosa; Gómez, Jordi

    2015-10-01

    The purpose of this work was to ascertain whether liver mRNA species share common structural features with hepatitis C virus (HCV) mRNA that allow them to support the RNase-P (pre-tRNA/processing enzyme) cleavage reaction in vitro. The presence of RNase-P competitive elements in the liver mRNA population was determined by means of biochemical techniques, and a set of sensitive mRNA species were identified through microarray screening. Cleavage specificity and substrate length requirement of around 200 nts, were determined for three mRNA species. One of these cleavage sites was found in interferon-alpha 5 (IFNA5) mRNA between specific base positions and with the characteristic RNase-P chemistry of cleavage. It was mapped within a cloverleaf-like structure revealed by a comparative structural analysis based on several direct enzymes and chemical probing methods of three RNA fragments of increasing size, and subsequently contrasted against site-directed mutants. The core region was coincident with the reported signal for the cytoplasmic accumulation region (CAR) in IFNAs. Striking similarities with the tRNA-like element of the antagonist HCV mRNA were found. In general, this study provides a new way of looking at a variety of viral tRNA-like motifs as this type of structural mimicry might be related to specific host mRNA species rather than, or in addition to, tRNA itself. PMID:25900662

  6. Structure of an Rrp6-RNA exosome complex bound to poly(A) RNA

    SciTech Connect

    Wasmuth, Elizabeth V.; Januszyk, Kurt; Lima, Christopher D.

    2014-08-20

    The eukaryotic RNA exosome processes and degrades RNA by directing substrates to the distributive or processive 3' to 5' exoribonuclease activities of Rrp6 or Rrp44, respectively. The non-catalytic nine-subunit exosome core (Exo9) features a prominent central channel. Although RNA can pass through the channel to engage Rrp44, it is not clear how RNA is directed to Rrp6 or whether Rrp6 uses the central channel. Here we report a 3.3 Å crystal structure of a ten-subunit RNA exosome complex from Saccharomyces cerevisiae composed of the Exo9 core and Rrp6 bound to single-stranded poly(A) RNA. The Rrp6 catalytic domain rests on top of the Exo9 S1/KH ring above the central channel, the RNA 3' end is anchored in the Rrp6 active site, and the remaining RNA traverses the S1/KH ring in an opposite orientation to that observed in a structure of a Rrp44-containing exosome complex. Solution studies with human and yeast RNA exosome complexes suggest that the RNA path to Rrp6 is conserved and dependent on the integrity of the S1/KH ring. Although path selection to Rrp6 or Rrp44 is stochastic in vitro, the fate of a particular RNA may be determined in vivo by the manner in which cofactors present RNA to the RNA exosome.

  7. The hybrid state of tRNA binding is an authentic translation elongation intermediate

    PubMed Central

    Dorner, Silke; Brunelle, Julie L; Sharma, Divya; Green, Rachel

    2006-01-01

    The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA–transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyl-tRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative ‘hybrid’ state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre–steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation. PMID:16501572

  8. A new way to see RNA

    PubMed Central

    Keating, Kevin S.; Humphris, Elisabeth L.; Pyle, Anna Marie

    2015-01-01

    Unlike proteins, the RNA backbone has numerous degrees of freedom (eight, if one counts the sugar pucker), making RNA modeling, structure building and prediction a multidimensional problem of exceptionally high complexity. And yet RNA tertiary structures are not infinite in their structural morphology; rather, they are built from a limited set of discrete units. In order to reduce the dimensionality of the RNA backbone in a physically reasonable way, a shorthand notation was created that reduced the RNA backbone torsion angles to two (η and θ, analogous to ϕ and ψ in proteins). When these torsion angles are calculated for nucleotides in a crystallographic database and plotted against one another, one obtains a plot analogous to a Ramachandran plot (the η/θ plot), with highly populated and unpopulated regions. Nucleotides that occupy proximal positions on the plot have identical structures and are found in the same units of tertiary structure. In this review, we describe the statistical validation of the η/θ formalism and the exploration of features within the η/θ plot. We also describe the application of the η/θ formalism in RNA motif discovery, structural comparison, RNA structure building and tertiary structure prediction. More than a tool, however, the η/θ formalism has provided new insights into RNA structure itself, revealing its fundamental components and the factors underlying RNA architectural form. PMID:21729350

  9. The Origins of the RNA World

    PubMed Central

    Robertson, Michael P; Joyce, Gerald F

    2012-01-01

    The general notion of an “RNA World” is that, in the early development of life on the Earth, genetic continuity was assured by the replication of RNA and genetically encoded proteins were not involved as catalysts. There is now strong evidence indicating that an RNA World did indeed exist before DNA- and protein-based life. However, arguments regarding whether life on Earth began with RNA are more tenuous. It might be imagined that all of the components of RNA were available in some prebiotic pool, and that these components assembled into replicating, evolving polynucleotides without the prior existence of any evolved macromolecules. A thorough consideration of this “RNA-first” view of the origin of life must reconcile concerns regarding the intractable mixtures that are obtained in experiments designed to simulate the chemistry of the primitive Earth. Perhaps these concerns will eventually be resolved, and recent experimental findings provide some reason for optimism. However, the problem of the origin of the RNA World is far from being solved, and it is fruitful to consider the alternative possibility that RNA was preceded by some other replicating, evolving molecule, just as DNA and proteins were preceded by RNA. PMID:20739415

  10. Coarse-grained modelling of supercoiled RNA

    NASA Astrophysics Data System (ADS)

    Matek, Christian; Šulc, Petr; Randisi, Ferdinando; Doye, Jonathan P. K.; Louis, Ard A.

    2015-12-01

    We study the behaviour of double-stranded RNA under twist and tension using oxRNA, a recently developed coarse-grained model of RNA. Introducing explicit salt-dependence into the model allows us to directly compare our results to data from recent single-molecule experiments. The model reproduces extension curves as a function of twist and stretching force, including the buckling transition and the behaviour of plectoneme structures. For negative supercoiling, we predict denaturation bubble formation in plectoneme end-loops, suggesting preferential plectoneme localisation in weak base sequences. OxRNA exhibits a positive twist-stretch coupling constant, in agreement with recent experimental observations.

  11. MicroRNA biogenesis pathways in cancer

    PubMed Central

    Lin, Shuibin; Gregory, Richard I.

    2016-01-01

    MicroRNAs (miRNAs) are critical regulators of gene expression. Amplification and overexpression of individual ‘oncomiRs’ or genetic loss of tumour suppressor miRNAs are associated with human cancer and are sufficient to drive tumorigenesis in mouse models. Furthermore, global miRNA depletion caused by genetic and epigenetic alterations in components of the miRNA biogenesis machinery is oncogenic. This, together with the recent identification of novel miRNA regulatory factors and pathways, highlights the importance of miRNA dysregulation in cancer. PMID:25998712

  12. Quality control of chemically damaged RNA.

    PubMed

    Simms, Carrie L; Zaher, Hani S

    2016-10-01

    The "central dogma" of molecular biology describes how information contained in DNA is transformed into RNA and finally into proteins. In order for proteins to maintain their functionality in both the parent cell and subsequent generations, it is essential that the information encoded in DNA and RNA remains unaltered. DNA and RNA are constantly exposed to damaging agents, which can modify nucleic acids and change the information they encode. While much is known about how cells respond to damaged DNA, the importance of protecting RNA has only become appreciated over the past decade. Modification of the nucleobase through oxidation and alkylation has long been known to affect its base-pairing properties during DNA replication. Similarly, recent studies have begun to highlight some of the unwanted consequences of chemical damage on mRNA decoding during translation. Oxidation and alkylation of mRNA appear to have drastic effects on the speed and fidelity of protein synthesis. As some mRNAs can persist for days in certain tissues, it is not surprising that it has recently emerged that mRNA-surveillance and RNA-repair pathways have evolved to clear or correct damaged mRNA. PMID:27155660

  13. MicroRNA expression profiling using microarrays.

    PubMed

    Love, Cassandra; Dave, Sandeep

    2013-01-01

    MicroRNAs are small noncoding RNAs which are able to regulate gene expression at both the transcriptional and translational levels. There is a growing recognition of the role of microRNAs in nearly every tissue type and cellular process. Thus there is an increasing need for accurate quantitation of microRNA expression in a variety of tissues. Microarrays provide a robust method for the examination of microRNA expression. In this chapter, we describe detailed methods for the use of microarrays to measure microRNA expression and discuss methods for the analysis of microRNA expression data. PMID:23666707

  14. Specific RNA binding to ordered phospholipid bilayers

    PubMed Central

    Janas, Tadeusz; Janas, Teresa; Yarus, Michael

    2006-01-01

    We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions. PMID:16641318

  15. The RNA World and its origins

    NASA Technical Reports Server (NTRS)

    Schwartz, A. W.

    1995-01-01

    The theory of the "RNA World" states that the first molecular systems to display the properties of self-replication and evolution were RNA molecules. The origin of life not only depended crucially upon this event, but RNA molecules can even be viewed as the first "living" things. In recent years this theory has gained ascendancy over competing ideas and is now largely accepted by biologists as the most satisfactory explanation for the origin of life. The reasons for this development will be reviewed and the problem of the origin of the first RNA molecules will be discussed.

  16. The relationship between RNA catalytic processes

    NASA Astrophysics Data System (ADS)

    Cedergren, Robert; Lang, B. Franz; Gravel, Denis

    1988-09-01

    Proposals that an RNA-based genetic system preceeded DNA, stem from the ability of RNA to store genetic information and to promote simple catalysis. However, to be a valid basis for the RNA world, RNA catalysis must demonstrate or be related to intrinsic chemical properties which could have existed in primordial times. We analyze this question by first classifying RNA catalysis and related processes according to their mechanism. We define: (A) thedisjunct nucleophile class which leads to 5'-phosphates. These include Group I and II intron splicing, nuclear mRNA splicing and RNase P reactions. Although Group I introns and its excision mechanism is likely to have existed in primordial times, present-day examples have arisen independently in different phyla much more recently. Comparative methodology indicates that RNase P catalysis originated before the divergence of the major kingdoms. In addition, alldisjunct nucleophile reactions can be interrelated by a proposed mechanism involving a distant 2-OH nucleophile. (B) theconjunct nucleophile class leading to 3'-phosphates. This class is composed of self-cleaving RNAs found in plant viruses and the newt. We propose that tRNA splicing is related to this mechanism rather than the previous one. The presence of introns in tRNA genes of eukaryotes and archaebacteria supports the idea that tRNA splicing predates the divergence of these cell types.

  17. RNA-Seq: revelation of the messengers.

    PubMed

    Van Verk, Marcel C; Hickman, Richard; Pieterse, Corné M J; Van Wees, Saskia C M

    2013-04-01

    Next-generation RNA-sequencing (RNA-Seq) is rapidly outcompeting microarrays as the technology of choice for whole-transcriptome studies. However, the bioinformatics skills required for RNA-Seq data analysis often pose a significant hurdle for many biologists. Here, we put forward the concepts and considerations that are critical for RNA-Seq data analysis and provide a generic tutorial with example data that outlines the whole pipeline from next-generation sequencing output to quantification of differential gene expression. PMID:23481128

  18. RNA silencing movement in plants.

    PubMed

    Mermigka, Glykeria; Verret, Frédéric; Kalantidis, Kriton

    2016-04-01

    Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant, respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review. PMID:26297506

  19. Base de linhas moleculares para síntese espectral estelar

    NASA Astrophysics Data System (ADS)

    Milone, A.; Sanzovo, G.

    2003-08-01

    A análise das abundâncias quí micas fotosféricas em estrelas do tipo solar ou tardia, através do cálculo teórico de seus espectros, emprega a espectroscopia de alta resolução e necessita de uma base representativa de linhas atômicas e moleculares com suas respectivas constantes bem determinadas. Nesse trabalho, utilizamos como ponto de partida as extensas listas de linhas espectrais de sistemas eletrônicos de algumas moléculas diatômicas compiladas por Kurucz para a construção de uma base de linhas moleculares para a sí ntese espectral estelar. Revisamos as determinações dos fatores rotacionais de Honl-London das forças de oscilador das linhas moleculares, para cada banda vibracional de alguns sistemas eletrônicos, seguindo a regra usual de normalização. Usamos as forças de oscilador eletrônicas da literatura. Os fatores vibracionais de Franck-Condon de cada banda foram especialmente recalculados empregando-se novas constantes moleculares. Reproduzimos, com êxito, as absorções espectrais de determinadas bandas eletrônicas-vibracionais das espécies moleculares C12C12, C12N14 e Mg24H em espectros de estrelas de referência como o Sol e Arcturus.

  20. Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

    PubMed Central

    Lakatos, Lóránt; Csorba, Tibor; Pantaleo, Vitantonio; Chapman, Elisabeth J; Carrington, James C; Liu, Yu-Ping; Dolja, Valerian V; Calvino, Lourdes Fernández; López-Moya, Juan José; Burgyán, József

    2006-01-01

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. PMID:16724105

  1. Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors.

    PubMed

    Lakatos, Lóránt; Csorba, Tibor; Pantaleo, Vitantonio; Chapman, Elisabeth J; Carrington, James C; Liu, Yu-Ping; Dolja, Valerian V; Calvino, Lourdes Fernández; López-Moya, Juan José; Burgyán, József

    2006-06-21

    RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation. PMID:16724105

  2. Hyaluronic acid-siRNA conjugate/reducible polyethylenimine complexes for targeted siRNA delivery.

    PubMed

    Jang, Yeon Lim; Ku, Sook Hee; Jin, So; Park, Jae Hyung; Kim, Won Jong; Kwon, Ick Chan; Kim, Sun Hwa; Jeong, Ji Hoon

    2014-10-01

    The clinical applications of therapeutic siRNA remain as a challenge due to the lack of efficient delivery system. In the present study, hyaluronic acid-siRNA conjugate (HA-SS-siRNA)/reducible polyethylenimine (BPEI1.2k-SS) complexes were developed to efficiently deliver the siRNA to HA receptor abundant region with the improved siRNA stability. HA and siRNA were conjugated with disulfide bonds, which are cleavable in cytoplasm. The synthesized HA-SS-siRNA was further complexed with BPEI1.2k-SS, resulting in the formation of spherical nanostructures with approximately 190 nm of size and neutral surface charge. HA-SS-siRNA/BPEI1.2k-SS complexes exhibited the improved stability against serum proteins or polyanions. These complexes were successfully translocated into intracellular region via HA receptor-mediated endocytosis, and silenced target gene expression. PMID:25942799

  3. RNA-mediated RNA degradation in transgene- and virus-induced gene silencing.

    PubMed

    Metzlaff, Michael

    2002-10-01

    In the 'RNA world' hypothesis it is postulated that RNA was the first genetic molecule. Recent discoveries in gene silencing research on plants, fungi and animals show that RNA indeed plays a key role not only in controlling invading nucleic acids, like viruses and transposable elements, but also in regulating the expression of transgenes and endogenous genes. Double-stranded RNAs were identified to be the triggering structures for the induction of a specific and highly efficient RNA silencing system, in which enzyme complexes, like Dicer and RISC, facilitate as 'molecular machines' the processing of dsRNA into characteristic small RNA species. RNA silencing can be transmitted rapidly from silenced to non-silenced cells by short and long distance signaling. There is evidence that at least one component of the signal is a specific, degradation-resistant RNA. PMID:12452426

  4. Exploring the RNA editing potential of RNA-Seq data by ExpEdit.

    PubMed

    D'Antonio, Mattia; Picardi, Ernesto; Castrignanò, Tiziana; D'Erchia, Anna Maria; Pesole, Graziano

    2015-01-01

    Revealing the impact of A-to-I RNA editing in RNA-Seq experiments is relevant in humans because RNA editing can influence gene expression. In addition, its deregulation has been linked to a variety of human diseases. Exploiting the RNA editing potential in complete RNA-Seq datasets, however, is a challenging task. Indeed, no dedicated software is available, and sometimes deep computational skills and appropriate hardware resources are required. To explore the impact of known RNA editing events in massive transcriptome sequencing experiments, we developed the ExpEdit web service application. In the present work, we provide an overview of ExpEdit as well as methodologies to investigate known RNA editing in human RNA-Seq datasets. PMID:25577388

  5. Integrated microRNA-mRNA analyses reveal OPLL specific microRNA regulatory network using high-throughput sequencing.

    PubMed

    Xu, Chen; Chen, Yu; Zhang, Hao; Chen, Yuanyuan; Shen, Xiaolong; Shi, Changgui; Liu, Yang; Yuan, Wen

    2016-01-01

    Ossification of the posterior longitudinal ligament (OPLL) is a genetic disorder which involves pathological heterotopic ossification of the spinal ligaments. Although studies have identified several genes that correlated with OPLL, the underlying regulation network is far from clear. Through small RNA sequencing, we compared the microRNA expressions of primary posterior longitudinal ligament cells form OPLL patients with normal patients (PLL) and identified 218 dysregulated miRNAs (FDR < 0.01). Furthermore, assessing the miRNA profiling data of multiple cell types, we found these dysregulated miRNAs were mostly OPLL specific. In order to decipher the regulation network of these OPLL specific miRNAs, we integrated mRNA expression profiling data with miRNA sequencing data. Through computational approaches, we showed the pivotal roles of these OPLL specific miRNAs in heterotopic ossification of longitudinal ligament by discovering highly correlated miRNA/mRNA pairs that associated with skeletal system development, collagen fibril organization, and extracellular matrix organization. The results of which provide strong evidence that the miRNA regulatory networks we established may indeed play vital roles in OPLL onset and progression. To date, this is the first systematic analysis of the micronome in OPLL, and thus may provide valuable resources in finding novel treatment and diagnostic targets of OPLL. PMID:26868491

  6. Relationships among the positive strand and double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases.

    PubMed Central

    Bruenn, J A

    1991-01-01

    The sequences of 50 RNA-dependent RNA polymerases (RDRPs) from 43 positive strand and 7 double strand RNA (dsRNA) viruses have been compared. The alignment permitted calculation of distances among the 50 viruses and a resultant dendrogram based on every amino acid, rather than just those amino acids in the conserved motifs. Remarkably, a large subgroup of these viruses, including vertebrate, plant, and insect viruses, forms a single cluster whose only common characteristic is exploitation of insect hosts or vectors. This similarity may be due to molecular constraints associated with a present and/or past ability to infect insects and/or to common descent from insect viruses. If common descent is important, as it appears to be, all the positive strand RNA viruses of eucaryotes except for the picornaviruses may have evolved from an ancestral dsRNA virus. Viral RDRPs appear to be inherited as modules rather than as portions of single RNA segments, implying that RNA recombination has played an important role in their dissemination. PMID:2014162

  7. Global analysis of the mammalian RNA degradome reveals widespread miRNA-dependent and miRNA-independent endonucleolytic cleavage

    PubMed Central

    Bracken, Cameron P.; Szubert, Jan M.; Mercer, Tim R.; Dinger, Marcel E.; Thomson, Daniel W.; Mattick, John S.; Michael, Michael Z.; Goodall, Gregory J.

    2011-01-01

    The Ago2 component of the RNA-induced silencing complex (RISC) is an endonuclease that cleaves mRNAs that base pair with high complementarity to RISC-bound microRNAs. Many examples of such direct cleavage have been identified in plants, but not in vertebrates, despite the conservation of catalytic capacity in vertebrate Ago2. We performed parallel analysis of RNA ends (PAREs), a deep sequencing approach that identifies 5′-phosphorylated, polyadenylated RNAs, to detect potential microRNA-directed mRNA cleavages in mouse embryo and adult tissues. We found that numerous mRNAs are potentially targeted for cleavage by endogenous microRNAs, but at very low levels relative to the mRNA abundance, apart from miR-151-5p-guided cleavage of the N4BP1 mRNA. We also find numerous examples of non-miRNA-directed cleavage, including cleavage of a group of mRNAs within a CA-repeat consensus sequence. The PARE analysis also identified many examples of adenylated small non-coding RNAs, including microRNAs, tRNA processing intermediates and various other small RNAs, consistent with adenylation being part of a widespread proof-reading and/or degradation pathway for small RNAs. PMID:21427086

  8. Taxas de eventos para as fontes astrofísicas do detector Mario Schenberg

    NASA Astrophysics Data System (ADS)

    Castro, C. S.; Araujo, J. C. N.; Miranda, O. D.; Aguiar, O. D.

    2003-08-01

    O detector de ondas gravitacionais Mario Schenberg será sensível a sinais que cheguem à Terra com amplitude h~10-21 e dentro da faixa em frequências que varia de 3,0 a 3,4 kHz. As principais fontes astrofísicas em condições de gerar um sinal detectável pela antena Schenberg são: colapsos estelares que produzam eventos do tipo supernova; instabilidades hidrodinâmicas em estrelas de nêutrons; excitação dos modos fluído (modos f) de estrelas de nêutrons; excitação dos primeiros modos quadrupolares de buracos negros com massa ~ 3,8 M¤; coalescências de estrelas de nêutrons e buracos negros em sistemas binários e, ainda, espiralações de mini-buracos negros. Neste trabalho nós determinamos as taxas de eventos para o Schenberg associadas a dois tipos de fontes: através da de-excitação dos modos f de estrelas de nêutrons e através da coalescência de mini-buracos negros de 0,5 M¤ (que atualmente têm sido colocados como possíveis candidatos a objetos massivos do halo Galáctico). Nós mostramos que esses tipos de fontes poderão produzir sinais em ondas gravitacionais com uma taxa em torno de um evento por ano dentro da banda do Schenberg.

  9. The ribosome triggers the stringent response by RelA via a highly distorted tRNA

    PubMed Central

    Agirrezabala, Xabier; Fernández, Israel S; Kelley, Ann C; Cartón, David Gil; Ramakrishnan, Venki; Valle, Mikel

    2013-01-01

    The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo-EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A-site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre. PMID:23877429

  10. The ribosome triggers the stringent response by RelA via a highly distorted tRNA.

    PubMed

    Agirrezabala, Xabier; Fernández, Israel S; Kelley, Ann C; Cartón, David Gil; Ramakrishnan, Venki; Valle, Mikel

    2013-09-01

    The bacterial stringent response links nutrient starvation with the transcriptional control of genes. This process is initiated by the stringent factor RelA, which senses the presence of deacylated tRNA in the ribosome as a symptom of amino-acid starvation to synthesize the alarmone (p)ppGpp. Here we report a cryo-EM study of RelA bound to ribosomes bearing cognate, deacylated tRNA in the A-site. The data show that RelA on the ribosome stabilizes an unusual distorted form of the tRNA, with the acceptor arm making contact with RelA and far from its normal location in the peptidyl transferase centre. PMID:23877429

  11. EF4 disengages the peptidyl-tRNA CCA end and facilitates back-translocation on the 70S ribosome.

    PubMed

    Zhang, Dejiu; Yan, Kaige; Liu, Guangqiao; Song, Guangtao; Luo, Jiejian; Shi, Yi; Cheng, Erchao; Wu, Shan; Jiang, Taijiao; Lou, Jizhong; Gao, Ning; Qin, Yan

    2016-02-01

    EF4 catalyzes tRNA back-translocation through an unknown mechanism. We report cryo-EM structures of Escherichia coli EF4 in post- and pretranslocational ribosomes (Post- and Pre-EF4) at 3.7- and 3.2-Å resolution, respectively. In Post-EF4, peptidyl-tRNA occupies the peptidyl (P) site, but the interaction between its CCA end and the P loop is disrupted. In Pre-EF4, the peptidyl-tRNA assumes a unique position near the aminoacyl (A) site, denoted the A site/EF4 bound (A/4) site, with a large displacement at its acceptor arm. Mutagenesis analyses suggest that a specific region in the EF4 C-terminal domain (CTD) interferes with base-pairing between the peptidyl-tRNA 3'-CCA and the P loop, whereas the EF4 CTD enhances peptidyl-tRNA interaction at the A/4 site. Therefore, EF4 induces back-translocation by disengaging the tRNA's CCA end from the peptidyl transferase center of the translating ribosome. PMID:26809121

  12. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

    PubMed Central

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-01-01

    Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo. PMID:22798264

  13. The structural basis of tRNA mimicry and conformational plasticity by a viral RNA

    PubMed Central

    Colussi, Timothy M.; Costantino, David A.; Hammond, John A.; Ruehle, Grant M.; Nix, Jay C.; Kieft, Jeffrey S.

    2014-01-01

    RNA is arguably the most functionally diverse biological macromolecule. In some cases a single discrete RNA sequence performs multiple roles and this can be conferred by a complex three-dimensional structure. This multifunctionality can also be driven or enhanced by the ability of a given RNA to assume different conformational (and therefore functional) states1. Despite its biological importance, a detailed structural understanding of the paradigm of RNA structure-driven multifunctionality is lacking. Examples to address this gap are found in single-stranded positive-sense RNA viruses, a prototype being the tRNA-like structure (TLS) found at the 3′ end of the Turnip Yellow Mosaic Virus (TYMV). This TLS not only acts like a tRNA to drive aminoacylation of the viral genomic RNA (gRNA)2-4, but also interacts with other structures in the gRNA's 3′ untranslated region5, contains the promoter for negative strand synthesis, and influences several infection-critical processes6. This TLS RNA can provide a glimpse into the structural basis of RNA multifunctionality and plasticity, but for decades its high-resolution structure has remained elusive. Here, we present the crystal structure of the complete TYMV TLS to 2.0 Å resolution. Globally, the RNA adopts a shape that mimics tRNA, but it uses a very different set of intramolecular interactions to achieve this shape. These interactions also allow the TLS to readily switch conformations. In addition, the TLS structure is ‘two-faced’: one ‘face’ closely mimics tRNA and drives aminoacylation, the other ‘face’ diverges from tRNA and enables additional functionality. The TLS is thus structured to perform several functions and interact with diverse binding partners, and we demonstrate its ability to specifically bind to ribosomes. PMID:24909993

  14. Noncoding flavivirus RNA displays RNA interference suppressor activity in insect and Mammalian cells.

    PubMed

    Schnettler, Esther; Sterken, Mark G; Leung, Jason Y; Metz, Stefan W; Geertsema, Corinne; Goldbach, Rob W; Vlak, Just M; Kohl, Alain; Khromykh, Alexander A; Pijlman, Gorben P

    2012-12-01

    West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3'-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses. PMID:23035235

  15. Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells

    PubMed Central

    Schnettler, Esther; Sterken, Mark G.; Leung, Jason Y.; Metz, Stefan W.; Geertsema, Corinne; Goldbach, Rob W.; Vlak, Just M.; Kohl, Alain

    2012-01-01

    West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses. PMID:23035235

  16. N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions.

    PubMed

    Liu, Nian; Dai, Qing; Zheng, Guanqun; He, Chuan; Parisien, Marc; Pan, Tao

    2015-02-26

    RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m(6)A-switch'. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology. PMID:25719671

  17. Mapping protein-RNA interactions by RCAP, RNA-cross-linking and peptide fingerprinting.

    PubMed

    Vaughan, Robert C; Kao, C Cheng

    2015-01-01

    RNA nanotechnology often feature protein RNA complexes. The interaction between proteins and large RNAs are difficult to study using traditional structure-based methods like NMR or X-ray crystallography. RCAP, an approach that uses reversible-cross-linking affinity purification method coupled with mass spectrometry, has been developed to map regions within proteins that contact RNA. This chapter details how RCAP is applied to map protein-RNA contacts within virions. PMID:25896007

  18. Coupling of RNA Polymerase II Transcription Elongation with Pre-mRNA Splicing.

    PubMed

    Saldi, Tassa; Cortazar, Michael A; Sheridan, Ryan M; Bentley, David L

    2016-06-19

    Pre-mRNA maturation frequently occurs at the same time and place as transcription by RNA polymerase II. The co-transcriptionality of mRNA processing has permitted the evolution of mechanisms that functionally couple transcription elongation with diverse events that occur on the nascent RNA. This review summarizes the current understanding of the relationship between transcriptional elongation through a chromatin template and co-transcriptional splicing including alternative splicing decisions that affect the expression of most human genes. PMID:27107644

  19. Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis.

    PubMed

    Mohn, Fabio; Handler, Dominik; Brennecke, Julius

    2015-05-15

    In animal gonads, PIWI-clade Argonaute proteins repress transposons sequence-specifically via bound Piwi-interacting RNAs (piRNAs). These are processed from single-stranded precursor RNAs by largely unknown mechanisms. Here we show that primary piRNA biogenesis is a 3'-directed and phased process that, in the Drosophila germ line, is initiated by secondary piRNA-guided transcript cleavage. Phasing results from consecutive endonucleolytic cleavages catalyzed by Zucchini, implying coupled formation of 3' and 5' ends of flanking piRNAs. Unexpectedly, Zucchini also participates in 3' end formation of secondary piRNAs. Its function can, however, be bypassed by downstream piRNA-guided precursor cleavages coupled to exonucleolytic trimming. Our data uncover an evolutionarily conserved piRNA biogenesis mechanism in which Zucchini plays a central role in defining piRNA 5' and 3' ends. PMID:25977553

  20. Evolution of catalytic RNA in the laboratory

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F.

    1992-01-01

    We are interested in the biochemistry of existing RNA enzymes and in the development of RNA enzymes with novel catalytic function. The focal point of our research program has been the design and operation of a laboratory system for the controlled evolution of catalytic RNA. This system serves as working model of RNA-based life and can be used to explore the catalytic potential of RNA. Evolution requires the integration of three chemical processes: amplification, mutation, and selection. Amplification results in additional copies of the genetic material. Mutation operates at the level of genotype to introduce variability, this variability in turn being expressed as a range of phenotypes. Selection operates at the level of phenotype to reduce variability by excluding those individuals that do not conform to the prevailing fitness criteria. These three processes must be linked so that only the selected individuals are amplified, subject to mutational error, to produce a progeny distribution of mutant individuals. We devised techniques for the amplification, mutation, and selection of catalytic RNA, all of which can be performed rapidly in vitro within a single reaction vessel. We integrated these techniques in such a way that they can be performed iteratively and routinely. This allowed us to conduct evolution experiments in response to artificially-imposed selection constraints. Our objective was to develop novel RNA enzymes by altering the selection constraints in a controlled manner. In this way we were able to expand the catalytic repertoire of RNA. Our long-range objective is to develop an RNA enzyme with RNA replicase activity. If such an enzyme had the ability to produce additional copies of itself, then RNA evolution would operate autonomously and the origin of life will have been realized in the laboratory.

  1. HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

    PubMed Central

    Götte, M; Fackler, S; Hermann, T; Perola, E; Cellai, L; Gross, H J; Le Grice, S F; Heumann, H

    1995-01-01

    Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA. Images PMID:7533725

  2. Molecular architecture of the ribosome-bound Hepatitis C Virus internal ribosomal entry site RNA.

    PubMed

    Yamamoto, Hiroshi; Collier, Marianne; Loerke, Justus; Ismer, Jochen; Schmidt, Andrea; Hilal, Tarek; Sprink, Thiemo; Yamamoto, Kaori; Mielke, Thorsten; Bürger, Jörg; Shaikh, Tanvir R; Dabrowski, Marylena; Hildebrand, Peter W; Scheerer, Patrick; Spahn, Christian M T

    2015-12-14

    Internal ribosomal entry sites (IRESs) are structured cis-acting RNAs that drive an alternative, cap-independent translation initiation pathway. They are used by many viruses to hijack the translational machinery of the host cell. IRESs facilitate translation initiation by recruiting and actively manipulating the eukaryotic ribosome using only a subset of canonical initiation factor and IRES transacting factors. Here we present cryo-EM reconstructions of the ribosome 80S- and 40S-bound Hepatitis C Virus (HCV) IRES. The presence of four subpopulations for the 80S•HCV IRES complex reveals dynamic conformational modes of the complex. At a global resolution of 3.9 Å for the most stable complex, a derived atomic model reveals a complex fold of the IRES RNA and molecular details of its interaction with the ribosome. The comparison of obtained structures explains how a modular architecture facilitates mRNA loading and tRNA binding to the P-site. This information provides the structural foundation for understanding the mechanism of HCV IRES RNA-driven translation initiation. PMID:26604301

  3. Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein-RNA complex

    SciTech Connect

    Alayyoubi, Maher; Leser, George P.; Kors, Christopher A.; Lamb, Robert A.

    2015-05-20

    Parainfluenza virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ~2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-Å resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the "rule of six" that governs the genome packaging of the Paramyxovirinae subfamily of viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.

  4. Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein–RNA complex

    PubMed Central

    Alayyoubi, Maher; Leser, George P.; Kors, Christopher A.; Lamb, Robert A.

    2015-01-01

    Parainfluenza virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-Å resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the “rule of six” that governs the genome packaging of the Paramyxovirinae subfamily of viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure. PMID:25831513

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

    PubMed

    Miao, Zhichao; Adamiak, Ryszard W; Blanchet, Marc-Frédérick; Boniecki, Michal; Bujnicki, Janusz M; Chen, Shi-Jie; Cheng, Clarence; Chojnowski, Grzegorz; Chou, Fang-Chieh; Cordero, Pablo; Cruz, José Almeida; Ferré-D'Amaré, Adrian R; Das, Rhiju; Ding, Feng; Dokholyan, Nikolay V; Dunin-Horkawicz, Stanislaw; Kladwang, Wipapat; Krokhotin, Andrey; Lach, Grzegorz; Magnus, Marcin; Major, François; Mann, Thomas H; Masquida, Benoît; Matelska, Dorota; Meyer, Mélanie; Peselis, Alla; Popenda, Mariusz; Purzycka, Katarzyna J; Serganov, Alexander; Stasiewicz, Juliusz; Szachniuk, Marta; Tandon, Arpit; Tian, Siqi; Wang, Jian; Xiao, Yi; Xu, Xiaojun; Zhang, Jinwei; Zhao, Peinan; Zok, Tomasz; Westhof, Eric

    2015-06-01

    This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/. PMID:25883046

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

    PubMed Central

    Miao, Zhichao; Adamiak, Ryszard W.; Blanchet, Marc-Frédérick; Boniecki, Michal; Bujnicki, Janusz M.; Chen, Shi-Jie; Cheng, Clarence; Chojnowski, Grzegorz; Chou, Fang-Chieh; Cordero, Pablo; Cruz, José Almeida; Ferré-D'Amaré, Adrian R.; Das, Rhiju; Ding, Feng; Dokholyan, Nikolay V.; Dunin-Horkawicz, Stanislaw; Kladwang, Wipapat; Krokhotin, Andrey; Lach, Grzegorz; Magnus, Marcin; Major, François; Mann, Thomas H.; Masquida, Benoît; Matelska, Dorota; Meyer, Mélanie; Peselis, Alla; Popenda, Mariusz; Purzycka, Katarzyna J.; Serganov, Alexander; Stasiewicz, Juliusz; Szachniuk, Marta; Tandon, Arpit; Tian, Siqi; Wang, Jian; Xiao, Yi; Xu, Xiaojun; Zhang, Jinwei; Zhao, Peinan; Zok, Tomasz; Westhof, Eric

    2015-01-01

    This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5–3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson–Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/. PMID:25883046

  7. The RNA sequence context defines the mechanistic routes by which yeast arginyl-tRNA synthetase charges tRNA.

    PubMed

    Sissler, M; Giegé, R; Florentz, C

    1998-06-01

    Arginylation of tRNA transcripts by yeast arginyl-tRNA synthetase can be triggered by two alternate recognition sets in anticodon loops: C35 and U36 or G36 in tRNA(Arg) and C36 and G37 in tRNA(Asp) (Sissler M, Giegé R, Florentz C, 1996, EMBO J 15:5069-5076). Kinetic studies on tRNA variants were done to explore the mechanisms by which these sets are expressed. Although the synthetase interacts in a similar manner with tRNA(Arg) and tRNA(Asp), the details of the interaction patterns are idiosyncratic, especially in anticodon loops (Sissler M, Eriani G, Martin F, Giegé R, Florentz C, 1997, Nucleic Acids Res 25:4899-4906). Exchange of individual recognition elements between arginine and aspartate tRNA frameworks strongly blocks arginylation of the mutated tRNAs, whereas full exchange of the recognition sets leads to efficient arginine acceptance of the transplanted tRNAs. Unpredictably, the similar catalytic efficiencies of native and transplanted tRNAs originate from different k(cat) and Km combinations. A closer analysis reveals that efficient arginylation results from strong anticooperative effects between individual recognition elements. Nonrecognition nucleotides as well as the tRNA architecture are additional factors that tune efficiency. Altogether, arginyl-tRNA synthetase is able to utilize different context-dependent mechanistic routes to be activated. This confers biological advantages to the arginine aminoacylation system and sheds light on its evolutionary relationship with the aspartate system. PMID:9622124

  8. MiRNA expression profile and miRNA-mRNA integrated analysis (MMIA) during podocyte differentiation.

    PubMed

    Li, Zhigui; Wang, Lifeng; Xu, Jing; Yang, Zhuo

    2015-06-01

    The podocyte is a prominent cell type, which encases the capillaries of glomerulus. Podocyte-selective deletion of Dicer or Drosha was reported to induce proteinuria and glomerulosclerosis, suggesting the essential role of microRNA (miRNA) in podocytes for renal function. However, no comprehensive miRNA expression or miRNA-mRNA integrated analysis (MMIA) can be found during podocyte differentiation. Herein, miRNA and mRNA microarrays are presented, which were carried out in differentiated and undifferentiated mouse podocyte cell lines (MPC5). A total of 50 abnormal miRNAs (26 down-regulated and 24 up-regulated) were identified in differentiated and undifferentiated podocytes. Using MMIA, 80 of the 743 mRNAs (>twofold change) were predicted for potential crosstalk with 30 miRNAs of the 50 abnormal miRNAs. In addition, the gene ontology of mRNAs and the pathway analysis of miRNAs revealed a new potential-regulated network during podocyte differentiation. The expressions of three remarkably changed miRNAs (miR-34c, miR-200a and miR-467e) and four mRNAs (Runx1t1, Atp2a2, Glrp1, and Mmp15), were randomly chosen for further validation by the quantitative real-time polymerase chain reaction, and their expression trends were consistent with the microarray data. Reference searching was also conducted to confirm our data and to find potential new molecules and miRNA-target pairs involved in the podocyte differentiation. The dual luciferase reporter assay for miR-200a/GLRX and let-7b/ARL4D confirmed the prediction of MMIA. The results of this study provide a detailed integration of mRNA and miRNA during podocyte differentiation. The molecular integration mode will open up new perspectives for a better understanding of the mechanism during podocyte differentiation. PMID:25433550

  9. RNA Quality Control as a Key to Suppressing RNA Silencing of Endogenous Genes in Plants.

    PubMed

    Liu, Lin; Chen, Xuemei

    2016-06-01

    RNA quality control of endogenous RNAs is an integral part of eukaryotic gene expression and often relies on exonucleolytic degradation to eliminate dysfunctional transcripts. In parallel, exogenous and selected endogenous RNAs are degraded through RNA silencing, which is a genome defense mechanism used by many eukaryotes. In plants, RNA silencing is triggered by the production of double-stranded RNAs (dsRNAs) by RNA-DEPENDENT RNA POLYMERASEs (RDRs) and proceeds through small interfering (si) RNA-directed, ARGONAUTE (AGO)-mediated cleavage of homologous transcripts. Many studies revealed that plants avert inappropriate posttranscriptional gene silencing of endogenous coding genes by using RNA surveillance mechanisms as a safeguard to protect their transcriptome profiles. The tug of war between RNA surveillance and RNA silencing ensures the appropriate partitioning of endogenous RNA substrates among these degradation pathways. Here we review recent advances on RNA quality control and its role in the suppression of RNA silencing at endogenous genes and discuss the mechanisms underlying the crosstalk among these pathways. PMID:27045817

  10. Nanoplasmonic probes of RNA folding and assembly during pre-mRNA splicing

    NASA Astrophysics Data System (ADS)

    Nguyen, Anh H.; Lee, Jong Uk; Sim, Sang Jun

    2016-02-01

    RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the pre-mature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ~29 nm, corresponding to intron removal and exon-2 and exon-3 connection to form the mRNA molecule, is measured by plasmonic coupling. The increased scattering intensity and surface-enhanced Raman scattering (SERS) fingerprinting reveal the clear dynamics of pre-mRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results suggest that the RNA splicing is successfully monitored with the nanoplasmonic system. Thus, this platform can be useful for studying RNA nanotechnology, biomolecular folding, alternative splicing, and maturation of microRNA.

  11. Fluorescence anisotropy: analysis of tRNA binding to the T box riboswitch antiterminator RNA.

    PubMed

    Zhou, S; Anupam, R; Hines, J V

    2015-01-01

    Fluorescence anisotropy can be utilized in drug discovery screening assays to identify compounds that disrupt medicinally important RNA-macromolecular complexes. Here we describe the application of this technique to monitor tRNA binding to T box riboswitch antiterminator RNA. PMID:25352143

  12. Covalent small-molecule-RNA complex formation enables cellular profiling of small-molecule-RNA interactions

    PubMed Central

    Guan, Lirui

    2013-01-01

    Won’t let you go! A strategy is described to design small molecules that react with their cellular RNA targets. This approach not only improves the activity of compounds targeting RNA in cell culture by ≈2500-fold but also enables cell-wide profiling of its RNA targets. PMID:23913698

  13. Covalent small-molecule-RNA complex formation enables cellular profiling of small-molecule-RNA interactions.

    PubMed

    Guan, Lirui; Disney, Matthew D

    2013-09-16

    Won't let you go! A strategy is described to design small molecules that react with their cellular RNA targets. This approach not only improves the activity of compounds targeting RNA in cell culture by a factor of about 2500 but also enables cell-wide profiling of its RNA targets. PMID:23913698

  14. VIRsiRNAdb: a curated database of experimentally validated viral siRNA/shRNA

    PubMed Central

    Thakur, Nishant; Qureshi, Abid; Kumar, Manoj

    2012-01-01

    RNAi technology has been emerging as a potential modality to inhibit viruses during past decade. In literature a few siRNA databases have been reported that focus on targeting human and mammalian genes but experimentally validated viral siRNA databases are lacking. We have developed VIRsiRNAdb, a manually curated database having comprehensive details of 1358 siRNA/shRNA targeting viral genome regions. Further, wherever available, information regarding alternative efficacies of above 300 siRNAs derived from different assays has also been incorporated. Important fields included in the database are siRNA sequence, virus subtype, target genome region, cell type, target object, experimental assay, efficacy, off-target and siRNA matching with reference viral sequences. Database also provides the users with facilities of advance search, browsing, data submission, linking to external databases and useful siRNA analysis tools especially siTarAlign which align the siRNA with reference viral genomes or user defined sequences. VIRsiRNAdb contains extensive details of siRNA/shRNA targeting 42 important human viruses including influenza virus, hepatitis B virus, HPV and SARS Corona virus. VIRsiRNAdb would prove useful for researchers in picking up the best viral siRNA for antiviral therapeutics development and also for developing better viral siRNA design tools. The database is freely available at http://crdd.osdd.net/servers/virsirnadb. PMID:22139916

  15. Methylated nucleosides in tRNA and tRNA methyltransferases

    PubMed Central

    Hori, Hiroyuki

    2014-01-01

    To date, more than 90 modified nucleosides have been found in tRNA and the biosynthetic pathways of the majority of tRNA modifications include a methylation step(s). Recent studies of the biosynthetic pathways have demonstrated that the availability of methyl group donors for the methylation in tRNA is important for correct and efficient protein synthesis. In this review, I focus on the methylated nucleosides and tRNA methyltransferases. The primary functions of tRNA methylations are linked to the different steps of protein synthesis, such as the stabilization of tRNA structure, reinforcement of the codon-anticodon interaction, regulation of wobble base pairing, and prevention of frameshift errors. However, beyond these basic functions, recent studies have demonstrated that tRNA methylations are also involved in the RNA quality control system and regulation of tRNA localization in the cell. In a thermophilic eubacterium, tRNA modifications and the modification enzymes form a network that responses to temperature changes. Furthermore, several modifications are involved in genetic diseases, infections, and the immune response. Moreover, structural, biochemical, and bioinformatics studies of tRNA methyltransferases have been clarifying the details of tRNA methyltransferases and have enabled these enzymes to be classified. In the final section, the evolution of modification enzymes is discussed. PMID:24904644

  16. The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing.

    PubMed

    Muppavarapu, Mridula; Huch, Susanne; Nissan, Tracy

    2016-04-01

    Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation. PMID:26918764

  17. Estudo de soluções locais e cosmológicas em teorias do tipo tensor-escalar

    NASA Astrophysics Data System (ADS)

    Silva E Costa, S.

    2003-08-01

    Teorias do tipo tensor-escalar são a mais simples extensão possí vel da Relatividade Geral. Nessas teorias, cujo modelo padrão é a teoria de Brans-Dicke, a curvatura do espaço-tempo, descrita por componentes tensoriais, aparece acoplada a um campo escalar que, de certo modo, representa uma variação na constante de acoplamento da gravitação. Tais teorias apresentam soluções locais e cosmológicas que, em determinados limites, recaem nas apresentadas pela Relatividade Geral, mas que em outros limites trazem novidades, tais como conseqüências observacionais da evolução de flutuações primordiais distintas daquelas previstas pela Relatividade Geral (ver, por ex., Nagata et al., PRD 66, p. 103510 (2002)). Graças a esta possibilidade de trazer à luz novidades em relação à gravitação, teorias do tipo tensor-escalar podem ser vistas como um interessante campo alternativo de pesquisas para soluções dos problemas de massa faltante (ou escura) e/ou energia escura. Seguindo tal linha, este trabalho, ainda em sua fase inicial, apresenta soluções gerais de teorias do tipo tensor-escalar para diversas situações, verificando-se em que consiste a divergência dessas soluções dos casos tradicionais possí veis na Relatividade Geral. Como exemplos das soluções aqui apresentadas pode-se destacar uma expressão geral para diferentes soluções cosmológicas englobando diferentes tipos de matéria (representados por diferentes equações de estado), e a expressão para uma solução local representando um buraco negro com rotação, similar à solução de Kerr da Relatividade Geral. Por fim, é importante ressaltar que, embora aqui apresentem-se poucos resultados novos, na literatura sobre o assunto a maior parte das soluções apresentadas limita-se a uns poucos casos especí ficos, tal como soluções cosmológicas apenas com curvatura nula, e que mesmo as soluções disponí veis são, em geral, pouco divulgadas e, portanto, pouco conhecidas, e

  18. Evolução química em galáxias compactas azuis (BCGs)

    NASA Astrophysics Data System (ADS)

    Lanfranchi, G. A.; Matteucci, F.

    2003-08-01

    Neste trabalho, a formação estelar e evolução quí mica em galáxias Compactas Azuis (Blue Compact Galaxies - BCGs) foram estudadas através da comparação de previsões de modelos de evolução quí mica a várias razões de abundância quí mica observadas nestas galáxias. Modelos detalhados com recentes dados de nucleossí ntese e que levam em consideração o papel desempenahdo por supernovas de ambos os tipos (II e Ia) na evolução galáctica foram desenvolvidos para as BCGs permitindo seguir a evolução de vários elementos quí micos (H, D, He, C, N, O, Mg, Si, S, Ca, e Fe). O modelo é caracterizado pelas prescrições adotadas para a formação estelar, a qual ocorre em vários surtos de atividade separados por longos perí odos quiescentes. Após ajustar os melhores modelos aos dados observacionais, as previsões destes modelos foram comparadas também a razões de abundância observadas em sistemas Damped Lyman alpha (DLAs) e a origem do N (primária ou secundária) foi discutida. Alguns dos resultados obtidos são: i) as razões de abundância observadas nas BCGs são reproduzidas por modelos com 2 a 7 surtos de formação estelar com eficiência entre n = 0.2-0.9 Gano-1; ii) os baixos valores de N/O observados nestas galáxias são um resultado natural de uma formação estelar em surtos; iii) os modelos para BCGs podem reproduzir os dados dos DLAs, iv) uma quantidade "baixa" de N primário produzido em estrelas de alta massa pode ser uma explicação para os baixos valores de [N/a] observados em DLAs.

  19. Posttranscriptional gene regulation by long noncoding RNA.

    PubMed

    Yoon, Je-Hyun; Abdelmohsen, Kotb; Gorospe, Myriam

    2013-10-01

    Eukaryotic cells transcribe a vast number of noncoding RNA species. Among them, long noncoding RNAs (lncRNAs) have been widely implicated in the regulation of gene transcription. However, examples of posttranscriptional gene regulation by lncRNAs are emerging. Through extended base-pairing, lncRNAs can stabilize or promote the translation of target mRNAs, while partial base-pairing facilitates mRNA decay or inhibits target mRNA translation. In the absence of complementarity, lncRNAs can suppress precursor mRNA splicing and translation by acting as decoys of RNA-binding proteins or microRNAs and can compete for microRNA-mediated inhibition leading to increased expression of the mRNA. Through these regulatory mechanisms, lncRNAs can elicit differentiation, proliferation, and cytoprotective programs, underscoring the rising recognition of lncRNA roles in human disease. In this review, we summarize the mechanisms of posttranscriptional gene regulation by lncRNAs identified until now. PMID:23178169

  20. A structural determinant required for RNA editing

    PubMed Central

    Tian, Nan; Yang, Yun; Sachsenmaier, Nora; Muggenhumer, Dominik; Bi, Jingpei; Waldsich, Christina; Jantsch, Michael F.; Jin, Yongfeng

    2011-01-01

    RNA editing by adenosine deaminases acting on RNAs (ADARs) can be both specific and non-specific, depending on the substrate. Specific editing of particular adenosines may depend on the overall sequence and structural context. However, the detailed mechanisms underlying these preferences are not fully understood. Here, we show that duplex structures mimicking an editing site in the Gabra3 pre-mRNA unexpectedly fail to support RNA editing at the Gabra3 I/M site, although phylogenetic analysis suggest an evolutionarily conserved duplex structure essential for efficient RNA editing. These unusual results led us to revisit the structural requirement for this editing by mutagenesis analysis. In vivo nuclear injection experiments of mutated editing substrates demonstrate that a non-conserved structure is a determinant for editing. This structure contains bulges either on the same or the strand opposing the edited adenosine. The position of these bulges and the distance to the edited base regulate editing. Moreover, elevated folding temperature can lead to a switch in RNA editing suggesting an RNA structural change. Our results indicate the importance of RNA tertiary structure in determining RNA editing. PMID:21427087

  1. In silico selection of RNA aptamers

    PubMed Central

    Chushak, Yaroslav; Stone, Morley O.

    2009-01-01

    In vitro selection of RNA aptamers that bind to a specific ligand usually begins with a random pool of RNA sequences. We propose a computational approach for designing a starting pool of RNA sequences for the selection of RNA aptamers for specific analyte binding. Our approach consists of three steps: (i) selection of RNA sequences based on their secondary structure, (ii) generating a library of three-dimensional (3D) structures of RNA molecules and (iii) high-throughput virtual screening of this library to select aptamers with binding affinity to a desired small molecule. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and developed a protocol for RNA 3D structure prediction. As verification, we tested the performance of in silico selection on a set of six known aptamer–ligand complexes. The structures of the native sequences for the ligands in the testing set were among the top 5% of the selected structures. The proposed approach reduces the RNA sequences search space by four to five orders of magnitude—significantly accelerating the experimental screening and selection of high-affinity aptamers. PMID:19465396

  2. Alternative applications for distinct RNA sequencing strategies

    PubMed Central

    Han, Leng; Vickers, Kasey C.; Samuels, David C.

    2015-01-01

    Recent advances in RNA library preparation methods, platform accessibility and cost efficiency have allowed high-throughput RNA sequencing (RNAseq) to replace conventional hybridization microarray platforms as the method of choice for mRNA profiling and transcriptome analyses. RNAseq is a powerful technique to profile both long and short RNA expression, and the depth of information gained from distinct RNAseq methods is striking and facilitates discovery. In addition to expression analysis, distinct RNAseq approaches also allow investigators the ability to assess transcriptional elongation, DNA variance and exogenous RNA content. Here we review the current state of the art in transcriptome sequencing and address epigenetic regulation, quantification of transcription activation, RNAseq output and a diverse set of applications for RNAseq data. We detail how RNAseq can be used to identify allele-specific expression, single-nucleotide polymorphisms and somatic mutations and discuss the benefits and limitations of using RNAseq to monitor DNA characteristics. Moreover, we highlight the power of combining RNA- and DNAseq methods for genomic analysis. In summary, RNAseq provides the opportunity to gain greater insight into transcriptional regulation and output than simply miRNA and mRNA profiling. PMID:25246237

  3. Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.

    PubMed

    Jourdain, Alexis A; Boehm, Erik; Maundrell, Kinsey; Martinou, Jean-Claude

    2016-03-14

    In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized "mitochondrial RNA granules," mitochondrial subdomains with an emerging role in the regulation of gene expression. PMID:26953349

  4. Next generation sequencing of viral RNA genomes

    PubMed Central

    2013-01-01

    Background With the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform. Results As representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers’ minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed. Conclusions The approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources. PMID:23822119

  5. Biomaterials for mRNA delivery.

    PubMed

    Islam, Mohammad Ariful; Reesor, Emma K G; Xu, Yingjie; Zope, Harshal R; Zetter, Bruce R; Shi, Jinjun

    2015-12-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  6. Biomaterials for mRNA Delivery

    PubMed Central

    Islam, Mohammad Ariful; Reesor, Emma K. G.; Xu, Yingjie; Zope, Harshal R.; Zetter, Bruce R.; Shi, Jinjun

    2015-01-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  7. RNA Encapsidation and Packaging in the Phleboviruses

    PubMed Central

    Hornak, Katherine E.; Lanchy, Jean-Marc; Lodmell, J. Stephen

    2016-01-01

    The Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Uukuniemi virus (UUKV), and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5–7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA) are synthesized. The interaction between the vRNA and the viral nucleocapsid (N) protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP) architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts are selectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses. PMID:27428993

  8. The NIH Extracellular RNA Communication Consortium

    PubMed Central

    Ainsztein, Alexandra M.; Brooks, Philip J.; Dugan, Vivien G.; Ganguly, Aniruddha; Guo, Max; Howcroft, T. Kevin; Kelley, Christine A.; Kuo, Lillian S.; Labosky, Patricia A.; Lenzi, Rebecca; McKie, George A.; Mohla, Suresh; Procaccini, Dena; Reilly, Matthew; Satterlee, John S.; Srinivas, Pothur R.; Church, Elizabeth Stansell; Sutherland, Margaret; Tagle, Danilo A.; Tucker, Jessica M.; Venkatachalam, Sundar

    2015-01-01

    The Extracellular RNA (exRNA) Communication Consortium, funded as an initiative of the NIH Common Fund, represents a consortium of investigators assembled to address the critical issues in the exRNA research arena. The overarching goal is to generate a multi-component community resource for sharing fundamental scientific discoveries, protocols, and innovative tools and technologies. The key initiatives include (a) generating a reference catalogue of exRNAs present in body fluids of normal healthy individuals that would facilitate disease diagnosis and therapies, (b) defining the fundamental principles of exRNA biogenesis, distribution, uptake, and function, as well as development of molecular tools, technologies, and imaging modalities to enable these studies, (c) identifying exRNA biomarkers of disease, (d) demonstrating clinical utility of exRNAs as therapeutic agents and developing scalable technologies required for these studies, and (e) developing a community resource, the exRNA Atlas, to provide the scientific community access to exRNA data, standardized exRNA protocols, and other useful tools and technologies generated by funded investigators. PMID:26320938

  9. MicroRNA: Mechanism of Gene Regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    MicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts through activation of a specific cellular pathway. The small RNA classified as miR are short sequences of 18-26 nucleotide long, encoded by nuclear genes with distinctive...

  10. Alternative applications for distinct RNA sequencing strategies.

    PubMed

    Han, Leng; Vickers, Kasey C; Samuels, David C; Guo, Yan

    2015-07-01

    Recent advances in RNA library preparation methods, platform accessibility and cost efficiency have allowed high-throughput RNA sequencing (RNAseq) to replace conventional hybridization microarray platforms as the method of choice for mRNA profiling and transcriptome analyses. RNAseq is a powerful technique to profile both long and short RNA expression, and the depth of information gained from distinct RNAseq methods is striking and facilitates discovery. In addition to expression analysis, distinct RNAseq approaches also allow investigators the ability to assess transcriptional elongation, DNA variance and exogenous RNA content. Here we review the current state of the art in transcriptome sequencing and address epigenetic regulation, quantification of transcription activation, RNAseq output and a diverse set of applications for RNAseq data. We detail how RNAseq can be used to identify allele-specific expression, single-nucleotide polymorphisms and somatic mutations and discuss the benefits and limitations of using RNAseq to monitor DNA characteristics. Moreover, we highlight the power of combining RNA- and DNAseq methods for genomic analysis. In summary, RNAseq provides the opportunity to gain greater insight into transcriptional regulation and output than simply miRNA and mRNA profiling. PMID:25246237

  11. RNA polymerase and the regulation of transcription

    SciTech Connect

    Reznikoff, W.S.; Gross, C.A.; Burgess, R.R.; Record, M.T.; Dahlberg, J.E.; Wickens, M.P.

    1987-01-01

    This book consists of eight sections, each containing several papers. The section titles are: RNA Polymerases; Transcription Initiation - Bacterial; Regulation of Bacterial Transcription Initiation; Stable RNA Synthesis in Eukaryotes: Chromatin Structure; Promoters; Enhancers; and the Global Control of Eukaryotic Transcription; Specific Eukaryotic Transcription Factors; Termination of Transcription; and Short Communications.

  12. RNA Encapsidation and Packaging in the Phleboviruses.

    PubMed

    Hornak, Katherine E; Lanchy, Jean-Marc; Lodmell, J Stephen

    2016-01-01

    The Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Uukuniemi virus (UUKV), and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5-7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA) are synthesized. The interaction between the vRNA and the viral nucleocapsid (N) protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP) architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts are selectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses. PMID:27428993

  13. Cytoplasmic mRNA turnover and ageing

    PubMed Central

    Borbolis, Fivos; Syntichaki, Popi

    2015-01-01

    Messenger RNA (mRNA) turnover that determines the lifetime of cytoplasmic mRNAs is a means to control gene expression under both normal and stress conditions, whereas its impact on ageing and age-related disorders has just become evident. Gene expression control is achieved at the level of the mRNA clearance as well as mRNA stability and accessibility to other molecules. All these processes are regulated by cis-acting motifs and trans-acting factors that determine the rates of translation and degradation of transcripts. Specific messenger RNA granules that harbor the mRNA decay machinery or various factors, involved in translational repression and transient storage of mRNAs, are also part of the mRNA fate regulation. Their assembly and function can be modulated to promote stress resistance to adverse conditions and over time affect the ageing process and the lifespan of the organism. Here, we provide insights into the complex relationships of ageing modulators and mRNA turnover mechanisms. PMID:26432921

  14. Isolation of total RNA from pollens.

    PubMed

    Bijli, K M; Singh, B P; Sridhara, S; Arora, N

    2001-05-01

    Isolation of total RNA from plant materials has been difficult, due to the presence of complex organic substances and the associated pigmentation. In fact, there is a dearth of standardized protocols for isolating total RNA from pollens. To find a simple and reliable method for isolating total RNA from pollen, four methods, viz. phenol/SDS (PS), guanidine HCl (GH), tri-reagent (TR), and modified SDS-betaME (SB) were tested with fresh pollen of Ricinus communis (procured at -70 degrees C) and pollen dried at 30-37 degrees C. The quality and quantity of RNA was superior for the material processed at -70 degrees C. SB gave the highest RNA yield (2.35 mg/g, OD260/280 >2.0), compared to other methods. The results obtained by the SB method were found to be comparable with the widely used tri-reagent method. This was validated with other pollens of Imperata cylindrica and Xanthium strumarium. The yield obtained from graded amounts of pollen was consistent with SB, compared to the TR method. The RNA isolated by SB gave good quality mRNA for synthesizing cDNA. The SDS-betaME method is simple, efficient, and uses less expensive reagents. Hence, we recommend the modified SDS-betaME method for isolating total RNA from pollens. PMID:11426703

  15. Changing genetic information through RNA editing

    NASA Technical Reports Server (NTRS)

    Maas, S.; Rich, A.

    2000-01-01

    RNA editing, the post-transcriptional alteration of a gene-encoded sequence, is a widespread phenomenon in eukaryotes. As a consequence of RNA editing, functionally distinct proteins can be produced from a single gene. The molecular mechanisms involved include single or multiple base insertions or deletions as well as base substitutions. In mammals, one type of substitutional RNA editing, characterized by site-specific base-modification, was shown to modulate important physiological processes. The underlying reaction mechanism of substitutional RNA editing involves hydrolytic deamination of cytosine or adenosine bases to uracil or inosine, respectively. Protein factors have been characterized that are able to induce RNA editing in vitro. A supergene family of RNA-dependent deaminases has emerged with the recent addition of adenosine deaminases specific for tRNA. Here we review the developments that have substantially increased our understanding of base-modification RNA editing over the past few years, with an emphasis on mechanistic differences, evolutionary aspects and the first insights into the regulation of editing activity.

  16. In silico selection of RNA aptamers.

    PubMed

    Chushak, Yaroslav; Stone, Morley O

    2009-07-01

    In vitro selection of RNA aptamers that bind to a specific ligand usually begins with a random pool of RNA sequences. We propose a computational approach for designing a starting pool of RNA sequences for the selection of RNA aptamers for specific analyte binding. Our approach consists of three steps: (i) selection of RNA sequences based on their secondary structure, (ii) generating a library of three-dimensional (3D) structures of RNA molecules and (iii) high-throughput virtual screening of this library to select aptamers with binding affinity to a desired small molecule. We developed a set of criteria that allows one to select a sequence with potential binding affinity from a pool of random sequences and developed a protocol for RNA 3D structure prediction. As verification, we tested the performance of in silico selection on a set of six known aptamer-ligand complexes. The structures of the native sequences for the ligands in the testing set were among the top 5% of the selected structures. The proposed approach reduces the RNA sequences search space by four to five orders of magnitude--significantly accelerating the experimental screening and selection of high-affinity aptamers. PMID:19465396

  17. Lipid-Based Nanocarriers for RNA Delivery.

    PubMed

    Xue, Hui Yi; Guo, Pengbo; Wen, Wu-Cheng; Wong, Ho Lun

    2015-01-01

    RNA-interference (RNAi) agents such as small-interfering RNA (siRNA) and micro-RNA (miRNA) have strong potential as therapeutic agents for the treatment of a broad range of diseases such as malignancies, infections, autoimmune diseases and neurological diseases that are associated with undesirable gene expression. In recent years, several clinical trials of RNAi therapeutics especially siRNAs have been conducted with limited success so far. For systemic administration of these poorly permeable and easily degradable macromolecules, it is obvious that a safe and efficient delivery platform is highly desirable. Because of high biocompatibility, biodegradability and solid track record for clinical use, nanocarriers made of lipids and/or phospholipids have been commonly employed to facilitate RNA delivery. In this article, the key features of the major sub-classes of lipid-based nanocarriers, e.g. liposomes, lipid nanoparticles and lipid nanoemulsions, will be reviewed. Focus of the discussion is on the various challenges researchers face when developing lipid-based RNA nanocarriers, such as the toxicity of cationic lipids and issues related to PEGylated lipids, as well as the strategies employed in tackling these challenges. It is hoped that by understanding more about the pros and cons of these most frequently used RNA delivery systems, the pharmaceutical scientists, biomedical researchers and clinicians will be more successful in overcoming some of the obstacles that currently limit the clinical translation of RNAi therapy. PMID:26027572

  18. RNA Interference for Wheat Functional Gene Analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA interference (RNAi) refers to a common mechanism of RNA-based post-transcriptional gene silencing in eukaryotic cells. In model plant species such as Arabidopsis and rice, RNAi has been routinely used to characterize gene function and to engineer novel phenotypes. In polyploid species, this appr...

  19. RNA trafficking in parasitic plant systems.

    PubMed

    Leblanc, Megan; Kim, Gunjune; Westwood, James H

    2012-01-01

    RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs, and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host-parasite connections and the potential significance of host RNAs for the parasite. Additional research on host-parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking. PMID:22936942

  20. Lipid-Based Nanocarriers for RNA Delivery

    PubMed Central

    Xue, Hui Yi; Guo, Pengbo; Wen, Wu-Cheng; Wong, Ho Lun

    2015-01-01

    RNA-interference (RNAi) agents such as small-interfering RNA (siRNA) and micro-RNA (miRNA) have strong potential as therapeutic agents for the treatment of a broad range of diseases such as malignancies, infections, autoimmune diseases and neurological diseases that are associated with undesirable gene expression. In recent years, several clinical trials of RNAi therapeutics especially siRNAs have been conducted with limited success so far. For systemic administration of these poorly permeable and easily degradable macromolecules, it is obvious that a safe and efficient delivery platform is highly desirable. Because of high biocompatibility, biodegradability and solid track record for clinical use, nanocarriers made of lipids and/or phospholipids have been commonly employed to facilitate RNA delivery. In this article, the key features of the major sub-classes of lipid-based nanocarriers, e.g. liposomes, lipid nanoparticles and lipid nanoemulsions, will be reviewed. Focus of the discussion is on the various challenges researchers face when developing lipid-based RNA nanocarriers, such as the toxicity of cationic lipids and issues related to PEGylated lipids, as well as the strategies employed in tackling these challenges. It is hoped that by understanding more about the pros and cons of these most frequently used RNA delivery systems, the pharmaceutical scientists, biomedical researchers and clinicians will be more successful in overcoming some of the obstacles that currently limit the clinical translation of RNAi therapy. PMID:26027572

  1. RNA-Seq for Plant Pathogenic Bacteria.

    PubMed

    Kimbrel, Jeffrey A; Di, Yanming; Cumbie, Jason S; Chang, Jeff H

    2011-01-01

    The throughput and single-base resolution of RNA-Sequencing (RNA-Seq) have contributed to a dramatic change in transcriptomic-based inquiries and resulted in many new insights into the complexities of bacterial transcriptomes. RNA-Seq could contribute to similar advances in our understanding of plant pathogenic bacteria but it is still a technology under development with limitations and unknowns that need to be considered. Here, we review some new developments for RNA-Seq and highlight recent findings for host-associated bacteria. We also discuss the technical and statistical challenges in the practical application of RNA-Seq for studying bacterial transcriptomes and describe some of the currently available solutions. PMID:24710287

  2. RNA quaternary structure and global symmetry.

    PubMed

    Jones, Christopher P; Ferré-D'Amaré, Adrian R

    2015-04-01

    Many proteins associate into symmetric multisubunit complexes. Structural analyses suggested that, by contrast, virtually all RNAs with complex 3D structures function as asymmetric monomers. Recent crystal structures revealed that several biological RNAs exhibit global symmetry at the level of their tertiary and quaternary structures. Here we survey known examples of global RNA symmetry, including the true quaternary symmetry of the bacteriophage ϕ29 prohead RNA (pRNA) and the internal pseudosymmetry of the single-chain flavin mononucleotide (FMN), glycine, and cyclic di-AMP (c-di-AMP) riboswitches. For these RNAs, global symmetry stabilizes the RNA fold, coordinates ligand-RNA interactions, and facilitates association with symmetric binding partners. PMID:25778613

  3. MicroRNA profiling: approaches and considerations

    PubMed Central

    Pritchard, Colin C.; Cheng, Heather H.; Tewari, Muneesh

    2015-01-01

    MicroRNAs (miRNAs) are small RNAs (~22 nt long) that post-transcriptionally regulate the expression of thousands of genes in a broad range of organisms, in both normal physiologic and disease contexts. MiRNA expression profiling is gaining popularity because miRNAs, as key regulators in gene expression networks, can influence many biological processes and have also shown promise as biomarkers for disease. Technological advances have enabled the development of various platforms for miRNA profiling, and an understanding of the strengths and pitfalls of different approaches can aid in the effective use of miRNA profiling for diverse applications. We review here the major considerations for carrying out and interpreting results of miRNA profiling studies, as well as current and emerging applications of miRNA profiling. PMID:22510765

  4. RNA quaternary structure and global symmetry

    PubMed Central

    Jones, Christopher P.; Ferré-D'Amaré, Adrian R.

    2015-01-01

    Many proteins associate into symmetric multisubunit complexes. Structural analyses suggested that, in contrast, virtually all RNAs with complex three-dimensional structures function as asymmetric monomers. Recent crystal structures revealed that several biological RNAs exhibit global symmetry at the level of their tertiary and quaternary structures. Here, we survey known examples of global RNA symmetry, including the true quaternary symmetry of the bacteriophage ϕ29 prohead RNA (pRNA), and the internal pseudosymmetry of the single-chain flavin mononucleotide (FMN), glycine, and cyclic diadenosine monophosphate (c-di-AMP) riboswitches. For these RNAs, global symmetry stabilizes the RNA fold, coordinates ligand-RNA interactions, and facilitates association with symmetric binding partners. PMID:25778613

  5. Identifying and Characterizing Hfq-RNA Interactions

    PubMed Central

    Faner, M.A.; Feig, A.L.

    2013-01-01

    To regulate stress responses and virulence, bacteria use small regulatory RNAs (sRNAs). These RNAs can up or down regulate target mRNAs through base pairing by influencing ribosomal access and RNA decay. A large class of these sRNAs, called trans-encoded sRNAs, requires the RNA binding protein Hfq to facilitate base pairing between the regulatory RNA and its target mRNA. The resulting network of regulation is best characterized in E. coli and S. typhimurium, but the importance of Hfq dependent sRNA regulation is recognized in a diverse population of bacteria. In this review we present the approaches and methods used to discover Hfq binding RNAs, characterize their interactions and elucidate their functions. PMID:23707622

  6. Revealing protein–lncRNA interaction

    PubMed Central

    Colantoni, Alessio; Helmer-Citterich, Manuela

    2016-01-01

    Long non-coding RNAs (lncRNAs) are associated to a plethora of cellular functions, most of which require the interaction with one or more RNA-binding proteins (RBPs); similarly, RBPs are often able to bind a large number of different RNAs. The currently available knowledge is already drawing an intricate network of interactions, whose deregulation is frequently associated to pathological states. Several different techniques were developed in the past years to obtain protein–RNA binding data in a high-throughput fashion. In parallel, in silico inference methods were developed for the accurate computational prediction of the interaction of RBP–lncRNA pairs. The field is growing rapidly, and it is foreseeable that in the near future, the protein–lncRNA interaction network will rise, offering essential clues for a better understanding of lncRNA cellular mechanisms and their disease-associated perturbations. PMID:26041786

  7. Single Guide RNA Library Design and Construction.

    PubMed

    Wang, Tim; Lander, Eric S; Sabatini, David M

    2016-01-01

    This protocol describes how to generate a single guide RNA (sgRNA) library for use in genetic screens. There are many online tools available for predicting sgRNA sequences with high target specificity and/or cleavage activity. Here, we refer the user to genome-wide sgRNA sequence predictions that we have developed for both the human and mouse and that are available from the Broad Institute website. Once a set of target genes and corresponding sgRNA sequences has been identified, customized oligonucleotide pools can be rapidly synthesized by a number of commercial vendors. Thereafter, as described here, the oligonucleotides can be efficiently cloned into an appropriate lentiviral expression vector backbone. The resulting plasmid pool can then be packaged into lentiviral particles and used to generate knockouts in any cell line of choice. PMID:26933249

  8. RNA-exporting machine in action

    SciTech Connect

    2011-01-01

    A tiny motor tasked with one of nature's biggest jobs is now better understood. The molecular machinery that helps export messenger RNA from a cell's nucleus has been structurally mapped at the Advanced Light Source, a synchrotron located at the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab). In this simulation, Gle1 (yellow) binds Dbp5 (green and blue-grey) causing Dbp5 to release RNA (orange). The movie begins with Dbp5 bound to RNA and then transitions to the Gle1-Dbp5 state, the step that jettisons RNA. The simulation then plays in reverse to mimic RNA and ATP dependent closing of Dbp5 and release of Gle1 (Movie courtesy of Karsten Weis's and James Berger's labs)

  9. Forms and Functions of Telomerase RNA

    NASA Astrophysics Data System (ADS)

    Collins, Kathleen

    Telomerase adds single-stranded telomeric DNA repeats to chromosome ends. Unlike other polymerases involved in genome replication, telomerase synthe¬sizes DNA without use of a DNA template. Instead, the enzyme active site copies a template carried within the integral RNA subunit of the telomerase ribonucleo-protein (RNP) complex. In addition to providing a template, telomerase RNA has non-template motifs with critical functions in the catalytic cycle of repeat synthesis. In its complexity of structure and function, telomerase RNA resembles the non-coding RNAs of RNP machines like the ribosome and spliceosome that evolved from catalytic RNAs of the RNA World. However, unlike these RNPs, telomerase evolved its RNP identity after advent of the Protein World. Insights about telomer-ase have broad significance for understanding non-coding RNA biology as well as chromosome end maintenance and human disease.

  10. Modeling the Thermoproteaceae RNase P RNA

    PubMed Central

    Chan, Patricia P.; Brown, James W.; Lowe, Todd M.

    2012-01-01

    The RNA component of the RNase P complex is found throughout most branches of the tree of life and is principally responsible for removing the 5′ leader sequence from pre-tRNA transcripts during tRNA maturation. RNase P RNA has a number of universal core features, however variations in sequence and structure found in homologs across the tree of life require multiple Rfam covariance search models to detect accurately. We describe a new Rfam search model to enable efficient detection of the diminutive archaeal Type T RNase P RNAs, which are missed by existing Rfam models. Using the new model, we establish effective score detection thresholds, and detect four new RNase P RNA genes in recently completed genomes from the crenarchaeal family Thermoproteaceae. PMID:23018780

  11. RNA-Seq for Plant Pathogenic Bacteria

    PubMed Central

    Kimbrel, Jeffrey A.; Di, Yanming; Cumbie, Jason S.; Chang, Jeff H.

    2011-01-01

    The throughput and single-base resolution of RNA-Sequencing (RNA-Seq) have contributed to a dramatic change in transcriptomic-based inquiries and resulted in many new insights into the complexities of bacterial transcriptomes. RNA-Seq could contribute to similar advances in our understanding of plant pathogenic bacteria but it is still a technology under development with limitations and unknowns that need to be considered. Here, we review some new developments for RNA-Seq and highlight recent findings for host-associated bacteria. We also discuss the technical and statistical challenges in the practical application of RNA-Seq for studying bacterial transcriptomes and describe some of the currently available solutions. PMID:24710287

  12. Promoter analysis of influenza virus RNA polymerase.

    PubMed Central

    Parvin, J D; Palese, P; Honda, A; Ishihama, A; Krystal, M

    1989-01-01

    Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonuclease-primed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes. Images PMID:2585601

  13. The structural basis of transfer RNA mimicry and conformational plasticity by a viral RNA.

    PubMed

    Colussi, Timothy M; Costantino, David A; Hammond, John A; Ruehle, Grant M; Nix, Jay C; Kieft, Jeffrey S

    2014-07-17

    RNA is arguably the most functionally diverse biological macromolecule. In some cases a single discrete RNA sequence performs multiple roles, and this can be conferred by a complex three-dimensional structure. Such multifunctionality can also be driven or enhanced by the ability of a given RNA to assume different conformational (and therefore functional) states. Despite its biological importance, a detailed structural understanding of the paradigm of RNA structure-driven multifunctionality is lacking. To address this gap it is useful to study examples from single-stranded positive-sense RNA viruses, a prototype being the tRNA-like structure (TLS) found at the 3' end of the turnip yellow mosaic virus (TYMV). This TLS not only acts like a tRNA to drive aminoacylation of the viral genomic (g)RNA, but also interacts with other structures in the 3' untranslated region of the gRNA, contains the promoter for negative-strand synthesis, and influences several infection-critical processes. TLS RNA can provide a glimpse into the structural basis of RNA multifunctionality and plasticity, but for decades its high-resolution structure has remained elusive. Here we present the crystal structure of the complete TYMV TLS to 2.0 Å resolution. Globally, the RNA adopts a shape that mimics tRNA, but it uses a very different set of intramolecular interactions to achieve this shape. These interactions also allow the TLS to readily switch conformations. In addition, the TLS structure is 'two faced': one face closely mimics tRNA and drives aminoacylation, the other face diverges from tRNA and enables additional functionality. The TLS is thus structured to perform several functions and interact with diverse binding partners, and we demonstrate its ability to specifically bind to ribosomes. PMID:24909993

  14. Four RNA families with functional transient structures.

    PubMed

    Zhu, Jing Yun A; Meyer, Irmtraud M

    2015-01-01

    Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5' flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5' UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM. All

  15. Four RNA families with functional transient structures

    PubMed Central

    Zhu, Jing Yun A; Meyer, Irmtraud M

    2015-01-01

    Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5′ flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5′ UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM

  16. Size, shape, and flexibility of RNA structures

    NASA Astrophysics Data System (ADS)

    Hyeon, Changbong; Dima, Ruxandra I.; Thirumalai, D.

    2006-11-01

    Determination of sizes and flexibilities of RNA molecules is important in understanding the nature of packing in folded structures and in elucidating interactions between RNA and DNA or proteins. Using the coordinates of the structures of RNA in the Protein Data Bank we find that the size of the folded RNA structures, measured using the radius of gyration RG, follows the Flory scaling law, namely, RG=5.5N1/3Å, where N is the number of nucleotides. The shape of RNA molecules is characterized by the asphericity Δ and the shape S parameters that are computed using the eigenvalues of the moment of inertia tensor. From the distribution of Δ, we find that a large fraction of folded RNA structures are aspherical and the distribution of S values shows that RNA molecules are prolate (S>0). The flexibility of folded structures is characterized by the persistence length lp. By fitting the distance distribution function P(r ), that is computed using the coordinates of the folded RNA, to the wormlike chain model we extracted the persistence length lp. We find that lp≈1.5N0.33Å which might reflect the large separation between the free energies that stabilize secondary and tertiary structures. The dependence of lp on N implies that the average length of helices should increase as the size of RNA grows. We also analyze packing in the structures of ribosomes (30S, 50S, and 70S) in terms of RG, Δ, S, and lp. The 70S and the 50S subunits are more spherical compared to most RNA molecules. The globularity in 50S is due to the presence of an unusually large number (compared to 30S subunit) of small helices that are stitched together by bulges and loops. Comparison of the shapes of the intact 70S ribosome and the constituent particles suggests that folding of the individual molecules might occur prior to assembly.

  17. Small RNA in the nucleus: the RNA-chromatin ping-pong

    PubMed Central

    Olovnikov, Ivan; Aravin, Alexei A.; Toth, Katalin Fejes

    2012-01-01

    Eukaryotes use several classes of small RNA molecules to guide diverse protein machineries to target messenger RNA. The role of small RNA in post-transcriptional regulation of mRNA stability and translation is now well established. Small RNAs can also guide sequence-specific modification of chromatin structure and thus contribute to establishment and maintenance of distinct chromatin domains. In this review we summarize the model for the inter-dependent interaction between small RNA and chromatin that has emerged from studies on fission yeast and plants. We focus on recent results that link a distinct class of small RNAs, the piRNAs, to chromatin regulation in animals. PMID:22349141

  18. RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine.

    PubMed

    Delatte, Benjamin; Wang, Fei; Ngoc, Long Vo; Collignon, Evelyne; Bonvin, Elise; Deplus, Rachel; Calonne, Emilie; Hassabi, Bouchra; Putmans, Pascale; Awe, Stephan; Wetzel, Collin; Kreher, Judith; Soin, Romuald; Creppe, Catherine; Limbach, Patrick A; Gueydan, Cyril; Kruys, Véronique; Brehm, Alexander; Minakhina, Svetlana; Defrance, Matthieu; Steward, Ruth; Fuks, François

    2016-01-15

    Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila. PMID:26816380

  19. On Ensino de Astronomia: Desafios para Implantação

    NASA Astrophysics Data System (ADS)

    Faria, R. Z.; Voelzke, M. R.

    2008-09-01

    Em 2002 o ensino de Astronomia foi proposto como um dos temas estruturadores pelos Parâmetros Curriculares Nacionais e sugerido como facilitador para que o aluno compreendesse a Física como construção humana e parte do seu mundo vivencial, mas raramente seus conceitos foram ensinados. A presente pesquisa discute dois aspectos relacionados à abordagem de Astronomia. O primeiro aspecto é se ela está sendo abordada pelos professores do Ensino Médio e o segundo, aborda a maneira como ela está sendo ensinada. Optou-se pela aplicação de um questionário a partir do 2° semestre de 2006 e durante o ano de 2007 com professores que ministram a disciplina de Física, os quais trabalham em escolas estaduais em Rio Grande da Serra, Ribeirão Pires e Mauá no estado São Paulo. Dos 66,2% dos professores que responderam ao questionário nos municípios de Rio Grande da Serra, Ribeirão Pires e Mauá, 57,4% não aplicaram nenhum tópico de astronomia, 70,2% não utilizaram laboratório, 89,4% não utilizaram qualquer tipo de programa computacional, 83,0% nunca fizeram visitas com alunos a museus e planetários e 38,3% não indicaram qualquer tipo de livro ou revista referente à astronomia aos seus alunos. Mesmo considerando a Astronomia um conteúdo potencialmente significativo, esta não fez parte dos planejamentos escolares. Portanto são necessárias propostas que visem estratégias para a educação continuada dos professores como, por exemplo, cursos específicos sobre o ensino em Astronomia.

  20. microRNA: Diagnostic Perspective

    PubMed Central

    Faruq, Omar; Vecchione, Andrea

    2015-01-01

    Biomarkers are biological measures of a biological state. An ideal marker should be safe and easy to measure, cost efficient, modifiable with treatment, and consistent across gender and ethnic groups. To date, none of the available biomarkers satisfy all of these criteria. In addition, the major limitations of these markers are low specificity, sensitivity, and false positive results. Recently identified, microRNAs (miRNAs) are endogenous, evolutionarily conserved small non-coding RNA (about 22–25 nt long), also known as micro-coordinators of gene expression, which have been shown to be an effective tools to study the biology of diseases and to have great potential as novel diagnostic and prognostic biomarkers with high specificity and sensitivity. In fact, it has been demonstrated that miRNAs play a pivotal role in the regulation of a wide range of developmental and physiological processes and their deficiencies have been related to a number of disease. In addition, miRNAs are stable and can be easily isolated and measured from tissues and body fluids. In this review, we provide a perspective on emerging concepts and potential usefulness of miRNAs as diagnostic markers, emphasizing the involvement of specific miRNAs in particular tumor types, subtypes, cardiovascular diseases, diabetes, infectious diseases, and forensic test. PMID:26284247

  1. MicroRNA and Metastasis.

    PubMed

    Ma, L

    2016-01-01

    Noncoding RNAs are important regulatory molecules of cellular processes. MicroRNAs (miRNAs) are small noncoding RNAs that bind to complementary sequences in the 3' untranslated region of target mRNAs, leading to degradation of the target mRNAs and/or inhibition of their translation. Some miRNAs are essential for normal animal development; however, many other miRNAs are dispensable for development but play a critical role in pathological conditions, including tumorigenesis and metastasis. miRNA genes often reside at fragile chromosome sites and are deregulated in cancer. Some miRNAs function as oncogenes or tumor suppressors, collectively termed "oncomirs." Specific metastasis-regulating miRNAs, collectively termed "metastamirs," govern molecular processes and pathways in malignant progression in either a tumor cell-autonomous or a cell-nonautonomous manner. Recently, exosome-transferred miRNAs have emerged as mediators of the tumor-stroma cross talk. In this chapter, we focus on the functions, mechanisms of action, and therapeutic potential of miRNAs, particularly oncomirs and metastamirs. PMID:27613133

  2. Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

    PubMed Central

    Liu, Nian; Parisien, Marc; Dai, Qing; Zheng, Guanqun; He, Chuan; Pan, Tao

    2013-01-01

    N6-methyladenosine (m6A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m6A demethylases and cell-type and cell-state-dependent m6A patterns indicate that m6A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m6A modification include mRNA splicing, export, stability, and immune tolerance; but m6A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m6A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m6A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m6A modification. We applied the method to determine the m6A status at several sites in two human lncRNAs and three human mRNAs and found that m6A fraction varies between 6% and 80% among these sites. We also found that many m6A candidate sites in these RNAs are however not modified. The precise determination of m6A status in a long noncoding RNA also enables the identification of an m6A-containing RNA structural motif. PMID:24141618

  3. Conformational selection and induced fit for RNA polymerase and RNA/DNA hybrid backtracked recognition

    PubMed Central

    Wu, Jian; Ye, Wei; Yang, Jingxu; Chen, Hai-Feng

    2015-01-01

    RNA polymerase catalyzes transcription with a high fidelity. If DNA/RNA mismatch or DNA damage occurs downstream, a backtracked RNA polymerase can proofread this situation. However, the backtracked mechanism is still poorly understood. Here we have performed multiple explicit-solvent molecular dynamics (MD) simulations on bound and apo DNA/RNA hybrid to study backtracked recognition. MD simulations at room temperature suggest that specific electrostatic interactions play key roles in the backtracked recognition between the polymerase and DNA/RNA hybrid. Kinetics analysis at high temperature shows that bound and apo DNA/RNA hybrid unfold via a two-state process. Both kinetics and free energy landscape analyses indicate that bound DNA/RNA hybrid folds in the order of DNA/RNA contracting, the tertiary folding and polymerase binding. The predicted Φ-values suggest that C7, G9, dC12, dC15, and dT16 are key bases for the backtracked recognition of DNA/RNA hybrid. The average RMSD values between the bound structures and the corresponding apo ones and Kolmogorov-Smirnov (KS) P-test analyses indicate that the recognition between DNA/RNA hybrid and polymerase might follow an induced fit mechanism for DNA/RNA hybrid and conformation selection for polymerase. Furthermore, this method could be used to relative studies of specific recognition between nucleic acid and protein. PMID:26594643

  4. SUMOylation of TARBP2 regulates miRNA/siRNA efficiency

    PubMed Central

    Chen, Cheng; Zhu, Changhong; Huang, Jian; Zhao, Xian; Deng, Rong; Zhang, Hailong; Dou, Jinzhuo; Chen, Qin; Xu, Ming; Yuan, Haihua; Wang, Yanli; Yu, Jianxiu

    2015-01-01

    Small RNA-induced gene silencing is essential for post-transcriptional regulation of gene expression; however, it remains unclear how miRNA/siRNA efficiency is regulated. Here we show that TARBP2 is SUMOylated at K52, which can be enhanced by its phosphorylation. This modification can stabilize TARBP2 via repressing its K48-linked ubiquitination. We find that TARBP2 SUMOylation does not influence the overall production of mature miRNAs, but it regulates miRNA/siRNA efficiency. SUMOylated TARBP2 recruits Ago2 to constitute the RNA-induced silencing complex (RISC)-loading complex (RLC), and simultaneously promotes more pre-miRNAs to load into the RLC. Consequently, Ago2 is stabilized and miRNAs/siRNAs bound by TARBP2/Dicer is effectively transferred to Ago2. Thus, these processes lead to the formation of the effective RISC for RNA interference (RNAi). Collectively, our data suggest that SUMOylation of TARBP2 is required for regulating miRNA/siRNA efficiency, which is a general mechanism of miRNA/siRNA regulation. PMID:26582366

  5. The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing

    PubMed Central

    Linnstaedt, Sarah D.; Kasprzak, Wojciech K.; Shapiro, Bruce A.; Casey, John L.

    2006-01-01

    RNA editing plays a critical role in the life cycle of hepatitis delta virus (HDV). The host editing enzyme ADAR1 recognizes specific RNA secondary structure features around the amber/W site in the HDV antigenome and deaminates the amber/W adenosine. A previous report suggested that a branched secondary structure is necessary for editing in HDV genotype III. This branched structure, which is distinct from the characteristic unbranched rod structure required for HDV replication, was only partially characterized, and knowledge concerning its formation and stability was limited. Here, we examine the secondary structures, conformational dynamics, and amber/W site editing of HDV genotype III RNA using a miniaturized HDV genotype III RNA in vitro. Computational analysis of this RNA using the MPGAfold algorithm indicated that the RNA has a tendency to form both metastable and stable unbranched secondary structures. Moreover, native polyacrylamide gel electrophoresis demonstrated that this RNA forms both branched and unbranched rod structures when transcribed in vitro. As predicted, the branched structure is a metastable structure that converts readily to the unbranched rod structure. Only branched RNA was edited at the amber/W site by ADAR1 in vitro. The structural heterogeneity of HDV genotype III RNA is significant because not only are both conformations of the RNA functionally important for viral replication, but the ratio of the two forms could modulate editing by determining the amount of substrate RNA available for modification. PMID:16790843

  6. RNA exosome regulated long non-coding RNA transcription controls super-enhancer activity

    PubMed Central

    Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson; Lim, Junghyun; Kazadi, David; Sun, Jianbo; Federation, Alexander; Chao, Jaime; Elliott, Oliver; Liu, Zhi-Ping; Economides, Aris N.; Bradner, James E.; Rabadan, Raul; Basu, Uttiya

    2015-01-01

    We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem (ES) cells by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3’ regulatory region super-enhancer function. CRISPRCas9 mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3’regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers, by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function. PMID:25957685

  7. Resected RNA pseudoknots and their recognition by histidyl-tRNA synthetase

    PubMed Central

    Felden, Brice; Giegé, Richard

    1998-01-01

    Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3′-CCAOH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase. Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs. Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs. This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides. The combination of identity elements found in tRNAHis species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes. The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2–3 orders of magnitude. Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions. PMID:9724720

  8. Resected RNA pseudoknots and their recognition by histidyl-tRNA synthetase.

    PubMed

    Felden, B; Giegé, R

    1998-09-01

    Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3'-CCAOH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase. Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs. Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs. This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides. The combination of identity elements found in tRNAHis species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes. The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2-3 orders of magnitude. Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions. PMID:9724720

  9. Self-assembled Messenger RNA Nanoparticles (mRNA-NPs) for Efficient Gene Expression

    PubMed Central

    Kim, Hyejin; Park, Yongkuk; Lee, Jong Bum

    2015-01-01

    Although mRNA has several advantages over plasmid DNA when delivered into cells for gene expression, mRNA transfection is a very rare occurrence in gene delivery. This is mainly because of the labile nature of RNA, resulting in a low expression level of the desired protein. In this study, self-assembled mRNA nanoparticles (mRNA-NPs) packed with multiple repeats of mRNA were synthesized to achieve efficient gene expression. This approach required only a one-step process to synthesize particles with a minimal amount of plasmid DNA to produce the RNA transcripts via rolling circle transcription. Moreover, there are no concerns for cytotoxicity which can be caused by chemical condensates because mRNA-NPs are made entirely of mRNA. An examination of the cells transfected with the mRNA-NPs encoding the green fluorescence protein (GFP) confirmed that the mRNA-NPs can be used as a novel platform for effective gene delivery. PMID:26235529

  10. In vitro RNA synthesis by infectious pancreatic necrosis virus-associated RNA polymerase.

    PubMed

    Mertens, P P; Jamieson, P B; Dobos, P

    1982-03-01

    The presence of an RNA-dependent RNA polymerase was demonstrated in purified infectious pancreatic necrosis virus (IPNV). The enzyme was active in vitro without any pretreatment of the virus. Optimum activity was shown at 30 degrees C, pH 8 and in the presence of 6 mM-magnesium ions. Approx. 50% of the polymerase product remained associated with the dsRNA template of the virions. The remainder was found as extravirion ssRNA broken down to 5S to 7S fragments by virus-associated RNase(s). Although the addition of bentonite considerably reduced the amount of RNA synthesized, it protected the ssRNA product from degradation. This, in turn, permitted the synthesis of small amounts of ssRNA, which when analysed by sucrose gradient centrifugation or polyacrylamide gel electrophoresis behaved identically to the 24S single-stranded virus mRNA produced in infected cells. The virion polymerase was not stimulated by S-adenosyl-L-methionine or the addition of cellular or capped reovirus ssRNA. Several other modifications of the assay system were tried in an attempt to increase 24S RNA synthesis, but with little success. When [3H]uridine-labelled virus was used in the polymerase reaction, some labelled 24S ssRNA was obtained, indicating that in vitro transcription may proceed by a semi-conservative (displacement) mechanism. PMID:6175731

  11. A comprehensive comparison of general RNA-RNA interaction prediction methods.

    PubMed

    Lai, Daniel; Meyer, Irmtraud M

    2016-04-20

    RNA-RNA interactions are fast emerging as a major functional component in many newly discovered non-coding RNAs. Basepairing is believed to be a major contributor to the stability of these intermolecular interactions, much like intramolecular basepairs formed in RNA secondary structure. As such, using algorithms similar to those for predicting RNA secondary structure, computational methods have been recently developed for the prediction of RNA-RNA interactions.We provide the first comprehensive comparison comprising 14 methods that predict general intermolecular basepairs. To evaluate these, we compile an extensive data set of 54 experimentally confirmed fungal snoRNA-rRNA interactions and 102 bacterial sRNA-mRNA interactions. We test the performance accuracy of all methods, evaluating the effects of tool settings, sequence length, and multiple sequence alignment usage and quality.Our results show that-unlike for RNA secondary structure prediction-the overall best performing tools are non-comparative energy-based tools utilizing accessibility information that predict short interactions on this data set. Furthermore, we find that maintaining high accuracy across biologically different data sets and increasing input lengths remains a huge challenge, causing implications forde novotranscriptome-wide searches. Finally, we make our interaction data set publicly available for future development and benchmarking efforts. PMID:26673718

  12. Hairpin dsRNA does not trigger RNA interference in Candida albicans cells.

    PubMed

    Staab, Janet F; White, Theodore C; Marr, Kieren A

    2011-01-01

    RNA interference/silencing mechanisms triggered by double-stranded RNA (dsRNA) have been described in many eukaryotes, including fungi. These mechanisms have in common small RNA molecules (siRNAs or microRNAs) originating from dsRNAs that, together with the effector protein Argonaute, mediate silencing. The genome of the fungal pathogen Candida albicans harbours a well-conserved Argonaute and a non-canonical Dicer, essential members of silencing pathways. Prototypical siRNAs are detected as members of the C. albicans transcriptome, which is potential evidence of RNA interference/silencing pathways in this organism. Surprisingly, expression of a dsRNA a hairpin ADE2 dsRNA molecule to interfere with the endogenous ADE2 mRNA did not result in down-regulation of the message or produce adenine auxotrophic strains. Cell free assays showed that the hairpin dsRNA was a substrate for the putative C. albicans Dicer, discounting the possibility that the nature of the dsRNA trigger affects silencing functionality. Our results suggested that unknown cellular events govern the functionality of siRNAs originating from transgenes in RNA interference/silencing pathways in C. albicans. PMID:20737430

  13. The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes

    PubMed Central

    Haac, Mary Etna; Anderson, Michelle A.E.; Eggleston, Heather; Myles, Kevin M.; Adelman, Zach N.

    2015-01-01

    Aedes aegypti mosquitoes vector several arboviruses of global health significance, including dengue viruses and chikungunya virus. RNA interference (RNAi) plays an important role in antiviral immunity, gene regulation and protection from transposable elements. Double-stranded RNA binding proteins (dsRBPs) are important for efficient RNAi; in Drosophila functional specialization of the miRNA, endo-siRNA and exo-siRNA pathway is aided by the dsRBPs Loquacious (Loqs-PB, Loqs-PD) and R2D2, respectively. However, this functional specialization has not been investigated in other dipterans. We were unable to detect Loqs-PD in Ae. aegypti; analysis of other dipteran genomes demonstrated that this isoform is not conserved outside of Drosophila. Overexpression experiments and small RNA sequencing following depletion of each dsRBP revealed that R2D2 and Loqs-PA cooperate non-redundantly in siRNA production, and that these proteins exhibit an inhibitory effect on miRNA levels. Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production. Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer. We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways. PMID:25765650

  14. SUMOylation of TARBP2 regulates miRNA/siRNA efficiency.

    PubMed

    Chen, Cheng; Zhu, Changhong; Huang, Jian; Zhao, Xian; Deng, Rong; Zhang, Hailong; Dou, Jinzhuo; Chen, Qin; Xu, Ming; Yuan, Haihua; Wang, Yanli; Yu, Jianxiu

    2015-01-01

    Small RNA-induced gene silencing is essential for post-transcriptional regulation of gene expression; however, it remains unclear how miRNA/siRNA efficiency is regulated. Here we show that TARBP2 is SUMOylated at K52, which can be enhanced by its phosphorylation. This modification can stabilize TARBP2 via repressing its K(48)-linked ubiquitination. We find that TARBP2 SUMOylation does not influence the overall production of mature miRNAs, but it regulates miRNA/siRNA efficiency. SUMOylated TARBP2 recruits Ago2 to constitute the RNA-induced silencing complex (RISC)-loading complex (RLC), and simultaneously promotes more pre-miRNAs to load into the RLC. Consequently, Ago2 is stabilized and miRNAs/siRNAs bound by TARBP2/Dicer is effectively transferred to Ago2. Thus, these processes lead to the formation of the effective RISC for RNA interference (RNAi). Collectively, our data suggest that SUMOylation of TARBP2 is required for regulating miRNA/siRNA efficiency, which is a general mechanism of miRNA/siRNA regulation. PMID:26582366

  15. RNA exosome-regulated long non-coding RNA transcription controls super-enhancer activity.

    PubMed

    Pefanis, Evangelos; Wang, Jiguang; Rothschild, Gerson; Lim, Junghyun; Kazadi, David; Sun, Jianbo; Federation, Alexander; Chao, Jaime; Elliott, Oliver; Liu, Zhi-Ping; Economides, Aris N; Bradner, James E; Rabadan, Raul; Basu, Uttiya

    2015-05-01

    We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem cells (ESCs) by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast number of long non-coding RNAs (lncRNAs) and enhancer RNAs (eRNAs) with emergent functionality. Unexpectedly, eRNA-expressing regions accumulate R-loop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNA-expressing element (lncRNA-CSR) engaged in long-range DNA interactions and regulating IgH 3' regulatory region super-enhancer function. CRISPR-Cas9-mediated ablation of lncRNA-CSR transcription decreases its chromosomal looping-mediated association with the IgH 3' regulatory region super-enhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers by resolving deleterious transcription-coupled secondary DNA structures, while also regulating long-range super-enhancer chromosomal interactions important for cellular function. PMID:25957685

  16. Kinetic models of the interference of gene transcription to ncRNA and mRNA

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2011-06-01

    The experiments indicate that the transcription of genes into ncRNA can positively or negatively interfere with transcription into mRNA. We propose two kinetic models describing this effect. The first model is focused on the ncRNA-induced chromatin modification facilitating the transcription of the downstream gene into mRNA. The second model includes the competition between the transcription into ncRNA and the binding of activator to a regulatory site of the downstream gene transcribed into mRNA. Our analysis based on the mean-field kinetic equations and Monte Carlo simulations shows the likely dependences of the transcription rate on RNA polymerase concentration in situations with different rate-limiting steps. Our models can also be used to scrutinize the dependence of the transcription rate on other kinetic parameters. Our kinetic Monte Carlo simulations show that the first model predicts stochastic bursts in the mRNA formation provided that the transcription into ncRNA is slow, while the second model predicts in addition anti-phase stochastic bursts in the mRNA and ncRNA formation provided that that the protein attachment to and detachment from a regulatory site is slow.

  17. Aminoacylation of tRNA with phosphoserine for synthesis of cysteinyl-tRNA(Cys).

    PubMed

    Zhang, Chun-Mei; Liu, Cuiping; Slater, Simon; Hou, Ya-Ming

    2008-05-01

    Cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii synthesizes Cys-tRNA(Cys) by an indirect pathway, whereby O-phosphoseryl-tRNA synthetase (SepRS) acylates tRNA(Cys) with phosphoserine (Sep), and Sep-tRNA-Cys-tRNA synthase (SepCysS) converts the tRNA-bound phosphoserine to cysteine. We show here that M. jannaschii SepRS differs from CysRS by recruiting the m1G37 modification as a determinant for aminoacylation, and in showing limited discrimination against mutations of conserved nucleotides. Kinetic and binding measurements show that both SepRS and SepCysS bind the reaction intermediate Sep-tRNA(Cys) tightly, and these two enzymes form a stable binary complex that promotes conversion of the intermediate to the product and sequesters the intermediate from binding to elongation factor EF-1alpha or infiltrating into the ribosome. These results highlight the importance of the protein binary complex for efficient synthesis of Cys-tRNA(Cys). PMID:18425141

  18. RNA Synthesis by in Vitro Selected Ribozymes for Recreating an RNA World

    PubMed Central

    Martin, Lyssa L.; Unrau, Peter J.; Müller, Ulrich F.

    2015-01-01

    The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme’s possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory. PMID:25610978

  19. Escherichia coli proline tRNA: structure and recognition sites for prolyl-tRNA synthetase.

    PubMed

    Hasegawa, T; Yokogawa, T

    2000-01-01

    A major proline tRNA was purified from bulk Escherichia coli A19 tRNA by affinity chromatography with a biotinylated DNA probe. Its nucleotide sequence including modified nucleotides was determined by the post-labelling technique. In order to study the recognition sites of this proline tRNA for prolyl-tRNA synthetase, various mutant transcripts were prepared using an in vitro transcription system with T7 RNA polymerase. Based on the results of in vitro kinetic analyses of mutant transcripts, it was concluded that the second and third letters, G35 and G36, of the anticodon, G37 of the anticodon loop, the discriminator base A73, G72 of the acceptor stem, G49 and U17A that existed in the corner of an L-shaped structure are the recognition sites of proline tRNA for prolyl-tRNA synthetase. PMID:12903242

  20. Origins of Life and the RNA World: Evolution of RNA-Replicase Recognition

    NASA Astrophysics Data System (ADS)

    Guogas, Laura; Hogle, James; Gehrke, Lee

    2004-06-01

    Central to understanding the origin of life is the elucidation of the first replication mechanism. The RNA World hypothesis suggests that the first self-replicating molecules were RNAs and that DNA later superceded RNA as the genetic material. RNA viruses were not subjected to the same evolutionary pressures as cellular organisms; consequently, they likely possess remnants of earlier replication strategies. Our laboratory investigates how members of the RNA virus family Bromoviridae can have structurally distinct 3' end tags yet are specifically recognized by conserved replication enzymes. This work addresses the idea that 3' tRNA tails were functionally replaced in some viruses by an RNA-protein complex. These viruses may serve as a timeline for the transition from the RNA world to DNA and protein based life.

  1. RNA-RNA interactions in gene regulation: the coding and noncoding players.

    PubMed

    Guil, Sonia; Esteller, Manel

    2015-05-01

    The past few years have witnessed an exciting increase in the richness and complexity of RNA-mediated regulatory circuitries, including new types of RNA-RNA interaction that underlie key steps in gene expression control in an organized and probably hierarchic system to dictate final protein output. Both small (especially miRNAs) and long coding (lc) and noncoding (nc) RNAs contain structural domains that can sense and bind other RNAs via complementary base pairing. The versatility of the interaction confers multiple roles to RNA-RNA hybrids, from control of RNA biogenesis to competition for common targets. Here, we focus on the emerging evidence around RNA networks and their impact on gene expression regulation in light of recent breakthroughs around the crosstalk between coding RNAs and ncRNAs. PMID:25818326

  2. Coupling pre-mRNA processing to transcription on the RNA factory assembly line

    PubMed Central

    Lee, Kuo-Ming; Tarn, Woan-Yuh

    2013-01-01

    It has been well-documented that nuclear processing of primary transcripts of RNA polymerase II occurs co-transcriptionally and is functionally coupled to transcription. Moreover, increasing evidence indicates that transcription influences pre-mRNA splicing and even several post-splicing RNA processing events. In this review, we discuss the issues of how RNA polymerase II modulates co-transcriptional RNA processing events via its carboxyl terminal domain, and the protein domains involved in coupling of transcription and RNA processing events. In addition, we describe how transcription influences the expression or stability of mRNAs through the formation of distinct mRNP complexes. Finally, we delineate emerging findings that chromatin modifications function in the regulation of RNA processing steps, especially splicing, in addition to transcription. Overall, we provide a comprehensive view that transcription could integrate different control systems, from epigenetic to post-transcriptional control, for efficient gene expression. PMID:23392244

  3. RNA Secondary Structure Modulates FMRP's Bi-Functional Role in the MicroRNA Pathway.

    PubMed

    Kenny, Phillip; Ceman, Stephanie

    2016-01-01

    MicroRNAs act by post-transcriptionally regulating the gene expression of 30%-60% of mammalian genomes. MicroRNAs are key regulators in all cellular processes, though the mechanism by which the cell activates or represses microRNA-mediated translational regulation is poorly understood. In this review, we discuss the RNA binding protein Fragile X Mental Retardation Protein (FMRP) and its role in microRNA-mediated translational regulation. Historically, FMRP is known to function as a translational suppressor. However, emerging data suggests that FMRP has both an agonistic and antagonistic role in regulating microRNA-mediated translational suppression. This bi-functional role is dependent on FMRP's interaction with the RNA helicase Moloney leukemia virus 10 (MOV10), which modifies the structural landscape of bound mRNA, therefore facilitating or inhibiting its association with the RNA-Induced Silencing Complex. PMID:27338369

  4. Who Watches the Watchmen: Roles of RNA Modifications in the RNA Interference Pathway

    PubMed Central

    Xhemalce, Blerta

    2016-01-01

    RNA levels are widely thought to be predictive of RNA function. However, the existence of more than a hundred chemically distinct modifications of RNA alone is a major indication that these moieties may impart distinct functions to subgroups of RNA molecules that share a primary sequence but display distinct RNA “epigenetic” marks. RNAs can be modified on many sites, including 5′ and 3′ ends, the sugar phosphate backbone, or internal bases, which collectively provide many opportunities for posttranscriptional regulation through a variety of mechanisms. Here, we will focus on how modifications on messenger and microRNAs may affect the process of RNA interference in mammalian cells. We believe that taking RNA modifications into account will not only advance our understanding of this crucial pathway in disease and cancer but will also open the path to exploiting the enzymes that “write” and “erase” them as targets for therapeutic drug development. PMID:27441695

  5. Evolution of RNA-Based Networks.

    PubMed

    Stadler, Peter F

    2016-01-01

    RNA molecules have served for decades as a paradigmatic example of molecular evolution that is tractable both in in vitro experiments and in detailed computer simulation. The adaptation of RNA sequences to external selection pressures is well studied and well understood. The de novo innovation or optimization of RNA aptamers and riboswitches in SELEX experiments serves as a case in point. Likewise, fitness landscapes building upon the efficiently computable RNA secondary structures have been a key toward understanding realistic fitness landscapes. Much less is known, however, on models in which multiple RNAs interact with each other, thus actively influencing the selection pressures acting on them. From a computational perspective, RNA-RNA interactions can be dealt with by same basic methods as the folding of a single RNA molecule, although many details become more complicated. RNA-RNA interactions are frequently employed in cellular regulation networks, e.g., as miRNA bases mRNA silencing or in the modulation of bacterial mRNAs by small, often highly structured sRNAs. In this chapter, we summarize the key features of networks of replicators. We highlight the differences between quasispecies-like models describing templates copied by an external replicase and hypercycle similar to autocatalytic replicators. Two aspects are of importance: the dynamics of selection within a population, usually described by conventional dynamical systems, and the evolution of replicating species in the space of chemical types. Product inhibition plays a key role in modulating selection dynamics from survival of the fittest to extinction of unfittest. The sequence evolution of replicators is rather well understood as approximate optimization in a fitness landscape for templates that is shaped by the sequence-structure map of RNA. Some of the properties of this map, in particular shape space covering and extensive neutral networks, give rise to evolutionary patterns such as drift

  6. A minimalist mitochondrial threonyl-tRNA synthetase exhibits tRNA-isoacceptor specificity during proofreading.

    PubMed

    Zhou, Xiao-Long; Ruan, Zhi-Rong; Wang, Meng; Fang, Zhi-Peng; Wang, Yong; Chen, Yun; Liu, Ru-Juan; Eriani, Gilbert; Wang, En-Duo

    2014-12-16

    Yeast mitochondria contain a minimalist threonyl-tRNA synthetase (ThrRS) composed only of the catalytic core and tRNA binding domain but lacking the entire editing domain. Besides the usual tRNA(Thr)2, some budding yeasts, such as Saccharomyces cerevisiae, also contain a non-canonical tRNA(Thr)1 with an enlarged 8-nucleotide anticodon loop, reprograming the usual leucine CUN codons to threonine. This raises interesting questions about the aminoacylation fidelity of such ThrRSs and the possible contribution of the two tRNA(Thr)s during editing. Here, we found that, despite the absence of the editing domain, S. cerevisiae mitochondrial ThrRS (ScmtThrRS) harbors a tRNA-dependent pre-transfer editing activity. Remarkably, only the usual tRNA(Thr)2 stimulated pre-transfer editing, thus, establishing the first example of a synthetase exhibiting tRNA-isoacceptor specificity during pre-transfer editing. We also showed that the failure of tRNA(Thr)1 to stimulate tRNA-dependent pre-transfer editing was due to the lack of an editing domain. Using assays of the complementation of a ScmtThrRS gene knockout strain, we showed that the catalytic core and tRNA binding domain of ScmtThrRS co-evolved to recognize the unusual tRNA(Thr)1. In combination, the results provide insights into the tRNA-dependent editing process and suggest that tRNA-dependent pre-transfer editing takes place in the aminoacylation catalytic core. PMID:25414329

  7. Combining miRNA and mRNA Expression Profiles in Wilms Tumor Subtypes

    PubMed Central

    Ludwig, Nicole; Werner, Tamara V.; Backes, Christina; Trampert, Patrick; Gessler, Manfred; Keller, Andreas; Lenhof, Hans-Peter; Graf, Norbert; Meese, Eckart

    2016-01-01

    Wilms tumor (WT) is the most common childhood renal cancer. Recent findings of mutations in microRNA (miRNA) processing proteins suggest a pivotal role of miRNAs in WT genesis. We performed miRNA expression profiling of 36 WTs of different subtypes and four normal kidney tissues using microarrays. Additionally, we determined the gene expression profile of 28 of these tumors to identify potentially correlated target genes and affected pathways. We identified 85 miRNAs and 2107 messenger RNAs (mRNA) differentially expressed in blastemal WT, and 266 miRNAs and 1267 mRNAs differentially expressed in regressive subtype. The hierarchical clustering of the samples, using either the miRNA or mRNA profile, showed the clear separation of WT from normal kidney samples, but the miRNA pattern yielded better separation of WT subtypes. A correlation analysis of the deregulated miRNA and mRNAs identified 13,026 miRNA/mRNA pairs with inversely correlated expression, of which 2844 are potential interactions of miRNA and their predicted mRNA targets. We found significant upregulation of miRNAs-183, -301a/b and -335 for the blastemal subtype, and miRNAs-181b, -223 and -630 for the regressive subtype. We found marked deregulation of miRNAs regulating epithelial to mesenchymal transition, especially in the blastemal subtype, and miRNAs influencing chemosensitivity, especially in regressive subtypes. Further research is needed to assess the influence of preoperative chemotherapy and tumor infiltrating lymphocytes on the miRNA and mRNA patterns in WT. PMID:27043538

  8. The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu.

    PubMed

    Shanmugam, Raghuvaran; Aklujkar, Muktak; Schäfer, Matthias; Reinhardt, Richard; Nickel, Olaf; Reuter, Gunter; Lovley, Derek R; Ehrenhofer-Murray, Ann; Nellen, Wolfgang; Ankri, Serge; Helm, Mark; Jurkowski, Tomasz P; Jeltsch, Albert

    2014-06-01

    Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes. PMID:24711368

  9. The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu

    PubMed Central

    Shanmugam, Raghuvaran; Aklujkar, Muktak; Schäfer, Matthias; Reinhardt, Richard; Nickel, Olaf; Reuter, Gunter; Lovley, Derek R.; Ehrenhofer-Murray, Ann; Nellen, Wolfgang; Ankri, Serge; Helm, Mark; Jurkowski, Tomasz P.; Jeltsch, Albert

    2014-01-01

    Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes. PMID:24711368

  10. Structure of the E. coli ribosome-EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM.

    PubMed

    Fischer, Niels; Neumann, Piotr; Konevega, Andrey L; Bock, Lars V; Ficner, Ralf; Rodnina, Marina V; Stark, Holger

    2015-04-23

    Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3 Å. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9 Å resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 Å resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8 Å), but provides more detailed information (2.65 Å) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes. PMID:25707802

  11. Effect of RNA Integrity Determined With the Agilent 2100 Bioanalyzer on Bacterial RNA Quantification with RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA integrity is critical for successful RNA quantification. The level of integrity required differs among sources and extraction procedures and has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from D. dadantii. The i...

  12. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    1991-01-01

    SP6 bacteriophage RNA polymerase is produced by cultivating a new microorganism (particularly new strains of Escherichia coli) harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene and recovering SP6 bacteriophage RNA polymerase from the culture broth. SP6 bacteriophage RNA polymerase gene is provided as are new microorganisms harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene.

  13. Bioengineered Nanoparticles for siRNA delivery

    PubMed Central

    Kozielski, Kristen L.; Tzeng, Stephany Y.; Green, Jordan J.

    2014-01-01

    Short interfering RNA (siRNA) has been an important laboratory tool in the last two decades and has allowed researchers to better understand the functions of non-protein-coding genes through RNA interference (RNAi). Although RNAi holds great promise for this purpose as well as for treatment of many diseases, efforts at using siRNA have been hampered by the difficulty of safely and effectively introducing it into cells of interest, both in vitro and in vivo. To overcome this challenge, many biomaterials and nanoparticles (NPs) have been developed and optimized for siRNA delivery, often taking cues from the DNA delivery field, although different barriers exist for these two types of molecules. In this review, we discuss general properties of biomaterials and nanoparticles that are necessary for effective nucleic acid delivery. We also discuss specific examples of bioengineered materials, including lipid-based NPs, polymeric NPs, inorganic NPs, and RNA-based NPs, which clearly illustrate the problems and successes in siRNA delivery. PMID:23821336

  14. Use of DNA, RNA, and Chimeric Templates by a Viral RNA-Dependent RNA Polymerase: Evolutionary Implications for the Transition from the RNA to the DNA World

    PubMed Central

    Siegel, Robert W.; Bellon, Laurent; Beigelman, Leonid; Kao, C. Cheng

    1999-01-01

    All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position −11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few conditions were needed for an ancestral RdRp to replicate DNA genomes. PMID:10400735

  15. Systematic discovery of Xist RNA binding proteins

    PubMed Central

    Chu, Ci; Zhang, Qiangfeng Cliff; da Rocha, Simão Teixeira; Flynn, Ryan A.; Bharadwaj, Maheetha; Calabrese, J. Mauro; Magnuson, Terry; Heard, Edith; Chang, Howard Y.

    2015-01-01

    Summary Noncoding RNAs (ncRNAs) function with associated proteins to effect complex structural and regulatory outcomes. To reveal the composition and dynamics of specific noncoding RNA- protein complexes (RNPs) in vivo, we developed comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS). ChIRP-MS analysis of four ncRNAs captures key protein interactors, including a U1-specific link to the 3′ RNA processing machinery. Xist, an essential lncRNA for X-chromosome inactivation (XCI), interacts with 81 proteins from chromatin modification, nuclear matrix, and RNA remodeling pathways. The Xist RNA-protein particle assembles in two steps coupled with the transition from pluripotency to differentiation. Specific interactors include HnrnpK that participates in Xist-mediated gene silencing and histone modifications, but not Xist localization and Drosophila Split ends homolog Spen that interacts via the A-repeat domain of Xist and is required for gene silencing. Thus, Xist lncRNA engages with proteins in a modular and developmentally controlled manner to coordinate chromatin spreading and silencing. PMID:25843628

  16. Nonenzymatic microorganism identification based on ribosomal RNA

    NASA Astrophysics Data System (ADS)

    Ives, Jeffrey T.; Pierini, Alicia M.; Stokes, Jeffrey A.; Wahlund, Thomas M.; Read, Betsy; Bechtel, James H.; Bronk, Burt V.

    1999-11-01

    Effective defense against biological warfare (BW) agents requires rapid, fieldable and accurate systems. For micro- organisms like bacteria and viruses, ribosomal RNA (rRNA) provides a valuable target with multiple advantages of species specificity and intrinsic target amplification. Vegetative and spore forms of bacteria contain approximately 104 copies of rRNA. Direct detection of rRNA copies can eliminate some of the interference and preparation difficulties involved in enzymatic amplification methods. In order to apply the advantages of rRNA to BW defense, we are developing a fieldable system based on 16S rRNA, physical disruption of the micro-organism, solid phase hybridization, and fluorescence detection. Our goals include species-specific identification, complete operation from raw sample to identification in 15 minutes or less, and compact, fieldable instrumentation. Initial work on this project has investigated the lysis and hybridization steps, the species-specificity of oligonucleotides probes, and the development of a novel electromagnetic method to physically disrupt the micro- organisms. Target bacteria have been Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis). Continuing work includes further development of methods to rapidly disrupt the micro-organisms and release the rRNA, improved integration and processing, and extension to bacterial and mammalian viruses like MS2 and vesicular stomatitis virus.

  17. High-Resolution Shadowing of Transfer RNA

    PubMed Central

    Abermann, Reinhard J.; Yoshikami, Doju

    1972-01-01

    High-resolution shadowing with metals that melt at high temperatures was used to study macromolecules. Molecules of transfer RNA shadowed with tantalum-tungsten are readily visualized in an electron microscope. Mounting procedures for tRNA were perfected that reproducibly gave uniform distributions of both monomeric and dimeric tRNA particles, and allowed a statistical assessment of their gross shapes and sizes. Monomeric tRNA yielded a fairly homogeneous population of rod-shaped particles, with axial dimensions of about 40 × 85 Å. Dimers of yeast alanine tRNA held together by hydrogen bonds and dimers constructed by covalent linkage of the amino-acid acceptor (3′-) termini of monomers both gave slightly more heterogeneous populations of particles. Yet, their structures were also basically rod shaped, with their lengths ranging to about twice that of the monomer; this result indicates an end-to-end arrangement of the monomeric units within both dimers. These results suggest that the amino-acid acceptor terminus and the anticodon region are at the ends of the rod-shaped, dehydrated tRNA monomer visible by electron microscopy, consistent with the generally accepted view of tRNA structure in solution suggested by other workers using other methods. This study demonstrates that high-resolution shadowing with tantalum-tungsten provides a means to examine the three-dimensional structures of relatively small biological macromolecules. Images PMID:4504373

  18. [The cellular receptors of exogenous RNA].

    PubMed

    Reniewicz, Patryk; Zyzak, Joanna; Siednienko, Jakub

    2016-01-01

    One of the key determinants of survival for organisms is proper recognition of exogenous and endogenous nucleic acids. Therefore, high eukaryotes developed a number of receptors that allow for discrimination between friend or foe DNA and RNA. Appearance of exogenous RNA in cytoplasm provides a signal of danger and triggers cellular responses that facilitate eradication of a pathogen. Recognition of exogenous RNA is additionally complicated by fact that large amount of endogenous RNA is present in cytoplasm Thus, number of different receptors, found in eukaryotic cells, is able to recognize that nucleic acid. First group of those receptors consist endosomal Toll like receptors, namely TLR3, TLR7, TLR8 and TLR13. Those receptors recognize RNA released from pathogens that enter the cell by endocytosis. The second group includes cytoplasmic sensors like PKR and the family of RLRs comprised of RIG-I, MDA5 and LGP2. Cytoplasmic receptors recognize RNA from pathogens invading the cell by non-endocytic pathway. In both cases binding of RNA by its receptors results in activation of the signalling cascades that lead to the production of interferon and other cytokines. PMID:27117110

  19. Stacking interactions in PUF-RNA complexes

    SciTech Connect

    Yiling Koh, Yvonne; Wang, Yeming; Qiu, Chen; Opperman, Laura; Gross, Leah; Tanaka Hall, Traci M; Wickens, Marvin

    2012-07-02

    Stacking interactions between amino acids and bases are common in RNA-protein interactions. Many proteins that regulate mRNAs interact with single-stranded RNA elements in the 3' UTR (3'-untranslated region) of their targets. PUF proteins are exemplary. Here we focus on complexes formed between a Caenorhabditis elegans PUF protein, FBF, and its cognate RNAs. Stacking interactions are particularly prominent and involve every RNA base in the recognition element. To assess the contribution of stacking interactions to formation of the RNA-protein complex, we combine in vivo selection experiments with site-directed mutagenesis, biochemistry, and structural analysis. Our results reveal that the identities of stacking amino acids in FBF affect both the affinity and specificity of the RNA-protein interaction. Substitutions in amino acid side chains can restrict or broaden RNA specificity. We conclude that the identities of stacking residues are important in achieving the natural specificities of PUF proteins. Similarly, in PUF proteins engineered to bind new RNA sequences, the identity of stacking residues may contribute to 'target' versus 'off-target' interactions, and thus be an important consideration in the design of proteins with new specificities.

  20. Structure of yeast Argonaute with guide RNA

    SciTech Connect

    Nakanishi, Kotaro; Weinberg, David E.; Bartel, David P.; Patel, Dinshaw J.

    2012-06-26

    The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 {angstrom} crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded and processed by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2{prime}-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage.