Comparative analysis of non-autonomous effects of tasiRNAs and miRNAs in Arabidopsis thaliana

In plants, small interfering RNAs (siRNAs) can trigger a silencing signal that may spread within a tissue to adjacent cells or even systemically to other organs. Movement of the signal is initially limited to a few cells, but in some cases the signal can be amplified and travel over larger distances. How far silencing initiated by other classes of plant small RNAs (sRNAs) than siRNAs can extend has been less clear. Using a system based on the silencing of the CH42 gene, we have tracked the mobility of silencing signals initiated in phloem companion cells by artificial microRNAs (miRNA) and trans-acting siRNA (tasiRNA) that have the same primary sequence. In this system, both the ta-siRNA and the miRNA act at a distance. Non-autonomous effects of the miRNA can be triggered by several different miRNA precursors deployed as backbones. While the tasiRNA also acts non-autonomously, it has a much greater range than the miRNA or hairpin-derived siRNAs directed against CH42, indicating that biogenesis can determine the non-autonomous effects of sRNAs. In agreement with this hypothesis, the silencing signals initiated by different sRNAs differ in their genetic requirements

Origins and Evolution of MicroRNA Genes in Plant Species

MicroRNAs (miRNAs) are among the most important regulatory elements of gene expression in animals and plants. However, their origin and evolutionary dynamics have not been studied systematically. In this paper, we identified putative miRNA genes in 11 plant species using the bioinformatic technique and examined their evolutionary changes. Our homology search indicated that no miRNA gene is currently shared between green algae and land plants. The number of miRNA genes has increased substantially in the land plant lineage, but after the divergence of eudicots and monocots, the number has changed in a lineage-specific manner. We found that miRNA genes have originated mainly by duplication of preexisting miRNA genes or protein-coding genes. Transposable elements also seem to have contributed to the generation of species-specific miRNA genes. The relative importance of these mechanisms in plants is quite different from that in Drosophila species, where the formation of hairpin structures in the genomes seems to be a major source of miRNA genes. This difference in the origin of miRNA genes between plants and Drosophila may be explained by the difference in the binding to target mRNAs between plants and animals. We also found that young miRNA genes are less conserved than old genes in plants as well as in Drosophila species. Yet, nearly half of the gene families in the ancestor of flowering plants have been lost in at least one species examined. This indicates that the repertoires of miRNA genes have changed more dynamically than previously thought during plant evolution

Identification of conserved and novel microRNAs that are responsive to heat stress in Brassica rapa

The species Brassica rapa includes various vegetable crops. Production of these vegetable crops is usually impaired by heat stress. Some microRNAs (miRNAs) in Arabidopsis have been considered to mediate gene silencing in plant response to abiotic stress. However, it remains unknown whether or what miRNAs play a role in heat resistance of B. rapa. To identify genomewide conserved and novel miRNAs that are responsive to heat stress in B. rapa, we defined temperature thresholds of non-heading Chinese cabbage (B. rapa ssp. chinensis) and constructed small RNA libraries from the seedlings that had been exposed to high temperature (46 °C) for 1 h. By deep sequencing and data analysis, we selected a series of conserved and novel miRNAs that responded to heat stress. In total, Chinese cabbage shares at least 35 conserved miRNA families with Arabidopsis thaliana. Among them, five miRNA families were responsive to heat stress. Northern hybridization and real-time PCR showed that the conserved miRNAs bra-miR398a and bra-miR398b were heat-inhibitive and guided heat response of their target gene, BracCSD1; and bra-miR156h and bra-miR156g were heat-induced and its putative target BracSPL2 was down-regulated. According to the criteria of miRNA and miRNA* that form a duplex, 21 novel miRNAs belonging to 19 miRNA families were predicted. Of these, four were identified to be heat-responsive by Northern blotting and/or expression analysis of the putative targets. The two novel miRNAs bra-miR1885b.3 and bra-miR5718 negatively regulated their putative target genes. 5′-Rapid amplification of cDNA ends PCR indicated that three novel miRNAs cleaved the transcripts of their target genes where their precursors may have evolved from. These results broaden our perspective on the important role of miRNA in plant responses to heat

Characterization of microRNAs Identified in a Table Grapevine Cultivar with Validation of Computationally Predicted Grapevine miRNAs by miR-RACE

BACKGROUND: Alignment analysis of the Vv-miRNAs identified from various grapevine cultivars indicates that over 30% orthologous Vv-miRNAs exhibit a 1-3 nucleotide discrepancy only at their ends, suggesting that this sequence discrepancy is not a random event, but might mainly derive from divergence of cultivars. With advantages of miR-RACE technology in determining precise sequences of potential miRNAs from bioinformatics prediction, the precise sequences of vv-miRNAs predicted computationally can be verified with miR-RACE in a different grapevine cultivar. This presents itself as a new approach for large scale discovery of precise miRNAs in different grapevine varieties. METHODOLOGY/PRINCIPAL FINDINGS: Among 88 unique sequences of Vv-miRNAs from bioinformatics prediction, 83 (96.3%) were successfully validated with MiR-RACE in grapevine cv. 'Summer Black'. All the validated sequences were identical to their corresponding ones obtained from deep sequencing of the small RNA library of 'Summer Black'. Quantitative RT-PCR analysis of the expressions levels of 10 Vv-miRNA/target gene pairs in grapevine tissues showed some negative correlation trends. Finally, comparison of Vv-miRNA sequences with their orthologs in Arabidopsis and study on the influence of divergent bases of the orthologous miRNAs on their targeting patterns in grapevine were also done. CONCLUSION: The validation of precise sequences of potential Vv-miRNAs from computational prediction in a different grapevine cultivar can be a new way to identify the orthologous Vv-miRNAs. Nucleotide discrepancy of orthologous Vv-miRNAs from different grapevine cultivars normally does not change their target genes. However, sequence variations of some orthologous miRNAs in grapevine and Arabidopsis can change their targeting patterns. These precise Vv-miRNAs sequences validated in our study could benefit some further study on grapevine functional genomics

Herkunft, Biogenese und nicht-Zellautonome Wirkungen Kleiner RNAs in Arabidopsis thaliana

The proper development, the reproduction, the response to environmental signals and even routine cellular processes of an organism depend on the proper regulation of its genome. As a consequence, cells have developed many strategies to control gene expression, ranging from the structure of chromosomes to the stability of proteins. One of the main regulators of gene activity in plants are the so-called small RNAs (sRNAs). These molecules are originated from a double stranded RNA, which is recognized and processed to 19-24 nt long sRNAs by Dicer-like (DCL) proteins. The sRNAs are then loaded into an effector complex called RISC, where they will confer specificity to the system. Gene silencing driven by sRNAs can occur both, at the transcriptional level (transcriptional gene silencing, TGS) or post-transcriptionally (post transcriptional gene silencing, PTGS). TGS is caused by DNA and/or histone methilation at the target sequence, while PTGS relies on the “slicing” of the target sequence or translation repression. sRNAs in plants can be divided in small interfering RNAs (siRNAs) or micro RNAs (miRNAs). siRNAs are originated from perfect double-stranded RNA, which can arise by different sources. On the other hand, miRNAs are processed from imperfect double-stranded RNAs that can be formed in molecules with self-complementarity. This PhD dissertation is divided in four different parts. In the first part, it is shown that random foldback structures in the genome, once transcribed, could be the source of new MIRNA genes. This scenario complements former theories, which were not sufficient to explain the origin of all new MIRNAs. In the second part of this thesis, the putative non-cell autonomy of miRNAs and trans-acting siRNAs (tasiRNA, a plant-specific class of siRNA) is investigated. By using synthetic molecules, it is shown that miRNAs can produce silencing which spread locally to a limited number of cells, while tasiRNA-triggered silencing is a long-range, cell-to-cell signal. Finally, the third and fourth parts discuss aspects related to the biogenesis of tasiRNAs. The importance and sufficiency of miR173 in starting transitivity is demonstrated and a new mechanism for gene silencing based on this finding is presented.Sowohl die Entwicklung eines Organismus, als auch seine Reproduktion, seine Reaktionen auf die Umwelt und seine zelluläre Prozesse beruhen auf der korrekten Regulierung seines Genoms. Folglich wurden im Laufe der Evolution verschiedene Strategien zur Kontrolle der Genexpression entwickelt, die von der Verpackung der Chromosomen bis hin zur Stabilität von Proteinen reichen. In Pflanzen sind unter den wichtigsten Regulatoren der Genaktivität die so genannten kleinen RNAs oder small RNAs, bzw. sRNAs. Diese Moleküle werden von dem sogenannten Dicer-like (DCL) Protein aus doppelsträngiger RNA hergestellt und in 19-24 Nukleotid-lange sRNAs prozessiert. Die sRNAs werden dann in einen Effektor-Komplex namens RISC geladen, und verleihen ihm so die Spezifität zur Erkennung bestimmter Nukleinsäuren (Zielsequenzen). Durch sRNAs gesteuerte Genregulation kann sowohl auf der transkriptionellen (transkriptionelles Gene-Silencing, TGS) als auch auf der post-transkriptionellen Ebene (post-transkriptionelles Gene-Silencing, PTGS) auftreten. Während beim TGS einzelne Bausteine der DNA und/oder Histone methyliert werden, werden beim PTGS Zielsequenzen zerstört oder inhibiert und damit die Biosynthese von Proteinen unterbrochen. In Pflanzen werden sRNAs in small interfering RNAs (siRNAs) und Mikro-RNAs (miRNAs) unterteilt. siRNAs werden aus doppelsträngiger RNA hergestellt, die verschiedenartiger Herkunft sein können. miRNAs werden aus RNA Molekülen, die interne Selbst-Komplementarität aufweisen, generiert. Diese Dissertation ist in vier verschiedene Abschnitte unterteilt. Im ersten Teil wird gezeigt, wie Expression von Strukturen mit zufälliger Selbstkomplementarität zu neuen MIRNA Genen führen kann. Diese Beobachtung komplementiert zuvor beschriebene Theorien, die nicht ausreichen um die Herkunft aller endogenen MIRNAs zu erklären, und trägt damit zum Gesamtbild über die Entstehung neuer MIRNAs bei. Im zweiten Teil dieser Arbeit wird die Wirkung von miRNAs und trans-acting siRNAs (tasiRNA, eine pflanzenspezifische Klasse von siRNAs) auf ihre Umgebung untersucht. Mit Hilfe von synthetischen Molekülen wird gezeigt, dass miRNAs ein auf wenige Zellschichten lokal begrenztes Gene Silencing initiieren, während tasiRNAs ein wesentlich weitläufigeres Silencing auslösen. Im dritten und vierten Teil werden die verschiedenen Aspekte der Biogenese von tasiRNAs diskutiert. Die Bedeutung und weit reichende Funktion des MIR173 Gen auf die Verstärkung von Gene Silencing, sowie eine neue, darauf basierende Methode für gezieltes Gen-Silencing werden vorgestellt

Mechanical behavior of steel pipes repaired with a hybrid laminate

Dissertação (mestrado)—Universidade de Brasília, Departamento de Engenharia Mecânica, Progrma de pós-graduação em Ciências Mecânicas, 2010.Em nível mundial, tubulações de aço desempenham papéis fundamentais nos diversos setores industriais. Contudo, qualquer tipo de duto está sujeito a desgastes em sua superfície devido à corrosão. Neste sentido, técnicas que viabilizem o reparo rápido e confiável de dutos são de fundamental importância para aumentar sua vida útil, prevenir acidentes, minimizar danos ambientais e permitir a manutenção da eficiência dos mesmos após serem reparados. Reparos com materiais compósitos estão tendo grande destaque no mercado internacional devido à sua alta eficiência, baixo peso e custo acessível. Estudos anteriores, presentes na literatura, sugerem que os materiais ideais para reparo de tubulações de aço são carbono/epóxi e vidro/epóxi, sendo que a solução mais vantajosa encontrada até o presente momento, em relação à configuração constituinte do reparo compósito em tubos de aço carbono, consiste em mesclar, no mesmo laminado, camadas de fibras de vidro com camadas de fibras de carbono, formando um reparo híbrido cujo dimensionamento ideal e número de lâminas de cada tipo de fibra devem ser cuidadosamente estudados para cada caso específico. Com o objetivo de obter informações de interesse tecnológico para setores industriais que necessitem executar manutenção preventiva em tubulações e realizar reparos emergenciais em caso de necessidade, obter-se-á soluções otimizadas de constituição e dimensionamento do reparo híbrido em material compósito, utilizado em tubos industriais de ampla aplicação no mercado atual. Para tanto, o trabalho consistirá em análises numéricas em dutos de aço (carbono), com diâmetro e comprimento nominais de 100 mm e 500 mm, respectivamente, que sofreram desbaste na região central, e foram reparados com compósitos de resina epóxi reforçada com tecidos híbridos (i.e. com camadas reforçadas com fibras de carbono e vidro-E). Com este trabalho foi possível verificar que o tubo reparado com material compósito híbrido conseguiu restaurar a rigidez e a resistência do tubo em aproximadamente 90%, sendo que a metodologia utilizada para selecionar as dimensões e os materiais adequados de reparo é rápida, eficiente, de baixo custo e depende sempre da situação específica desejada. Portanto, o reparo em análise está projetado para o pior caso de desbaste (60% da espessura original do tubo), para um tubo de aço 1010 e cujas pressões hidrostáticas internas não ultrapassem a pressão de falha calculada. _______________________________________________________________________________ ABSTRACTWorldwide, steel pipes play an important role in many industrial sectors. Meanwhile, any kind of duct is prone to suffer superficial damages due to corrosion. In this sense, techniques which allow fast and reliable repairs of ducts have a fundamental importance to increase the life cycle, avoid accidents, minimize environmental contaminations and to keep their efficiency after the repair. Composite repairs are very important in the international scenario due to their high efficiency, low weight and accessible cost. According to previous studies in literature carried out at UnB, the ideal composite materials to repair steel pipes are carbon/epóxi and glass/epóxi, composing an hybrid repair, that is the solution most advantageous until the present moment. This repair mixes, in the same laminate, glass fibre layers with carbon fibre layers. With the objective to obtain information of technological relevance to industrial sectors which need to carry out preventive maintenance and emergencial repairs, a methodology to allow the geometrical parameters and number of layers of the repair will be carefully studied. In this way, the work will consist of numerical analyses in carbon steel pipes, with 100 mm diameter and 500 mm in length, that are damaged in the central region, and had been repaired with a cold cure epoxy resin, adopted as matrix, and hybrid reinforcing layers (carbon and E-glass fiber layers). With this work it was possible to verify that the repaired pipe with hybrid material restored the rigidity and the resistance of the perfect pipe in 90%, finding the best methodology to select the dimensions and the adequate materials of repair and being the most efficient on low cost repair. Therefore, the repair in analysis is projected for the worst case of damage (60% of the original pipe thickness), for a steel pipe 1010 and whose internal hydrostatic pressures do not exceed the rupture pressure calculated

Gene Regulation Mediated by microRNA-Triggered Secondary Small RNAs in Plants

In plants, proper development and response to abiotic and biotic stimuli requires an orchestrated regulation of gene expression. Small RNAs (sRNAs) are key molecules involved in this process, leading to downregulation of their target genes. Two main classes of sRNAs exist, the small interfering RNAs (siRNAs) and microRNAs (miRNAs). The role of the latter class in plant development and physiology is well known, with many examples of how miRNAs directly impact the expression of genes in cells where they are produced, with dramatic consequences to the life of the plant. However, there is an aspect of miRNA biology that is still poorly understood. In some cases, miRNA targeting can lead to the production of secondary siRNAs from its target. These siRNAs, which display a characteristic phased production pattern, can act in cis, reinforcing the initial silencing signal set by the triggering miRNA, or in trans, affecting genes that are unrelated to the initial target. In this review, the mechanisms and implications of this process in the gene regulation mediated by miRNAs will be discussed. This work will also explore techniques for gene silencing in plants that are based on this unique pathway.</p

Downregulation of plant genes with miRNA-induced gene silencing

In plants, some microRNAs (miRNAs) can trigger the production of secondary small interfering RNAs (siRNAs) from their targets. miRNA-induced gene silencing (MIGS) exploits this unique feature to efficiently downregulate gene expression. The simple flanking of a sequence of interest with the target site for the miR173 (an miRNA able to trigger transitivity) is sufficient to start the production of secondary siRNAs and, consequently, silencing of the target gene. This technique can be easily adapted to promote gene silencing of more than one gene, even with those that share no sequence similarities. This chapter describes the necessary steps for designing and implementing the use of MIGS in plants.</p