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Both RIG-I and DRH-1 function in antiviral immunity through the activation of type-I interferon signaling in mammals and antiviral RNAi in C. elegans, respectively. This work investigates the function of DRH-1 in the production of virus-derived siRNAs (vsiRNAs). Deep sequencing of wild-type and drh-1 mutants challenged with Orsay Virus demonstrated that drh-1 mutants produced primary vsiRNAs that are dependent on DCR-1 and the dsRNA-binding protein RDE-4. However, these DRH-1 independent vsiRNAs are predominantly derived from the 5' terminal regions of both OrV RNA1 and RNA2 and were depleted from the internal regions of viral RNA. In contrast, wild-type and other mutant animals did not exhibit a bias for primary vsiRNA biogenesis from the 5' terminal regions. Mammalian RIG-I acts as an ATP-powered translocase, though the biological function of this activity is unknown. Therefore, DRH-1 may function to promote the production of primary vsiRNAs spreading from the 5' terminal regions to the internal and 3' regions of the viral RNA using translocase activity.
RNA interference (RNAi) acts as an antiviral defense mechanism in fungi, plants, nematodes, insects, and mammals. In antiviral RNAi, virus-specific double-stranded RNA is processed into small interfering RNAs (siRNAs) to guide specific viral RNA degradation by the RNAi machinery. Although antiviral RNAi is non cell-autonomous in plants, it is unknown if antiviral RNAi is also systemic in animals. In this dissertation, I characterized the nematode Caenorhabditis elegans mutants defective in systemic RNAi in their antiviral RNAi response induced by either the replication of a Flock house virus-derived replicon or the infection of Orsay virus. The results from these genetic studies provided evidence for the first time to support an antiviral function of systemic RNAi in animals. Comparison of the population of viral siRNAs by deep sequencing further revealed that C. elegans mutants with strong defects in systemic antiviral RNAi were all partially defective in the biogenesis of the viral secondary siRNAs. A possible role for the viral siRNAs in systemic antiviral RNAi is discussed.
The model organism, Caenorhabditis elegans, has been used in basic research for the last 50 years, providing a wealth of knowledge in a range of fields spanning genetics, development, neurobiology, and computational biology just naming a few. However, only in the last ~15 years has this model organism been broadly considered for investigating host-pathogen interactions. During this time a number of bacterial, fungal, and microsporidial pathogens were shown to infect C. elegans; however, until recently, no natural viruses were known, resulting in a limited understanding of virus-host interactions in C. elegans. In lieu of a known C. elegans virus, several studies were conducted by establishing an unnatural virus infection or creating transgenic viral replicon systems. In 2011, we along with our collaborators reported the discovery of the first known viruses that naturally infect Caenorhabditis nematodes. This dissertation encompasses the identification and characterization of the first viruses known to naturally infect C. elegans and its closest known relative, C. briggsae. Using Next generation 454 pyrosequencing, a novel virus (Orsay virus) was found in a wild isolate of C. elegans (JU1580) and 2 novel viruses (Santeuil and Le Blanc viruses) were found in wild isolates of C. briggsae (JU1264 and JU1498). Phylogenetic analysis revealed the closest known relatives of Orsay, Santeuil, and Le Blanc were viruses in the family Nodaviridae: viruses that naturally infect insects (alphanodaviruses) and fishes (betanodaviruses). Comparative genome analysis revealed substantial differences in genome size and genome organization between the Caenorhabditis viruses and nodaviruses. For instance, the Caenorhabditis viruses have bigger genomes partly due to the presence of a larger 5' untranslated region (UTR) in the RNA1 segment and an additional open reading frame (ORF delta) in the RNA2 segment. While ORF delta has no known function, evidence demonstrates it is translated fused to the capsid protein by a slippery sequence-mediated frameshift. An immunofluorescence assay (IFA) was established targeting viral proteins in infected Caenorhabditis nematodes. Viral protein could be detected as early as 6 hours post-infection (hpi) and the number of infected animals steadily rose between 6 to 12 hpi. In both Orsay virus-infected C. elegans and Santeuil and Le Blanc virus-infected C. briggsae, the site of infection localized primarily to the intestinal tissue. Strikingly, only 1 to 6 cells of the total 20 nonregenerating intestinal cells were positive by IFA for viral protein, suggesting that infection is confined to a subset of the intestine. A transcriptional profile analysis was carried out to investigate which host genes in Caenorhabditis nematodes were responding to viral infection, yielding ~200 genes that were specifically induced by virus in C. elegans. These candidate genes were then individually subjected to RNA interference revealing several characterized and uncharacterized genes that conferred an increased infection phenotype upon knockdown and may play a previously undescribed role in antiviral defense. The discovery and characterization of these novel Caenorhabditis nematode viruses lay the groundwork for the introduction of C. elegans as a powerful model organism to study virus-host interactions.
Viruses and RNAi share an intricate relationship at many levels. RNAi is an important antiviral defense mechanism in plants and invertebrates, microRNAs – of viral or cellular origin – affect many aspects of virus biology, and replication of many, if not all, mammalian viruses can be suppressed by RNAi. Antiviral RNAi: Concepts, Methods, and Applications provides a collection of protocols for the analysis of viral small RNAs and natural antiviral RNAi responses as well as for the development and optimization of RNAi-based antiviral drugs. As RNAi is a central regulatory mechanism in the cell, the methods in this volume can also be applied out of the context of a virus infection. Divided into five convenient parts, this detailed volume reviews important basic concepts in the field of antiviral RNAi, provides experimental and bio-informatic tools for the analysis of small silencing RNAs, covers methods to biochemically dissect RNAi-based antiviral defense and viral counter-defense mechanisms, describes methods for the design, expression, and delivery of therapeutic antiviral siRNAs, and finally presents genome-wide RNAi approaches for the identification of factors involved in virus replication. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Antiviral RNAi: Concepts, Methods, and Applications serves as an ideal guide for both novice and experienced researchers alike striving to dissect the role of RNAi in the viral life cycle or to further boost the development of novel therapeutics and experimental tools based on RNAi technology.
This book summarizes the latest findings on the functions of microRNAs in the regulation of plant development and responses to the surrounding environment. MicroRNAs are an important class of molecules that can be found in diverse groups of organisms, including plants and animals, and the investigation of their roles is a highly dynamic and “hot” research topic. The respective chapters address four main aspects, namely: microRNA investigation and annotation, the regulatory roles of microRNAs in various developmental processes, in response to abiotic factors, and in the context of biotic stress response regulation. Systematically reviewing the most important findings in this field, the book offers an essential guide for undergraduate and graduate students, teachers, and plant science researchers. Due to the potential applications of microRNAs in crop breeding and plant protection, it also represents a valuable resource for scientists in academia and the private sector alike.
Recent progress in high-throughput technologies and genome wide transcriptome studies have lead to a significant scientific milestone of discovering non-coding RNAs (ncRNAs) which spans through a major portion of the genome. These RNAs most often act as riboregulators, and actively participate in the regulation of important cellular functions at the transcriptional and/or post-transcriptional levels rather than simply being an intermediated messenger between DNA and proteins. As the appreciation for the importance of ncRNAs continues to emerge, it is also increasingly clear that these play critical roles in gene regulatory processes during development and differentiation. Further, regulatory RNAs are useful biomarkers for diagnosis of diseases. Hence these RNA regulators are essential to the development of therapeutics. This book on “Regulatory RNAs” offers a comprehensive view on our current understanding of these regulatory RNAs viz. siRNA, miRNA, piRNA, snoRNA, long non-coding RNA, small RNA etc. It addresses both the biogenesis and mechanism of action of regulatory RNAs with a primary focus on their annotation, experimental methodologies (microarray, next-gen sequencing etc.) for their discovery, computational tools for their prediction, and above all, applications of these revolutionary regulatory molecules in understanding biological systems and diseases, including therapeutics. This comprehensive volume is intended for readers with research or teaching interests in ncRNA biology and will provide a major information resource on current research in the fast-moving fields of RNA and gene expression regulation. Cutting-edge and concise, “Regulatory RNAs: Basics, Methods and Applications” promises to support vital research in the field of regulatory RNAs, ever-continuing to grow rapidly and gain increasing importance in basic and translational biology.
In the first edition of Genetics and Molecular Biology, renowned researcher and award-winning teacher Robert Schleif produced a unique and stimulating text that was a notable departure from the standard compendia of facts and observations. Schleif's strategy was to present the underlying fundamental concepts of molecular biology with clear explanations and critical analysis of well-chosen experiments. The result was a concise and practical approach that offered students a real understanding of the subject. This second edition retains that valuable approach--with material thoroughly updated to include an integrated treatment of prokaryotic and eukaryotic molecular biology. Genetics and Molecular Biology is copiously illustrated with two-color line art. Each chapter includes an extensive list of important references to the primary literature, as well as many innovative and thought-provoking problems on material covered in the text or on related topics. These help focus the student's attention of a variety of critical issues. Solutions are provided for half of the problems. Praise for the first edition: "Schleif's Genetics and Molecular Biology... is a remarkable achievement. It is an advanced text, derived from material taught largely to postgraduates, and will probably be thought best suited to budding professionals in molecular genetics. In some ways this would be a pity, because there is also gold here for the rest of us... The lessons here in dealing with the information explosion in biology are that an ounce of rationale is worth a pound of facts and that, for educational value, there is nothing to beat an author writing about stuff he knows from theinside."--Nature. "Schleif presents a quantitative, chemically rigorous approach to analyzing problems in molecular biology. The text is unique and clearly superior to any currently available."--R.L. Bernstein, San Francisco State University. "The greatest strength is the author's ability to challenge the student to become involved and get below the surface."--Clifford Brunk, UCLA
The diversity of RNAs inside living cells is amazing. We have known of the more “classic” RNA species: mRNA, tRNA, rRNA, snRNA and snoRNA for some time now, but in a steady stream new types of molecules are being described as it is becoming clear that most of the genomic information of cells ends up in RNA. To deal with the enormous load of resulting RNA processing and degradation reactions, cells need adequate and efficient molecular machines. The RNA exosome is arising as a major facilitator to this effect. Structural and functional data gathered over the last decade have illustrated the biochemical importance of this multimeric complex and its many co-factors, revealing its enormous regulatory power. By gathering some of the most prominent researchers in the exosome field, it is the aim of this volume to introduce this fascinating protein complex as well as to give a timely and rich account of its many functions. The exosome was discovered more than a decade ago by Phil Mitchell and David Tollervey by its ability to trim the 3’end of yeast, S. cerevisiae, 5. 8S rRNA. In a historic account they laid out the events surrounding this identification and the subsequent birth of the research field. In the chapter by Kurt Januszyk and Christopher Lima the structural organization of eukaryotic exosomes and their evolutionary counterparts in bacteria and archaea are discussed in large part through presentation of structures.
The first of its kind, this laboratory handbook emphasizes diverse methods and technologies needed to investigate C. elegans, both as an integrated organism and as a model system for research inquiries in cell, developmental, and molecular biology, as well as in genetics and pharmacology. Four primary sections--Genetic and Culture Methods, Neurobiology, Cell and Molecular Biology, and Genomics and Informatics--reflect the cross-disciplinary nature of C. elegans research. Because C. elegans is a simple and malleable organism with a small genome and few cell types, it provides an elegant demonstr.
Now in four convenient volumes, Field’s Virology remains the most authoritative reference in this fast-changing field, providing definitive coverage of virology, including virus biology as well as replication and medical aspects of specific virus families.