Browsing by Subject "RNA Interference"
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Item The Biogenesis of Small Interfering RNA in Neurospora Crassa(2013-01-16) Chang, Shwu-Shin; Yi Liu, Ph.D.RNA interference is a well-conserved gene silencing mechanism in eukaryotes. It regulates various biological processes including development, genome defense and heterochromatin formation. RNAi is initiated by the production of dsRNA, which is processed by Dicer to produce small interfering RNA (siRNA). In the filamentous fungus, Neurospora crassa, two types of siRNA have been characterized. One is involved in transgene-induced silencing, termed quelling; the other type is induced by DNA damage and functions to slow down protein translation after DNA damage. Both of these siRNAs originate from repetitive sequences in the Neurospora genome. We show that the components of the homologous recombination (HR) machinery are required to generate these types of small RNA specifically at repetitive regions. Furthermore, chromatin remodeling and DNA replication enzymes are required for efficient HR activity and small RNA production. Lastly, we show that the two small RNA pathways are mechanistically similar by demonstrating that quelling-induced siRNA can also be induced upon DNA damage. Our results suggest that the small RNA biogenesis machinery is recruited specifically to the repetitive loci after homologous recombination, which may result in the formation of aberrant DNA structures. dsRNA not only triggers the RNAi pathway, but also initiates a signaling cascade that results in activating the transcription of ~60 genes, including the RNAi components, in Neurospora. The function of the dsRNA activated genes suggests that RNAi is part of a broad ancient host defense response against viral and transposon infection. A genetic screen has been designed to identify the components involved in this dsRNA triggered transcriptional response; several mutants have been identified and characterized.Item Host Modulagtors of the Death Response to Influenza a Infection(2012-07-17) Ward, Samuel Enoch; White, Michael A.Influenza A virus infects 5-20% of the population annually, resulting in ~35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here I employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human mucosal epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to decoding vulnerabilities in the command and control networks specified by influenza virulence factors. [Keywords: Influenza A, genomewide screen, RNAi, innate immunity, CHEK1, IFITM3, virus]Item The Mechanism of Double-Stranded RNA Response in Neurospora(2009-01-09) Choudhary, Swati; Liu, YiIn eukaryotic cells, recognition of double-stranded RNA (dsRNA) by the enzyme Dicer initiates the RNA interference (RNAi) pathway, resulting in post-transcriptional gene silencing. Argonaute proteins play a critical role in this conserved pathway, which is present in protists, fungi, plants and animals. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that the production of dsRNA triggers the transcriptional induction of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for efficient gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 post-transcriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. A genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes (DRAGs) in Neurospora. Our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections. In order to understand the signaling mechanisms underlying this dsRNA response, we undertook a study of the dsRNA response elements (dsREs) in the promoter regions of qde-2 and other DRAGs. We demonstrate that different regions of the qde-2 promoter orchestrate early and late transcriptional induction in response to dsRNA. In the qde-2 promoter, a GC-rich element and downstream CAAT repeats were found to be important for the early response. In addition, the GC-rich dsRE was found in the promoters of other DRAGs, and was sufficient for dsRNA-induced transcriptional response. These results suggest that these DRAGs share the transcriptional induction pathway triggered by dsRNA. Finally, we demonstrate that QDE-2 contains an additional 10KDa N-terminal RGG domain, which is important for binding small interfering RNAs (siRNAs) and therefore required for its stability as well as efficient RNAi.Item The Mechanism of RNA Interference in Neurospora(2007-08-08) Maiti, Mekhala; Liu, YiIn the canonical RNA interference (RNAi) pathway, small-interfering RNA (siRNA) duplexes generated by Dicer are incorporated into the RNA-induced-silencing complex (RISC), and subsequently converted to single-stranded siRNA. Generation of single stranded siRNA is a pre-requisite for recognition and cleavage of the target mRNA by Argonaute. In biochemical experiments, Argonaute generates single-stranded siRNA by cleaving the passenger strand of the siRNA duplex. Mutational analysis of Neurospora homologue of Argonaute-2, known as Quelling Deficient -2 (QDE-2), revealed that the endonuclease activity of QDE-2 is required for the generation of singlestranded siRNA in vivo. Further biochemical studies to understand the mechanism for removal of the nicked passenger strand from siRNA duplex, led to the identification of a novel QDE-2 interacting protein (QIP) with a putative exonuclease domain. Disruption of qip led to the impairment of RNAi and most of the siRNAs were accumulated in nickedduplex form. Furthermore, QIP functions as an exonuclease to remove the cleaved passenger strand in a QDE-2 dependent manner. Thus, the cleavage of the passenger strand by QDE2 and its subsequent removal by QIP are critical biochemical steps in Neurospora RNAi pathway. Quelling, an RNAi related phenomenon in Neurospora, is induced by multiple copies of transgene. It was proposed that QDE-1 (a RNA dependent RNA polymerase, RdRp) and QDE-3 (a RecQ helicase) functions in quelling pathway by generating double-stranded RNA (dsRNA) from transgenes. To further understand the importance of QDE-1 and QDE-3, quelling assays were performed in the qde-1ko and qde-3ko strains. In contrast to previous results, the requirement of QDE-1 and QDE-3 was bypassed when the transgene copy number was high. Moreover, gene silencing analyses using strains lacking all potential RdRps suggested that unlike in C.elegans and Arabidopsis, the amplification of secondary dsRNA or siRNA is largely absent in Neurospora. The search for potential regulatory mechanisms of RNAi components in Neurospora led to the identification of a dsRNA response pathway. Two key components of the Neurospora RNAi pathway, qde2 and dicer like protein-2 (dcl-2), are induced by dsRNA at transcriptional and posttranscriptional level. The induction of QDE-2 is required for efficient gene silencing, indicating the importance of this regulatory mechanism in RNAi pathway.Item Small RNAs Regulate Transcription by Interacting with Noncoding RNA Transcripts(2010-05-14) Schwartz, Jacob C.; Corey, DavidGeneral methods for controlling gene expression have long been appreciated as an attractive target in drug design. Recently, the Corey lab has demonstrated that short RNA duplexes designed to target the promoter region for human genes can inhibit or activate gene expression in a sequence dependent manner. The mechanism by which RNAs achieve promoter recognition has remained unclear. Sequence specific recognition could be achieved by (1) RNA hybridization to genomic DNA, or (2) RNA recognition of some uncharacterized RNA species. Promoter targeted duplex RNA has been shown to recruit argonaute proteins to the promoter DNA and these proteins are necessary for duplex RNAs to regulate transcription. Argonaute proteins are known to recognize RNA:RNA interactions. However, genes targeted with duplex RNAs have no characterized transcripts in their promoters. I tested the hypothesis that promoter RNA transcripts exist and serve as a substrate for short duplex RNAs to hybridize to and regulate gene expression of adjacent genes. I found previously undiscovered RNA transcripts expressed from the promoter of progesterone receptor (PR) using RT-PCR. Quantitative RT-PCR of the promoter RNA of PR reveals expression levels between 10 and 1000 fold lower than PR in T47D and MCF7 breast cancer cells. I have cloned three transcripts overlapping the promoter of PR from two cell lines – T47D and MCF7, each with unique splicing and transcription start sites. All of these transcripts initiate within the protein coding region of PR and run antisense to the gene PR. I have been able to show that the promoter transcripts can be immunoprecipitated with antibodies against the argonaute proteins in cells transfected with duplex RNAs targeting the promoter of PR but not in cells transfected with mismatched duplex RNAs. Also, biotinylated RNAs bind to and pull down these noncoding RNAs. Finally, knockdown of the antisense transcript with an antisense oligonucleotide prevent gene activation by duplex RNAs. Following this study, our lab uncovered that duplex RNAs can target beyond the 3' terminus of genes and silence or activate transcription. I further showed that this transcription regulation is mediated by argonaute binding to noncoding RNAs overlapping the 3' terminus of the genes, PR and BRCA1. The signal is transmitted from the 3' terminus to the gene promoter because the 5' and 3' ends of these genes are held in a chromatin loop, which I validated using a chromatin conformation capture assay. This brings the ends of the gene in close proximity to each other. Due to this interaction, short RNAs that bind a noncoding RNA at the 3' end of the gene also physically interacts with noncoding RNAs that associate with the gene promoter. This is confirmed by RNA immunoprecipitation of both transcripts with duplex RNAs targeting either the 5' or 3' ends of the gene. More than 20 years ago, it was found that proteins recognizing DNA at the 5’ end of genes could regulate transcription. This study presents a paradigm shift implicating noncoding RNAs at the 5' and 3' ends of genes can be recognized by proteins which activate or inhibit transcription of adjacent protein coding genes. Recent studies demonstrate an abundance of RNAs transcribed in human cells that do not code for protein. My results suggest a new model for duplex RNA recognition of gene promoters. Argonaute proteins loaded with one strand of the RNA duplex recognizes, through Watson-Crick base pairing, a noncoding transcript that is associated with chromatin at the promoter of the targeted gene. This RNA:RNA interaction in close proximity to the promoter mediates protein-protein interactions between argonaute and other factors on the promoter to turn off or on gene expression.