Browsing by Subject "RNA, Small Interfering"
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Item The Function and Mechanism of RNA Interference in Neurospora(2009-01-14) Lee, Heng-Chi; Liu, YiRNA interference (RNAi) is a conserved gene silencing mechanism important for various biological processes, including developmental timing, genome defense, and heterochromatin formation. RNAi is triggered by double stranded RNA (dsRNA), which is processed by Dicer to siRNA. siRNA is loaded onto RNA-induced silencing complex (RISC), in which an Argonaute family protein, guided by a siRNA, mediates the cleavage of homologous RNAs. In the filamentous fungus Neurospora, we show that dsRNA not only trigger RNAi, it also transcriptionally activates several key components of RNAi pathway, including qde-2 (an Argonaute) and dcl-2 (a Dicer). A genome wide identification of dsRNA activated genes suggests that RNAi is part of a broad ancient host-defense response against viral and transposon infections. Our research on qde-2 regulation also suggests a role of RNAi during DNA damage. We show that DNA damage induces qde-2 expression, and the purification of QDE-2 bound RNAs identifies a novel class of small RNAs named qiRNAs. qiRNAs are averaged 21 nt in length and are mostly derived from ribosomal DNA (rDNA) locus. Importantly, qiRNA biogenesis requires RNAi components and RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs during DNA repair. Further analysis suggests that the qiRNA contributes to the DNA damage checkpoints by inhibiting protein translation after DNA damage. To trigger RNAi against transgenes, it has been proposed that transgene- specific aberrant RNA (aRNA) is made and converted into dsRNA by RNA dependent RNA polymerase (RdRP). How aRNA is produced and specifically recognized by RdRP is not known. We show that QDE-1, a RdRP is also the DNA-dependent RNA polymerase (DdRP) that produces aRNA from ssDNA. QDE-1 is recruited to ssDNA by Replication Protein A (RPA) and QDE-3 (an RecQ helicase), both of them are also essential for aRNA production. Moreover, QDE-1 can produce dsRNA from ssDNA, a process facilitated by RPA. Our results provide a molecular mechanism of aRNA production in RNAi pathway.Item Human shRNA Library Screening to Dissect Pathways Involved In Telomerase Actions(2011-12-12) Hoshiyama, Hirotoshi; Shay, Jerry W.The minimal components of human telomerase are the human telomerase reverse transcriptase (hTERT) and the human telomerase template RNA (hTR). Although it is known that both components are minimally sufficient to reconstitute telomerase activity, the factors involved in any of the multiple steps of telomerase action such as telomerase assembly, telomerase recruitment to telomeres, and telomere extension/regulation are not well understood. There are a large numbers of proteins that have associations with telomerase, yet the functional roles of those in telomere maintenance and telomerase regulation are not well understood. Identifying novel proteins and pathways involved in any of these important telomerase-associated functions will be useful for identifying new targets for the development of novel inhibitors that block telomerase function in cancer cells. Therefore, my goal has been to develop methods to dissect these molecular pathways and identify functional factors involved in any step of telomerase actions. To accomplish this, I designed a selective screening system by exploiting lentiviral shRNA libraries and tetracycline inducible-hTERT cell lines that is hTR deficient but expressing mutant hTR. Thus, the overall strategy of the screening system may be considered a “synthetic rescue screen”. In brief this screen was set up to rescue cells from apoptotic death due to mutant sequence incorporation at telomeres by reducing the gene expressions with lentiviral shRNA libraries. This allows us to look a set of genes involved in pathways involved in functional aspects of telomerase actions, not based on structural association with telomerase. During the work, I have established multiple lines of inducible-hTERT cells to use in selective screening systems. I have also developed a method for rapid construction of high-complexity custom shRNA libraries for targeted screening and re-evaluate hundreds of primary candidate genes to identify smaller numbers of secondary candidate genes by removing false positives. In order to analyze the pooled shRNA screening result, I have developed a method for quantitative identification of half-hairpins from a pooled shRNA library based on the pGIPZ vector. Introducing multiplexing codes and refining sample preparation schemes resulted in the predicted ability to detect two-fold enrichments followed by massive parallel sequencing. Development of those methods allowed me to identify several candidate proteins, which may be involved in telomerase actions.Item To develop a small interfering Rna (siRNA) design and information resource to facilitate genetic manipulaton of human cells.(2004-05-25) Shah, Jyoti Khetsi; Minna, John D.Part I: Small interfering RNAs (siRNAs) have revolutionized our ability to study the effects of altering the expression of single genes in mammalian (and other) cells through targeted knockdown of gene expression. In the past, there were a set of rules designed to develop siRNA which worked efficiently in most cases. There was further refinement performed in these rules in some modern research analyses which attempted to address the question of what most closely determines siRNA functionality. I have designed and implemented a new software tool siRNA Information Resource ('sIR') that incorporates the most recent refinements in the design algorithm in order to provide fast and efficient siRNA design. sIR is a web-based computational tool which takes these existing rules for designing synthetic siRNAs and puts them in a software architecture that allows the researcher to design siRNAs for every gene. It also provides a database containing information about already developed siRNA and thus allows the researcher to access the siRNA information database consisting of siRNA information from literature and various other sources. This will ultimately help in future siRNA related discoveries. It also includes a scoring system which helps in rational selection of efficient siRNA. sIR was successfully validated using already designed and developed target siRNA sequences. Part II: One of the major problems in using chemotherapy to treat cancer is whether patients, whose tumors do not respond to one drug, would respond to another. Thus, it would be very useful if one could rationally select the appropriate chemotherapy for each patient's tumor. We are asking is whether tumor gene "expression signatures" detected by microarray analysis could identify a set of genes correlating with sensitivity or resistance to a particular drug. A large panel of breast cancer cell lines was tested with cisplatin, paclitaxel, vinorelbine, doxorubicin and gemcitabine, in vitro using a colorimetric assay to determine the concentration of drug that gives 50% growth inhibition (IC50). Gene expression profiles were also performed using Affymetrix chips and the two data sets were merged. It was found that a panel of ~100 genes were significantly up regulated (4 fold or more) for each drug in resistant cells. As an alternative approach, Pearson correlations between each gene expression data and each drug IC50 across all cell lines analyzed were determined. A positive correlation for a pair of gene and drug indicates the gene may be associated with resistance to the drug whereas a negative correlation would associate that gene with sensitivity to the drug. Some of these genes might be associated with the drug mechanism of action. We conclude that gene expression signatures do exist for individual breast tumor cell chemosensitivity and these could be of clinical significance.