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Single-Molecule Imaging Reveals That Argonaute Re-Shapes The Properties Of Its Nucleic Acid Guides: A Dissertation, William E. Salomon Dec 2015

Single-Molecule Imaging Reveals That Argonaute Re-Shapes The Properties Of Its Nucleic Acid Guides: A Dissertation, William E. Salomon

GSBS Dissertations and Theses

Small RNA silencing pathways regulate development, viral defense, and genomic integrity in all kingdoms of life. An Argonaute (Ago) protein, guided by a tightly bound, small RNA or DNA, lies at the core of these pathways. Argonaute uses its small RNA or DNA to find its target sequences, which it either cleaves or stably binds, acting as a binding scaffold for other proteins. We used Co-localization Single-Molecule Spectroscopy (CoSMoS) to analyze target binding and cleavage by Ago and its guide. We find that both eukaryotic and prokaryotic Argonaute proteins re-shape the fundamental properties of RNA:RNA, RNA:DNA, and DNA ...


Rna Interference By The Numbers: Explaining Biology Through Enzymology: A Dissertation, Liang Meng Wee Jun 2013

Rna Interference By The Numbers: Explaining Biology Through Enzymology: A Dissertation, Liang Meng Wee

GSBS Dissertations and Theses

Small silencing RNAs function in almost every aspect of cellular biology. Argonaute proteins bind small RNA and execute gene silencing. The number of Argonaute paralogs range from 5 in Drosophila melanogaster , 8 in Homo sapiens to an astounding 27 in Caenorhabditis elegans. This begs several questions: Do Argonaute proteins have different small RNA repertoires? Do Argonaute proteins behave differently? And if so, how are they functionally and mechanistically distinct?

To address these questions, we examined the thermodynamic, kinetic and functional properties of fly Argonaute1 (dAgo1), fly Argonaute2 (dAgo2) and mouse Argonaute2 (mAGO2). Our studies reveal that in fly, small RNA ...


The Study Of Two Strategies For Decreasing Mutant Huntingtin: Degradation By Puromycin Sensitive Aminopeptidase And Rna Interference: A Dissertation, Joanna Chaurette May 2013

The Study Of Two Strategies For Decreasing Mutant Huntingtin: Degradation By Puromycin Sensitive Aminopeptidase And Rna Interference: A Dissertation, Joanna Chaurette

GSBS Dissertations and Theses

Huntington’s disease (HD) is a fatal neurodegenerative disease caused by a CAG repeat expansion in exon 1 of the huntingtin gene, resulting in an expanded polyglutamine (polyQ) repeat in the huntingtin protein. Patients receive symptomatic treatment for motor, emotional, and cognitive impairments; however, there is no treatment to slow the progression of the disease, with death occurring 15-20 years after diagnosis. Mutant huntingtin protein interferes with multiple cellular processes leading to cellular dysfunction and neuronal loss. Due to the complexity of mutant huntingtin toxicity, many approaches to treating each effect are being investigated. Unfortunately, addressing one cause of toxicity ...


Therapeutic Silencing Of Mutant Huntingtin By Targeting Single Nucleotide Polymorphisms: A Dissertation, Edith L. Pfister Jul 2012

Therapeutic Silencing Of Mutant Huntingtin By Targeting Single Nucleotide Polymorphisms: A Dissertation, Edith L. Pfister

GSBS Dissertations and Theses

Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder. Invariably fatal, HD is caused by expansion of the CAG repeat region in exon 1 of the Huntingtin gene which creates a toxic protein with an extended polyglutamine tract 1. Silencing mutant Huntingtin messenger RNA (mRNA) is a promising therapeutic approach 2-6. The ideal silencing strategy would reduce mutant Huntingtin while leaving the wild-type mRNA intact. Unfortunately, targeting the disease causing CAG repeat expansion is difficult and risks targeting other CAG repeat containing genes.

We examined an alternative strategy, targeting single nucleotide polymorphisms (SNPs) in the Huntingtin mRNA. The ...


Cooperativity In Mammalian Rna Silencing: A Dissertation, Jennifer A. Broderick Jul 2011

Cooperativity In Mammalian Rna Silencing: A Dissertation, Jennifer A. Broderick

GSBS Dissertations and Theses

Argonaute proteins are the core component of an RNA silencing complex. The human genome encodes four Argonaute paralogs –Ago1, Ago2, Ago3 and Ago4– proteins that are guided to target mRNAs by microRNAs. More than 500 miRNAs are conserved between mammals, and each microRNA can repress hundreds of genes, regulating almost every cellular process. We still do not fully understand the molecular mechanisms by which miRNAs regulate gene expression. Although we understand many aspects of microRNA biogenesis and formation of the RNA-induced silencing complex, much less is known about the subsequent steps leading to target mRNA regulation.

Mammalian microRNAs rarely have ...


Roles Of Cellular Rna-Dependent Rna Polymerases In Endogenous Small Rna Pathways In Caenorhabditis Elegans: A Dissertation, Jessica J. Vasale Jun 2010

Roles Of Cellular Rna-Dependent Rna Polymerases In Endogenous Small Rna Pathways In Caenorhabditis Elegans: A Dissertation, Jessica J. Vasale

GSBS Dissertations and Theses

The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact ...


The Molecular Mechanisms Underlying The Polarized Distribution Of Drosophila Dscam In Neurons: A Dissertation, Shun-Jen Yang Oct 2008

The Molecular Mechanisms Underlying The Polarized Distribution Of Drosophila Dscam In Neurons: A Dissertation, Shun-Jen Yang

GSBS Dissertations and Theses

Neurons exhibit highly polarized structures, including two morphologically and functionally distinct domains, axons and dendrites. Dendrites and axons receive versus send information, and proper execution of each requires different sets of molecules. Differential distribution of membrane proteins in distinct neuronal compartments plays essential roles in neuronal functions. The major goal of my doctoral thesis was to study the molecular mechanisms that govern the differential distribution of membrane proteins in neurons, using the Drosophilalarval mushroom body (MB) as a model system.

My work was initiated by an observation of differential distribution of distinct Dscam isoforms in neurons. Dscam stands for ...


Small Rna Sorting In Drosophila Produces Chemically Distinct Functional Rna-Protein Complexes: A Dissertation, Michael D. Horwich Jun 2008

Small Rna Sorting In Drosophila Produces Chemically Distinct Functional Rna-Protein Complexes: A Dissertation, Michael D. Horwich

GSBS Dissertations and Theses

Small interfering RNAs (siRNAs), microRNAs (miRNAs), and piRNAs (piRNA) are conserved classes of small single-stranded ~21-30 nucleotide (nt) RNA guides that repress eukaryotic gene expression using distinct RNA Induced Silencing Complexes (RISCs). At its core, RISC is composed of a single-stranded small RNA guide bound to a member of the Argonaute protein family, which together bind and repress complementary target RNA. miRNAs target protein coding mRNAs—a function essential for normal development and broadly involved in pathways of human disease; small interfering RNAs (siRNA) defend against viruses, but can also be engineered to direct experimental or therapeutic gene silencing; piwi ...


Dissecting Small Rna Loading Pathway In Drosophila Melanogaster: A Dissertation, Tingting Du Jan 2008

Dissecting Small Rna Loading Pathway In Drosophila Melanogaster: A Dissertation, Tingting Du

GSBS Dissertations and Theses

In the preceding chapters, I have discussed my doctoral research on studying the siRNA loading pathway in Drosophila using both biochemical and genetic approaches. We established a gel shift system to identify the intermediate complexes formed during siRNA loading. We detected at least three complexes, named complex B, RISC loading complex (RLC) and RISC. Using kinetic modeling, we determined that the siRNA enters complex B and RLC early during assembly when it remains double-stranded, and then matures in RISC to generate Argonaute bearing only the single-stranded guide. We further characterized the three complexes. We showed that complex B comprises Dcr-1 ...


Understanding Assembly Of Ago2 Risc: The Rnai Enzyme: A Dissertation, Christian B. Matranga Sep 2007

Understanding Assembly Of Ago2 Risc: The Rnai Enzyme: A Dissertation, Christian B. Matranga

GSBS Dissertations and Theses

In 1990, Richard Jorgensen’s lab initiated a study to test if they could create a more vivid color petunia (Napoli et al. 1990). Their plan was to transform plants with the chalcone synthase transgene––the predicted rate limiting factor in the production of purple pigmentation. Much to their surprise, the transgenic plants, as well as their progeny, displayed a great reduction in pigmentation. This loss of endogenous function was termed “cosuppression” and it was thought that sequence-specific repression resulted from over-expression of the homologous transgene sequence. In 1998, Andrew Fire and Craig Mello described a phenomenon in which double ...


Interaction Of A Mammalian Virus With Host Rna Silencing Pathways: A Dissertation, Bradford Michael Stadler Mar 2007

Interaction Of A Mammalian Virus With Host Rna Silencing Pathways: A Dissertation, Bradford Michael Stadler

GSBS Dissertations and Theses

In the complex relationships of mammalian viruses with their hosts, it is currently unclear as to what role RNA silencing pathways play during the course of infection. RNA silencing-based immunity is the cornerstone of plant and invertebrate defense against viral pathogens, and examples of host defense mechanisms and numerous viral counterdefense mechanisms exist. Recent studies indicate that RNA silencing might also play an active role in the context of a mammalian virus infection. We show here that a mammalian virus, human adenovirus, interacts with RNA silencing pathways during infection, as the virus produces microRNAs (miRNAs) and regulates the expression of ...


The Argonaute Family Of Genes In Caenorhabditis Elegans: A Dissertation, Erbay Yigit Feb 2007

The Argonaute Family Of Genes In Caenorhabditis Elegans: A Dissertation, Erbay Yigit

GSBS Dissertations and Theses

Members of the Argonaute family of proteins, which interact with small RNAs, are the key players of RNAi and other related pathways. The C. elegans genome encodes 27 members of the Argonaute family. During this thesis research, we sought to understand the functions of the members of this gene family in C. elegans. Among the Argonaute family members, rde-1 and alg-1/2have previously been shown to be essential for RNAi and development, respectively. In this work, we wanted to assign functions to the remaining members of this large family of proteins.

Here, we describe the phenotype of 31 deletion ...


Rna Silencing Pathways In Schizosaccharomyces Pombe And Drosophila Melanogaster: A Dissertation, Alla A. Sigova Nov 2006

Rna Silencing Pathways In Schizosaccharomyces Pombe And Drosophila Melanogaster: A Dissertation, Alla A. Sigova

GSBS Dissertations and Theses

RNA silencing is an evolutionary conserved sequence-specific mechanism of regulation of gene expression. RNA interference (RNAi), a type of RNA silencing in animals, is based on recognition and endonucleolytic cleavage of target mRNA complimentary in sequence to 21-nucleotide (nt) small RNA guides, called small interfering RNAs (siRNAs). Another class of 21-nt small RNAs, called micro RNAs (miRNAs), is endogenously encoded in eukaryotic genomes. Both production of siRNAs from long double-stranded RNA (dsRNA) and biogenesis of miRNAs from hairpin structures are governed by the ribonuclease III enzyme Dicer. Although produced as duplex molecules, siRNAs and miRNAs are assembled into effector complex ...


Identification Of Novel (RNai DeFicient) Genes In C. Elegans: A Dissertation, Chun-Chieh G. Chen Sep 2006

Identification Of Novel (RNai DeFicient) Genes In C. Elegans: A Dissertation, Chun-Chieh G. Chen

GSBS Dissertations and Theses

RNA interference or RNAi was first discovered as an experimental approach that induces potent sequence-specific gene silencing. Remarkably, subsequent studies on dissecting the molecular mechanism of the RNAi pathway reveal that RNAi is conserved in most eukaryotes. In addition, genes and mechanisms related to RNAi are employed to elicit the regulation of endogenous gene expression that controls a variety of important biological processes. To investigate the mechanism of RNAi in the nematode C. elegans, we performed genetic screens in search of RNAi deficient mutants (rde). Here I report the summary of the genetic screens in search of rde ...


Biochemical Mechanism Of Rna Interference In Higher Organisms: A Dissertation, Dianne S. Schwarz Aug 2005

Biochemical Mechanism Of Rna Interference In Higher Organisms: A Dissertation, Dianne S. Schwarz

GSBS Dissertations and Theses

RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene silencing pathway found in eukaryotes, in which 21-nucleotide, small interfering RNAs (siRNAs) guide destruction of a corresponding target mRNA. RNAi is a natural mechanism for both genome surveillance and gene regulation. Moreover, siRNAs can be transfected into cultured mammalian cells, causing the sequence-specific ‘knock down’ of an mRNA. My work in the Zamore lab has centered around the Drosophilain vitro system and cultured mammalian cells to study the RNA interference (RNAi) pathway. small interfering RNAs (siRNAs) are incorporated into the RNA-induced silencing complex (RISC), which culminates in the cleavage of ...


A Biochemical Dissection Of The Rna Interference Pathway In Drosophila Melanogaster: A Dissertation, Benjamin Haley Aug 2005

A Biochemical Dissection Of The Rna Interference Pathway In Drosophila Melanogaster: A Dissertation, Benjamin Haley

GSBS Dissertations and Theses

In diverse eukaryotic organisms, double-stranded RNA (dsRNA) induces robust silencing of cellular RNA cognate to either strand of the input dsRNA; a phenomenon now known as RNA interference (RNAi). Within the RNAi pathway, small, 21 nucleotide (nt) duplexed RNA, dubbed small interfering RNAs (siRNAs), derived from the longer input dsRNA, guide the RNA induced silencing complex (RISC) to destroy its target RNA. Due to its ability to silence virtually any gene, whether endogenous or exogenous, in a variety of model organisms and systems, RNAi has become a valuable laboratory tool, and is even being heralded as a potential therapy for ...


Hiv-1 Gene Expression: Transcriptional Regulation And Rna Interference Studies: A Dissertation, Ya-Lin Chiu Jan 2003

Hiv-1 Gene Expression: Transcriptional Regulation And Rna Interference Studies: A Dissertation, Ya-Lin Chiu

GSBS Dissertations and Theses

Gene expression of human immunodeficiency virus type-1 (HIV-1), which causes Acquired Immunodeficiency Syndrome (AIDS), is regulated at the transcriptional level, where negative factors can block elongation that is overcome by HIV Tat protein and P-TEFb. P-TEFb, a positive elongation transcription factor with two subunits, CDK9 and Cyclin T1 (CycT1), catalyzes Tat-dependent phosphorylation of Ser-5 in the Pol II C-terminal domain (CTD), allowing production of longer mRNAs. Ser-5 phosphorylation enables the CTD to recruit mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. This dissertation demonstrates that stable binding of Mce1 and cap methyltransferase to template-engaged Pol II depends on ...


Analysis Of Rna Interference In C. Elegans: A Dissertation, Alla Grishok Sep 2001

Analysis Of Rna Interference In C. Elegans: A Dissertation, Alla Grishok

GSBS Dissertations and Theses

RNA interference (RNAi) in the nematode Caenorhabditis elegans is a type of homology-dependent post-transcriptional gene silencing induced by dsRNA. This dissertation describes the genetic analysis of the RNA interference pathway and inheritance properties associated with this phenomenon. We demonstrate that the RNAi effect can be observed in the progeny of the injected animal for at least two generations. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-l and rde-4 are required for the formation of this interfering agent but are not needed for interference thereafter. In contrast, the rde-2 ...