WorldCat Identities

Fire, Andrew Zachary

Overview
Works: 26 works in 26 publications in 1 language and 28 library holdings
Roles: Thesis advisor
Publication Timeline
Key
Publications about  Andrew Zachary Fire Publications about Andrew Zachary Fire
Publications by  Andrew Zachary Fire Publications by Andrew Zachary Fire
Most widely held works by Andrew Zachary Fire
A role for the let-7 primary microRNA in target gene recognition and repression by Robin Deis Trujillo ( )
1 edition published in 2010 in English and held by 2 WorldCat member libraries worldwide
MicroRNA (miRNA) genes produce three noncoding RNA products: the long primary transcript (pri-miRNA), the ~70 nucleotide pre-miRNA, and the ~22-nt mature miRNA. Only the mature miRNA is considered to be the functional species of a miRNA gene in recognizing cognate target mRNAs and modulating their expression. However, mature miRNAs are processed from the primary transcript through sequential endonucleolytic steps. As a result, the mature miRNA sequence is present in all three RNA products of a miRNA gene. It has thus been intrinsically difficult to determine the contribution of each miRNA gene product to target repression. In fact, direct functional roles for pri- and pre-miRNAs have never been ruled out. Here we show that pri- and pre-miRNAs may not be mere transitory intermediates of mature miRNA biogenesis. We found that ectopic expression of the C. elegans miRNA gene let-7 (cel-let-7) in human culture cells results in the production of truncated pre- and mature miRNAs that lack the first two 5' nucleotides, one of which is the first nucleotide of the miRNA seed region (nucleotide SD1). We found this nucleotide to be required for repression of target reporters by cel-let-7 in these cells, demonstrating that pri-let-7 may have a direct role in target repression. Further, we show that the nucleotide sequence and structure of both the pri-/pre-let-7 loop and stem regions play a key role in miRNA gene function in reporter assays. In vitro and in vivo analyses indicated the significance of these regions may be in the mediation of a physical interaction between pri-let-7 and target RNAs. These observations suggest that regulatory information encoded in the structured pri-miRNAs, but absent from mature miRNAs, could be directly interpreted for target recognition and repression through RNA:RNA interaction. Intriguingly, some mutations in the loop nucleotide sequence also restored processing of the 5' ends of C. elegans pre- and mature let-7 in culture cells, demonstrating that the pri-/pre-miRNA loop region can also regulate the precision of mature miRNA biogenesis. Importantly, in the presence of functional pre- and mature let-7, cel-let-7 activity in target repression consists of both SD1-independent and SD1-dependent components, implying potential contributions by both pri- and mature let-7. Finally, we interrogated the effects of pri-/pre-let-7 loop mutations on their ability to rescue a let-7 loss-of-function mutant phenotype in C. elegans. Our results indicate decreased significance of these parameters in the control of worm vulval development, although context-dependent differences in mature miRNA biogenesis between heterologous culture and live animals may partially explain this discrepancy. Taken together the work presented here reveals a novel layer of regulatory complexity encoded in long primary miRNAs that may have broad implications in understanding the mechanisms by which miRNA genes control target expression
Systematic identification of mirna targets and the steps in gene expression regulated by mirnas by David Gillis Hendrickson ( )
1 edition published in 2009 in English and held by 2 WorldCat member libraries worldwide
In the last decade, RNA interference (RNAi), the process by which small RNAs direct the post-transcriptional silencing of cognate mRNA transcripts has revolutionized longstanding paradigms about RNA function. In addition, researchers have harnessed this pathway for experimentally induced gene silencing in what is arguably one of the most important technological advances in modern biology. That disruption of the RNAi pathway results in aberrant development, cancer, and embryonic death suggesting that RNAi is an integral component of eukaryotic gene expression programming. Although RNAi is a vast and diverse pathway with marked distinctions between species, the basic organization has been established largely from work carried out in worms, flies, humans and mice. Double stranded RNA (dsRNA) inputs are "diced" by the class III ribonuclease Dicer into small dsRNA intermediates of ~21-22nt in length which are transferred to the RNA induced silencing complex (RISC) wherein the guide strand is selected and bound to an Argonaute (Ago) family protein. Target mRNAs are then recruited to RISC through Watson-Crick base pairing to the guide strand. Silencing of target transcripts can be directed by Ago mediated cleavage, or through Ago mediated recruitment of factors that induce translational inhibition and mRNA degradation. MicroRNAs (miRNAs) are the most common class of endogenous small silencing RNAs. Despite these advances, many of molecular details of RISC mediated gene silencing are poorly understood as current models are based on only a few miRNA:mRNA target pairs. Here, we present a method for systematic identification of specific miRNA targets. We demonstrate that immuno-affinity purification (IP) of Argonaute proteins is a viable method for isolating RISC associated miRNAs and mRNAs for identification using DNA microarrays. The strong enrichment of mRNAs with binding sites to the experimentally introduced miR-1 and miR-124 in Ago IPs from human embryonic kidney 293T cells (HEK293T) validates the utility of this method. Furthermore, mRNAs classified as targets of miR-1 and miR-124 using this approach behave like bona fide targets in that they exhibit significant down-regulation at the mRNA level. To learn about the steps in gene expression regulated by miRNAs, we simultaneously measured miR-124 mediated changes in Ago enrichment, mRNA abundance, and ribosome occupancy and ribosome density for ~8,000 genes. The translational parameters were used to estimate apparent changes in translational rate and were collected using standard polysome profiling in tandem with DNA microarrays and a novel gradient encoding scheme. We found that for the majority of the miR-124 targets, changes in mRNA concentration and apparent translation rate are concordant and that ~75% of the estimated change in protein levels could be accounted for by changes in mRNA abundance. Our data is most consistent with models of miRNA inhibition of translation initiation. To rule out miRNA mediated repressive mechanisms that would not be visible to our translational profiling (concordant reductions in translational initiation and elongation, co-translational proteolysis) we tested the protein levels for 13 targets by Western blot and found that our estimated changes in protein were nearly identical to the actual changes for 12/13 of the proteins measured. In addition, we observed a large dynamic range for miR-124 mediated down-regulation of mRNA abundance and apparent translation rate, and estimated protein abundance demonstrating the versatility of miRNA mediated regulation. The concordance between miR-124 specific changes in mRNA level and translation supports a model wherein these two regulatory outcomes are functionally linked in a sequential process or regulated by the same cis factors. We have also sought to learn about the RNAi pathway from a Dicer-centric perspective. We generated a library of Dicer truncations to test the contribution of Dicer's conserved protein domains to in vitro dicing reactions to learn about potentially interesting in vivo function as well as for increasing the efficacy of in vitro dicing as a gene silencing tool. We found that the domain of unknown function 283 (DUF283) may be important for proper spacing in dicing reactions and is part of Dicer's "molecular ruler". In addition we found that the ATPase/Helicase domains may inhibit Dicer activity and are dispensable for in vitro dicing, but may play a role in non-canonical substrate recognition
Transcription-factor occupancy at HOT regions quantitatively predicts RNA polymerase recruitment in five human cell lines by Joseph William Foley ( )
1 edition published in 2013 in English and held by 1 WorldCat member library worldwide
The human genome's nucleotide sequence contains a small amount of information relative to the complexity of the organism. The diversity of developmental programs and stimulus responses is instead enabled by a vast regulatory network of chemical modifications and protein interactions that determine the activity level of each genome element dynamically. Recent advances in molecular profiling techniques have led to the creation of large data sets that assay each regulatory interaction across the entire genome at once, with high sensitivity and precision. Here I introduce a new computational method, UniPeak, that synthesizes the output from any number of these experiments into a single straightforward analysis with rigorous quantitative measures. I then apply this method to the largest available data sets of genome regulation experiments, and integrate them with parallel experiments on genome activity. I discover and characterize novel regulatory interactions, and model how these interactions regulate the activity of genes
The rogue transcriptome using sequencing to track RNA regulation and identify unusual RNAs by Di Wu ( )
1 edition published in 2013 in English and held by 1 WorldCat member library worldwide
First, we take a look at the function of a specific type of RNA editing mediated by ADAR (adenosine deaminase that acts on RNAs). We test our hypotheses on ADAR activity and target specificity through transcriptome analysis in the context of exogenous RNAi response pathway in C. elegans. By examining of small RNA and mRNA populations in adar mutants and additional RNAi defective mutants, we show that the activity and specificity of the ADAR enzyme likely precludes the detection of its targets through conventional methods of sequence capture and analysis. Based on this understanding, we develop novel computational approaches for analysis of small RNA and mRNA populations and identify a broader set of ADAR targets, leading to a deeper understanding of the ADAR pathway, the conserved functionalities of the ADAR enzyme, and its relationship with the exogenous RNAi pathway in C. elegans. Second, we examine the dynamics of the transcriptome upon and after exposure to extreme heat. We hypothesize that the recovery response after removal from heat exposure might involve alternations in transcription regulation that are more complex than a reversal of the heat-shock induced effects, and that one such form of long-term regulation might involve the endogenous RNAi pathway and regulation via synthesis of endogenous siRNAs. By examining the dynamics of the C. elegans transcriptome during time-course of heat exposure and recovery, we reach a deeper understanding of heat shock biology and explore the relationship between endogenous siRNAs and their sequence complementary mRNA targets in the context of environmentally induced perturbance
Regulation of meiotic recombination DNA double-strand break formation and repair in C. elegans by Simona Rosu ( )
1 edition published in 2012 in English and held by 1 WorldCat member library worldwide
Sexually reproducing organisms undergo meiotic recombination to increase genetic diversity and to ensure correct segregation of chromosomes at the first meiotic division. During meiotic recombination, DNA double-strand breaks (DSBs) are deliberately induced, and a subset of the breaks are repaired as inter-homolog crossovers (COs). Inter-homolog COs provide physical connections between homologous chromosomes that enable them to segregate away from each other. Despite reliance on COs for chromosome segregation, few COs are made per chromosome pair, implying mechanisms for robust CO control. In addition, DNA breaks are potentially dangerous lesions, therefore DSB formation and repair must be tightly regulated, both to ensure the formation of COs, but also to protect against deleterious effects. This thesis provides new insights into mechanisms that regulate DSB formation and repair, using the nematode Caenorhabditis elegans as a model organism. CO regulation mechanisms were probed by monitoring the repair outcome of a DSB induced at a defined site at different stages of meiotic progression, in WT and mutant situations. This analysis uncovered a previously unappreciated level of control in restricting CO number, which is to limit the duration of access to the homolog as a repair template. In addition, this work showed that when no competing breaks are present, a single induced DSB is converted to a CO with high efficiency. This feature helps achieve CO assurance. Insights into regulation of DSB formation were uncovered from the identification and characterization of novel protein DSB-2. DSB-2 is required for break formation, but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of break formation, suggesting it acts to promote competence for DSB formation. Both approaches presented in this thesis uncovered evidence for negative feedback regulatory mechanisms, suggesting a model wherein formation of CO-eligible recombination intermediates signals shutdown of DSB formation as well as shutdown of inter-homolog access. The proposed regulatory networks provide a mechanism to both ensure sufficient breaks and COs are made, and to shut down these processes to prevent deleterious effects as meiosis progresses
In the right place at the right time understanding basic microrna biology through the control of developmental timing by lin-4 and let-7 in caenorhabditis elegans by Huibin Zhang ( )
1 edition published in 2012 in English and held by 1 WorldCat member library worldwide
MicroRNAs are an important class of short RNA molecules that regulate gene expression in diverse organisms. Despite their short history, extensive research on microRNAs has revealed how microRNAs are made, their regulatory mechanisms and functions in different biological processes. In addition, novel experimental techniques and computational algorithms have been developed to study microRNAs. Our knowledge of microRNAs is constantly evolving as newer studies turn up exceptions to canonical models established by early studies. This highlights how our understanding of microRNAs is far from complete and much work is still needed to investigate unexplored aspects of the microRNA world. The founding microRNAs, C. elegans lin-4 and let-7, have well-characterized heterochronic defects in mutant animals and their target genes have been genetically validated. Combined with powerful experimental tools available in C. elegans, the microRNA function of lin-4 and let-7 in developmental timing of C. elegans is an ideal in vivo model system for testing microRNA-related hypotheses. Using functional assays to evaluate lin-4 and let-7 rescue activities in transgenic animals, we have investigated several poorly understood aspects of microRNAs, from biogenesis to functional mechanism. In this work, we first demonstrated the cell autonomous nature of lin-4 in C. elegans temporal development. Through tissue/cell-specific expression of lin-4 and the use of tissue/cell-specific reporters and microRNA sponges, we have shown that lin-4 rescue activities were limited to sites of lin-4 expression, despite its broad endogenous expression and function in many tissues. Next, we showed that intron-containing microRNAs ("inc-miRs") are functional in C. elegans, raising the possibility that mature microRNAs need not be encoded as contiguous units in the genome. This result also highlights a blindspot in our current novel microRNA discovery methods that assume inc-miRs do not exist. Lastly, we investigated the partnership between microRNA "seed" and "non-seed" sequences in providing microRNA function during C. elegans temporal development. By assessing microRNA function in transgenic mutant animals expressing lin-4 or let-7 mutant microRNA, we found contrasting results between lin-4 and let-7. While the results suggest a functional requirement for lin-4 seed sequence, mutations in either let-7 seed or non-seed sequences provided similar levels of functional activities. Using C. elegans lin-4 and let-7 as an experimental model, this work has furthered our understanding on microRNA autonomy, microRNA biogenesis and functional partnership between microRNA seed and non-seed sequence
Bayesian assembly of reads from high throughput sequencing by Jonathan Daniel Laserson ( )
1 edition published in 2012 in English and held by 1 WorldCat member library worldwide
The high-throughput sequencing revolution allows us to take millions of noisy short reads from the DNA in a sample, essentially taking a snapshot of the genomic material in the sample. To recover the true genomes, these reads are assembled by algorithms exploiting their high coverage and overlap. I focus on two scenarios for sequence assembly. The first is de novo assembly, where the reads come from an unknown and diverse population of genomes. The second is variant assembly, where the reads come from short but clonally related genomes, only slightly mutated from each other. In both cases I use the same principled Bayesian approach to design an algorithm that uncovers the composition of the genomic sequences that produced the reads. I will demonstrate the algorithms' performance on real data taken from various metagenomic environments, as well as the immune system B cells. On that latter dataset, collected from 10 organ donors each providing 4 tissue samples, the results show evidence of clone migration between tissues and provide new insights on the organization of the immune system
Regulatory controls of microRNA genes beyond the mature microRNAs by Gwen Liu ( )
1 edition published in 2010 in English and held by 1 WorldCat member library worldwide
MicroRNA (miRNA) genes, which encode an abundant class of ~22-nucleotide (nt), evolutionarily conserved small RNAs, control a fundamental layer of genetic programs at the post-transcriptional level. However, little is known about how the activity of miRNA genes is regulated and how the regulatory information controlling their activity is encoded. Interestingly, mature miRNAs can often be classified into large families consisting of members with identical seeds (nucleotides 2 through 8 of the mature miRNAs) and highly homologous ~22-nt mature miRNA sequences, but with divergent sequences and structural elements beyond their mature miRNAs. Here we investigated whether members of a miRNA gene family that encode identical or nearly identical mature miRNAs are functionally interchangeable in vivo and if not, why? We compare the activities of the mir-181 gene family in promoting double positive T cell development and show that miRNA genes that encode identical to nearly identical mature miRNAs can have distinct activities. The differences in activity between mir-181a-1 and mir-181c are largely determined by their unique primary/precursor-miRNA (pri/pre-miRNA) loop nucleotides, and the differences in activity between mir-181a-1 and mir-181b-1 are determined by both the pri/pre-miRNA loop and stem. Furthermore, the organization of mir-181a-1/b-1 in a cluster is important for its full activity. We also show that mir-181 family members can differentially regulate target genes quantitatively, and that some target genes can be upregulated. Taken together, we have demonstrated that regulatory information encoded in a miRNA gene beyond the mature miRNA plays a critical role in controlling the activity of the miRNA gene, suggesting that miRNA family members could have evolved different functions through their divergent miRNA gene sequences and structural elements beyond their mature miRNAs. Although, proteins may have evolved to recognize the structural and sequence elements of the pri/pre-miRNAs, we suggest that the regulatory information encoded in the structured pri/pre-miRNA may be directly interpreted through target and pri/pre-miRNA interactions
Regulation and coordination of homologous pairing and synapsis during caenorhabditis elegans meiosis by Weibin Zhang ( )
1 edition published in 2012 in English and held by 1 WorldCat member library worldwide
For successful segregation of chromosomes during meiosis, chromosomes have to recognize and align with their correct homologous partners, and then stabilize the homologous alignment with assembly of a proteinaceous structure, called the synaptonemal complex (SC), between them. SC assembly is highly processive and cooperative, yet the SC structure does not distinguish between homologous and nonhomologous associations. Thus SC assembly has to be tightly regulated and coordinated with homologous pairing to ensure that the SC is stabilizing productive homologous associations. Moreover, homologous pairing is accompanied by dramatic chromosome movement and nuclear reorganization of chromosomes into a clustered configuration during C. elegans early meiotic prophase. Subsequently, the clustered chromosomes are redispersed into aligned homologs upon SC assembly. The exact mechanisms involved in regulation and coordination of homologous pairing and synapsis are poorly understood, and this thesis was aimed at gaining a better understanding of the coordination of these two inter-related meiotic events. We identified HAL-2 as a major player in this coordination. We demonstrated that HAL-2 promotes homologous pairing mainly by preventing detrimental effects of SC precursors (SYP proteins). Homologous pairing is not established in hal-2 mutants, and several markers indicative of pairing center-mediated chromosome movement are also absent in hal-2 mutants. Pairing centers (PCs) are cis-acting chromosomal sites that mediate chromosome movement by connecting chromosomes to cytoplasmic microtubules via the conserved SUN-1/ZYG-12 nuclear envelope-spanning complexes. hal-2 mutants also exhibit defective SC assembly, with SYP proteins being loaded inappropriately along single unpaired chromosomes in hal-2 mutants. Moreover markers of PC-mediated chromosome movement and function are coordinately restored in hal-2 mutants by the removal of SYP proteins. Combined with other data, these findings indicate that SYP proteins can inhibit homologous pairing and that HAL-2 promotes pairing largely by antagonizing this inhibition, thus allowing activation and regulation of PC function. Given that HAL-2 concentrates in the nucleoplasm of meiotic germ cells and colocalizes with SYP proteins in nuclear aggregates when SC assembly is prevented, we propose that HAL-2 functions to shepherd SYP proteins prior to licensing of SC assembly, preventing SC precursors from interacting inappropriately with chromosomes and allowing them to accumulate sufficiently for rapid cooperative assembly upon homology verification. In this thesis, we also identified and characterized me16, a hypomorphic allele of scc-3. SCC-3 is a conserved component of the cohesin complex, and the scc-3(me16) mutant was demonstrated to be a partial loss of function mutation by comparative analyses with a null mutant, scc-3(ku263). Besides reduced chromosomal localization of cohesin, scc-3(me16) mutants also display an extended region of clustered chromosomes, incomplete SC assembly and defective recombination. Further, analyses of nuclear aggregates that form in the scc-3(ku263) null mutants led to the findings that components of the lateral elements, including cohesin, colocalize into these nuclear aggregates and that this localization is not dependent on REC-8
Genome-wide probing of RNA structures by Yue Wan ( )
1 edition published in 2012 in English and held by 1 WorldCat member library worldwide
RNA plays an important role in regulating cellular gene expression. Its ability to fold into secondary and tertiary structures underlies the RNA's ability to function and be processed in every step of its life cycle, including transcription, splicing, cellular localization, translation and turnover of the RNA. However, structural information for most RNAs in the cells, such as mRNAs, is missing due to the low throughput nature of RNA structure probing and the traditional difficulty of probing long RNAs, making it difficult to assess the full impact of RNA structure in biology. To fulfill the need to understand the roles of RNA structure on a global scale, we developed for the first time a novel strategy termed Parallel Analysis of RNA Structure (PARS), which couples structure-specific probing to high throughput sequencing to simultaneously generate secondary structures for thousands of RNA species at single nucleotide resolution. Applying PARS to the yeast transcriptome identified distinct structural organization of mRNAs, including a three nucleotide periodicity in the coding region and increased accessibility near the start codon for translation. Furthermore, we demonstrated that PARS can be used to study dynamics by probing the yeast transcriptome across a temperature gradient to identify functional structural regions that have differential propensity to melt. PARS can be readily applied to different organisms and conditions, to identify structural determinants that regulate gene expression changes across organisms in different cellular states. This expansion of RNA structural data will likely enhance our understanding of how RNA structure translates into RNA function in cellular systems, as well as open doors to potentially targeting these structural elements to regulate cellular behavior in diseases
Identification of RNA regulatory information in the Saccharomyces cerevisiae transcriptome by Daniel Patrick Riordan ( )
1 edition published in 2011 in English and held by 1 WorldCat member library worldwide
The unique post-transcriptional behavior of each mRNA is thought to be largely determined by features present in its molecular sequence, representing a type of RNA regulatory code. However, the details by which distinct regulatory outcomes are programmed into the sequences of different transcripts are mostly unknown. We set out to identify features of yeast mRNAs that influence their post-transcriptional fates. Using bioinformatic and in vitro selection approaches, we characterized several RNA recognition elements involved in mediating specific interactions with individual yeast RNA-binding proteins (RBPs). Most of the RNA elements we uncovered were associated with significant mRNA expression changes and were phylogenetically conserved in related yeasts, providing insights into the function and evolution of the corresponding interactions. We also analyzed RNA-protein interaction sites for the yeast Puf3 RBP by high-throughput sequencing under different growth conditions. These results provided high-resolution experimental evidence for Puf3 binding at consensus RNA elements in the transcriptome, and enabled detailed comparisons of individual interaction sites. Finally, we developed complementary methods for transcriptome-wide mapping of potential sites of RNA 2'-O-methylation. Application of these methods to the yeast transcriptome successfully recovered known sites of RNA modification and suggested that ribose methylation of functionally-related transcripts may occur and influence the regulation of endogenous yeast mRNAs. Overall, these results contribute to understanding of how RNA sequence features help to specify global differences in gene expression characteristics
Characterization of an RNase III protein and its potential roles in the RNA interference pathway of the protozoan parasite, Entamoeba histolytica by Justine Michelle Pompey ( )
1 edition published in 2012 in English and held by 1 WorldCat member library worldwide
Entamoeba histolytica, a human intestinal parasite and a leading cause of death worldwide, contains a complex repertoire of endogenous small RNAs. Core elements of the RNAi machinery have been identified in the E. histolytica genome including three Argonaute proteins (EhAgo2-1, EhAgo2-2, and EhAgo2-3) and two genes with RNA-directed RNA polymerase (RdRP) domains. However, to date no canonical Dicer enzyme, an RNaseIII endonuclease responsible for cleaving double-stranded RNA (dsRNA) to 20-30nt small RNAs and a critical player in the RNAi pathway, has been identified in the E. histolytica genome. We conducted bioinformatics searches of the genome which revealed only one gene with an RNaseIII domain, EhRNaseIII. Interestingly, this candidate lacks the canonical Dicer structure - it is substantially smaller than other Dicers, contains only a single RNaseIII domain, and lacks PAZ or double-stranded RNA binding domain (dsRBD). Most eukaryotic Dicer proteins identified to date contain two RNaseIII domains, which form a heterodimer required for cleavage of the dsRNA, and contain PAZ or dsRBDs. We determined that EhRNaseIII exists as a homodimer in E. histolytica trophozoites. We sought to probe the contributions of EhRNaseIII, EhAgo2-2, and EhRdRP1 to the RNAi pathway in E. histolytica. Attempts to downregulate these genes using RNA-based methods including antisense mediated silencing and the production of gene-specific secondary small RNAs were unsuccessful. Expression of EhRNaseIII dominant-negative mutants also failed to impair production of small RNAs. In order to determine if EhRNaseIII was capable of generating small RNAs of the size observed in E. histolytica trophozoites, we conducted in vitro cleavage assays of dsRNA. Despite using multiple protein sources (E. histolytica whole cell lysate, immunoprecipitated EhRNaseIII, and recombinant EhRNaseIII protein) and two structurally distinct dsRNA substrates, we were unable to detect small RNA cleavage products. We further investigated whether EhRNaseIII was sufficient to act as a Dicer enzyme by co-expressing it with a dsRNA substrate in Saccharomyces cerevisiae, which lacks an RNAi pathway. However, no small RNAs were detected. We also assessed whether EhAgo2-2, which associates with 5'-polyphosphate small RNAs in E. histolytica, could mediate silencing using 5'-monophosphated small RNAs in S. cerevisiae. No downregulation in reporter transcript or protein was observed. These data indicate that EhRNaseIII is not a minimal Dicer enzyme and suggest that small RNA biogenesis may occur solely through a Dicer-independent small pathway in E. histolytica. These data also suggest that EhAgo2-2 may be specific for 5'-polyphosphate small RNAs. Future studies will continue to investigate the mechanism of small RNA-induced silencing in E. histolytica
ESTIMATION OF LYMPHOCYTE AGGREGATE AND INDIVIDUAL CLONAL ABUNDANCES FROM REPLICATE SEQUENCE LIBRARIES : lymphclon by Yi Liu ( Book )
1 edition published in 2013 in English and held by 1 WorldCat member library worldwide
An in vivo characterization of chromatin state and its relationship to the expression of foreign, non-integrating transgenic DNA in mouse liver by Lia Gracey ( )
1 edition published in 2012 in English and held by 1 WorldCat member library worldwide
The pursuit of gene therapy to treat a wide-spectrum of diseases holds great promise, but its application in the clinic is still blocked by several barriers. Gene therapy approaches that rely on a virus to carry genetic material are plagued by problems with a host immune response and difficulty reaching therapeutic doses. Nonviral approaches have the advantage that delivering naked DNA does not illicit the same significant immune response and therapeutic levels are often initially easily attained. But, one prominent barrier to nonviral gene therapy is the lack of in vivo sustained expression from a foreign transgene. In the early stages after delivery, robust expression can be achieved from transgenic DNA, but this expression is very quickly and efficiently silenced, with loss of the desired effect of gene replacement. Our work took a two-step approach to further our understanding of how to create a better expression vector, both for gene therapy purposes as well as basic scientific goals of sustaining expression from delivered transgenic DNA. First, we studied whether we could manipulate and control the chromatin structure that a delivered gene adopts in vivo via nucleosome positioning signals on plasmid DNA in mouse liver. We developed a selective hybridization assay that allowed us to use a high-throughput sequencing approach to more rapidly screen the nucleosome occupancy and positioning of numerous constructs with varied DNA elements. We were able to transiently control the initial chromatin structure, but other forces in the cell soon overcame the thermodynamic preferences of nucleosome formation. We now believe that the use of nucleosome eviction elements may be a more promising approach: it will likely be more feasible to exclude nucleosome formation using rigid DNA sequences instead of permanently positioning a nucleosome using DNA sequence alone. Secondly, we gained a more fundamental understanding of the mechanism(s) responsible for the silencing of episomal (non-integrating) DNA constructs in mouse liver. We studied the differences between plasmid DNA and minicircle DNA, which was derived from a parental plasmid but lacked bacterial backbone sequences. At 6 weeks after delivery, plasmid DNA was effectively silenced but there was still active expression from the minicircle. We used high-throughput sequencing approaches to quantitate the levels of transcription and the enrichment of various histone modifications and RNA polymerase II (Pol II) on plasmid and minicircle. We found that Pol II appeared to stall at the transcription start site on the plasmid but was present at the 5' and 3' ends of the gene on the minicircle. This observation may explain the 28-fold higher level of transcript produced by the minicircle versus the plasmid. Though we saw enrichment of activating and silencing histone modifications on both plasmid and minicircle (likely due to our analysis of a population of cells which at any given point could have a construct that is actively expressed or silenced) we observed a striking abundance of a well-characterized histone modification associated with silencing on the plasmid and not on the minicircle. These results represent a contribution to the fields of gene therapy and chromatin biology that will help us move in the direction of achieving safe, therapeutic, and sustained expression of transgenic DNA
Priming by streptococcus pneumoniae causes changes in gene expression in Drosophila melanogaster by Junaid Ziauddin ( )
1 edition published in 2013 in English and held by 1 WorldCat member library worldwide
Insects are typically described as having no memory yet they can be primed by past exposure to survive a specific pathogenic challenge. To determine how a primed immune response might work in the context of a transcriptional response to a pathogen, we performed a microarray analysis that identified genes modulated by priming during a S. pneumoniae infection in Drosophila melanogaster. We followed 22 transcripts at a high level of resolution using qRT-PCR of tight timelines of infected primed and naìˆve flies. We knocked down expression of 17 of these transcripts/genes and observed changes in survival and bacterial growth dynamics. We found that priming affected the transcriptional response in three ways. First, the basal levels of some transcripts changed permanently upon priming. Second, the induced maximal expression level of genes was altered between the first and second infection. Third, there were changes in sensitivity where genes were induced at earlier times in the infection in the primed flies. We hypothesized that these changes are evolved to adapt, where the fly's immunity changes in a fashion that allows it to respond more effectively to that infection in subsequent exposures. We highlight 5 genes that follow our gene induction model and show that when knocked down, primed flies exhibit survival and bacterial growth phenotypes that demonstrate their important role in the primed response. These data show how a simple signaling network can possess memory which allows it to respond more effectively to threats based upon past experiences. We also added Serratia marcescens to the repertoire of pathogens that induce the primed immune response. We showed that this memory lasts for the life of the fly and the Toll pathway is necessary for the response. We demonstrated that Serratia is not cleared in primed flies despite longer survival times, indicating a tolerance effect. We found the highest protection to be species specific and nutrient deprivation to negatively impact priming. It is the first time gram-negative bacteria have been characterized in the primed response and the first time tolerance-based priming against any pathogen has been demonstrated in Drosophila
The role of the 5'-3' exoribonuclease Xrn2 in RNA virus infection by Cecilia D Sedano ( )
1 edition published in 2014 in English and held by 1 WorldCat member library worldwide
Hepatitis C virus (HCV) is a small, hepatotropic, RNA virus that can establish persistent infections leading to chronic hepatitis and hepatocellular carcinoma (HCC). With an estimated 2% of the world's population infected, HCV is a serious global health problem. Curiously, this virus has the unique feature of depending on the liver-specific, microRNA miR-122. In the current model, miR-122 interacts with the 5'-end of the HCV RNA genome, protecting it from hitherto unknown degradation machinery. The work described in this dissertation highlights a novel cytoplasmic antiviral function for the mammalian 5'-3' exoribonuclease Xrn2, and enzyme best known for its role in RNA polymerase II transcription termination. We discovered that Xrn2 modulates HCV RNA abundance by destabilizing the viral RNA genome. Importantly, during sequestration of miR-122, Xrn2 depletion restored HCV RNA abundance, suggesting that Xrn2 depletion eliminates the miR-122 requirement for viral RNA stability. Thus, Xrn2 is an antiviral effector so potent that HCV has evolved a mechanism to evade it, through the subversion of miR-122 to form a protective oligomeric complex at the 5' end of the viral genome. To determine if Xrn2's antiviral function is specific to HCV, we studied its role in poliovirus (PV) infection. Our experiments showed that Xrn2 interacts with PV RNA early during infection, and that Xrn2 activity may be suppressed by proteolytic cleavage that is mediated by the PV protease 2A. This work revealed an unprecedented role for Xrn2 and highlighted the broad function of this novel antiviral factor in RNA virus infection. In this dissertation, we also investigated the regulation of noncoding RNAs in HCV infection. Sucrose-gradient analysis of miR-122 during HCV infection uncovered that this highly conserved miRNA and its isomiR variants form unique complexes, which are specifically modulated during infection. This highlights the ability of HCV to modulate the engagement of specific miR-122-complexes, and the distinct functions in gene regulation these complexes may play. We also examined the role of Xrn2 in mammalian long noncoding RNA (lncRNA) degradation, and their regulation during HCV infection, by performing lncRNA microarrays. Our analyses will provide important insights into the biological functions of lncRNAs and help us identify novel therapeutic targets for HCV infection and HCC. Taken together, the work described here has uncovered a novel cytoplasmic antiviral function for Xrn2 in RNA virus infection. Our research highlights the potential of studying lncRNAs and miRNA variants during viral infection, which can lead the way for the discovery of new biomarkers and therapeutics
Molecular signatures of virome-host interactions by Poornima Parameswaran ( )
1 edition published in 2010 in English and held by 1 WorldCat member library worldwide
Small RNAs that are 19-30 nucleotides in length use the genetic information encoded in their sequence to effect gene regulation in a sequence-directed manner. Infections with RNA viruses in plants, worms and flies generate short viral-derived RNAs ("vsRNAs") that map to the genome of the infecting virus. The regulated production of these vsRNAs and their engagement by the immune apparatus is essential for inhibiting viral growth, making vsRNAs important components of antiviral immunity in these organisms. RNA virus-derived vsRNA-mediated gene silencing is yet to be demonstrated in mammalian systems. We investigated diverse RNA virus-mammalian systems, and characterized changes in small RNA populations that occur during viral infection in animal cells using high-throughput sequencing. Due to the large number of samples to be analyzed, we designed DNA barcodes to 'tag' RNA samples from individual experiments, which facilitated sequencing in parallel from multiple samples. Our work demonstrated the generality of RNA virus-derived vsRNA production, and the ability of the cellular short RNA apparatus to engage these vsRNAs in worms during Flock House Virus replication, and in mammalian cells during infections with Hepatitis C, Polio, Dengue, Vesicular Stomatitis or West Nile virus. In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host-encoded small RNA (miRNA) profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We found evidence for populations of vsRNAs that exist as duplexed small interfering RNAs ("siRNAs; " effectors of gene silencing) with zero to three nucleotide 3' overhangs. We also observed strand-selective loading of siRNAs onto Argonaute complexes, which are mediators of gene silencing. We quantitated the capacity of these HCVrep-derived vsRNAs to down-regulate target mRNAs in a sequence-specific manner in mammalian systems. We found that abundant HCVrep vsRNAs are not capable of mediating robust silencing (i.e. 2-fold or higher) of Luciferase reporters that have been engineered with vsRNA targets. Additionally, over-expression of siRNAs corresponding to five abundant vsRNAs failed to enhance silencing of Hepatitis C Virus mRNA. These results complement each other and suggest that in mammalian hosts, the virus may coexist with an abundant population of vsRNAs. Questions still remain as to whether robust gene silencing may be achieved by the cooperative action of abundant vsRNAs, or if abundant vsRNAs specifically inhibited from functioning in gene silencing, or alternatively, if they have novel roles in pathways distinct from gene silencing
COSA-1, a meiotic crossover site associated protein by Rayka Yokoo ( )
1 edition published in 2011 in English and held by 1 WorldCat member library worldwide
The formation of crossovers during meiosis is important both for the reassortment of genetic traits, and for creating a physical connection between homologous chromosomes to ensure faithful segregation. This thesis explores the role of a novel protein, COSA-1 (crossover site associated-1), in forming crossovers in the nematode C. elegans. cosa-1 was isolated in a screen for dead embryos, signaling missegregation of the autosomes, and a high incidence of males, signaling missegregation of the X chromosome. Interestingly, obvious orthologs of COSA-1 are only present in metazoans with the notable exception of Drosophilids. Thus no obvious orthologs of COSA-1 exist in the common model organisms, S. cerevisiae, S. pombe, D. melanogaster, and A. thaliana, making C. elegans one of the few organisms in which COSA-1 could have been isolated and studied. Yet, COSA-1 is conserved into humans suggesting the study of COSA-1 will have implications for human reproduction. Not only does C. elegans cosa-1 play a crucial role in promoting the formation of crossovers, evidenced by the lack of chiasmata in the cosa-1 mutant, GFP::COSA-1 exhibits an interesting localization pattern to presumptive crossover sites. In particular, unlike other crossover promoting proteins MSH-5 and ZHP-3, GFP::COSA-1 does not exhibit an initial broader localization but instead localizes to just 6 foci, 1 focus for the 1 crossover on each of the 6 chromosomes. This localization of GFP::COSA-1 to 6 foci was robust under various conditions. Mathematical modeling of the relationship between irradiation dose and COSA-1 foci revealed a relationship between obligate crossover, the phenomenon in which at least one crossover is formed per chromosome, and crossover interference, the phenomenon in which the formation of a crossover inhibits the formation of other crossovers nearby. In addition, analysis of GFP::COSA-1 foci in mutants that form extra crossovers, in particular rtel-1 and dpy-28, revealed a separation between the number of GFP::COSA-1 foci, 6, and the number of COs made, more than 6, suggesting that interference is still operating on GFP::COSA-1foci. Thus GFP::COSA-1 may serve as a better readout for crossover interference than crossovers per se. The ability to visualize GFP::COSA-1 in live worms holds great potential for elucidating the mechanisms involved in crossover interference, crossover distribution, and crossover designation
In vitro transcription studies of adenovirus by Andrew Zachary Fire ( Book )
1 edition published in 1983 in English and held by 1 WorldCat member library worldwide
Modulation of hepatitis C virus RNA abundance by the liver-specific microRNA miR-122 by Erica Machlin Cox ( )
1 edition published in 2012 in English and held by 1 WorldCat member library worldwide
Hepatitis C virus (HCV) is a global health problem, infecting approximately 2% of the world's population. The virus is hepatotropic, replicating in liver cells, and its only known hosts are humans and chimpanzees. HCV is an unusual virus in that it requires the liver-specific host microRNA (miRNA) miR-122 for HCV RNA accumulation. Though the precise mechanism by which miR-122 upregulates HCV RNA is still under investigation, it is known that miR-122 must bind to two adjacent sites in the 5' end of the HCV genome. In this dissertation, a stepwise mutational analysis of the entire sequence of miR-122 was performed to identify residues important for HCV RNA accumulation. All mutant miRNAs were tested in canonical miRNA reporter assays and in HCV RNA accumulation assays. The identities of two nucleotides within miR-122, at positions 15 and 16, were shown to be dispensable for canonical miRNA and siRNA activity but required for HCV RNA accumulation. Compensatory mutations in the HCV genome upstream of the original binding sites uncovered supplementary binding sites for nucleotides 15 and 16 of miR-122. This analysis led to a new model for miR-122-HCV RNA interactions. To further define the requirements of HCV for miR-122, we investigated whether the predecessor of mature miR-122, a long hairpin precursor designated pre-miR-122, was also able to mediate HCV RNA accumulation. The function of pre-miR-122 was tested in miRNA, siRNA, and HCV RNA accumulation assays. Inhibition of pre-miR-122 processing was achieved by substituting deoxyribonucleotides into the loop of pre-miR-122 to prevent Dicer-mediated cleavage. Full-length pre-miR-122 was demonstrated to be functional in miRNA and siRNA assays and to be sufficient for HCV RNA accumulation. Pre-miR-122 also required traditional components of the RNA-induced silencing complex (RISC) for activity. Taken together, this research has uncovered novel requirements of miR-122 for HCV RNA accumulation. Components shown to be dispensable for canonical miRNA interactions were necessary for this unusual microRNA-target RNA interaction. Uncovering hepatitis C virus's stringent requirements for the mature and precursor forms of miR-122 will pave the way for new antiviral therapies targeting a host factor
 
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