http://hdl.handle.net/1721.1/7785 Fri, 24 May 2013 08:08:43 GMT 2013-05-24T08:08:43Z http://hdl.handle.net/1721.1/72822 Conservation of exon scrambling in human and mouse Hamilton, Monica L. (Monica Lauren) Exon scrambling is a phenomenon in which the exons of an mRNA transcript are spliced in an order inconsistent with that of the genome. In this thesis, I present a computational analysis of scrambled exons in human and mouse. RNA-seq data was mapped to the genome and all unaligned reads were subsequently mapped to a database of all possible exon-exon junctions. Eight conserved genes were found to undergo scrambled splicing in both species. In several cases, not only the gene was conserved, but the particular exons involved were conserved as well. Reading frame was preserved in just over half of the events, indicating that although some transcripts may be translated into protein, some may be non-functional or may play a regulatory role. The introns flanking scrambled exons were significantly longer than average, providing clues to the mechanism for this abnormal splicing pattern. The results of this study demonstrate that presence of scrambled transcripts in the cell is infrequent, but can be conserved over tens of millions of years of evolution, suggesting it has a biological function. Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.; Cataloged from PDF version of thesis.; Includes bibliographical references (p. 21-23). Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/1721.1/72822 2012-01-01T00:00:00Z http://hdl.handle.net/1721.1/70395 Physiological IRE-1-XBP-1 and PEK-1 signaling in Caenorhabditis elegans larval development and immunity Kinkel, Stephanie A The endoplasmic reticulum (ER) is responsible for the folding and processing of approximately one third of proteins in eukaryotic cells, and homeostasis in this compartment is tightly regulated. The Unfolded Protein Response, or UPR, is activated in response to perturbations in protein folding in the ER, collectively termed ER stress. This compensatory mechanism, mediated by IRE-1, PERK- 1/PEK-1 and ATF-6 in metazoans, resolves an overcrowded ER lumen in part through the increase of protein degradation and folding. Typical studies focus on activation of the UPR in response to characterized chemical agents that potently alter ER function or protein stability and folding, leaving physiological or native roles for the UPR relatively uncharacterized. Richardson et al previously demonstrated a role for the UPR in innate immunity in C elegans. Here, in an effort to understand this role, we demonstrate that intestinal expression of XBP-1 is sufficient to overcome PMK-1 dependent larval lethality on a lawn of pathogenic Pseudomonas aeruginosa. Further, we demonstrate that XBP-1 deficiency results in constitutive ER stress even in the absence of pathogenic infection. This elevated ER stress is reflected by increased activities of both IRE- 1 and PEK-1 under physiological conditions. Our data suggest that negative feedback loops involving the activation of IRE-1-XBP-1 and PEK-1 pathways serve essential roles not only at the extremes of ER stress but also in the maintenance of ER homeostasis under physiological conditions. Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.; Cataloged from PDF version of thesis.; Includes bibliographical references (p. 27-29). Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/1721.1/70395 2012-01-01T00:00:00Z http://hdl.handle.net/1721.1/68432 MicroRNA targeting in mus musculus and Caenorhabditis elegans Lafkas, Ginamarie N MicroRNAs (miRNAs) are small, approximately 22 nucleotide RNAs that regulate gene expression post-transcriptionally by base-pairing to complementary sites in the target mRNA. The first miRNA, lin-4, was discovered in 1993 in Caenorhabditis elegans; since then hundreds of miRNAs have been identified in C. elegans, Drosophila melanogaster, plants, mouse, and humans, where they approach a number equivalent to 1-2% of the protein-coding genes. With the exception of plants, miRNAs most commonly regulate targets by imperfectly pairing to 3' untranslated regions (UTRs), leading to translational repression or mRNA destabilization. The microRNA miR-196 is encoded at three paralogous locations in the HoxA, B, and C clusters in mammals and has conserved complementarity to the 3'UTRs of Hoxb8, Hoxc8, and Hoxa7; in particular, miR-1 96 has complete complementarity to Hoxb8 with the exception of a single G:U wobble. In 2004, Yekta et al., were able to detect RNA fragments diagnostic of miR-1 96-directed cleavage of Hoxb8 transcript in mouse embryos, and cell culture experiments showed down-regulation of Hoxb8, Hoxc8, Hoxd8, and Hoxa7. To address the biological significance of miR-196 mediated repression of Hox genes in vivo, we attempted to generate targeted transgenic mice for the Hoxa7, b8 and c8 genes. These mice would allow us to study the resulting phenotypic and molecular consequences and determine the impact this regulation has on establishment of the anterior-posterior axis in the developing embryo as well as its role in defining the expression boundaries of the individual Hox genes in vivo. The lin-4 miRNA plays a role in regulating the heterochronic genes involved in larval development in C. elegans. We used luciferase assays to test the efficacy of the seven proposed lin-4 binding sites in the lin-14 3'UTR and to determine if lin-4 is capable of recognizing and mediating repression through them. The wild-type lin-14 3'UTR was compared with mutant UTRs in which the lin-4 target sites were mutated. We determined that the three canonical 8mer sites are functional, as expected, and that at least one of the four additional sites is also recognized by lin-4 and contributes to the overall repression of the lin-14 3'UTR. Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.; Cataloged from PDF version of thesis.; Includes bibliographical references (p. 45-52). Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/1721.1/68432 2011-01-01T00:00:00Z http://hdl.handle.net/1721.1/65751 Identification of toxoplasma gondii genes involved in the strain-specific modulation of IL-12 cytokine secretion Mc Kell, Renée Abba Toxoplasma gondii is an intracellular pathogen that causes life-threatening toxoplasmosis in developing fetuses and immune-compromised individuals. An immune response to a Toxoplasma infection is characterized by the stimulation of high levels of interleukin- 12 (IL- 12), followed by the production of interferon-y (IFN-y) by immune cells. Although IFN-y is the main mediator of resistance to Toxoplasma infection, Toxoplasma is able to manipulate the immune response through the regulation of IL- 12 production. Toxoplasma has been shown to modulate the induction of IL-12 in a strain-specific manner. An infection with a type II strain, but not type I and III strains, induce high levels of IL- 12 production by macrophages in vitro. Previous studies have implicated two Toxoplasma genes that play a role in this strainspecific difference in IL- 12 production. A rhoptry protein kinase, ROP 16, from type I and III strains, was found to be crucial in the suppression of IL-12. A type II dense granule protein, GRA 15, induces IL-12 through NF-KB activation. I have screened F1 progeny from a type III x type II cross for IL-12 induction and NF-KB activation. My preliminary experiments indicate that there are other Toxoplasma factors involved in the strain-specific inhibition of IL-12, and this inhibition has a genetic basis. To understand the role of IL-12 regulation by Toxoplasma, I intend to (i) identify novel Toxoplasma genes involved in the inhibition of IL- 12 secretion (ii) test and characterize the effects of the Toxoplasma protein ROP38 on IL-12 signaling and (iii) determine the target/s of Toxoplasma inhibition of IL-12 production. Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.; Cataloged from PDF version of thesis.; Includes bibliographical references (p. 15-18). Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/1721.1/65751 2011-01-01T00:00:00Z