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dc.contributor.authorHoloch, Daniel Benjaminen_US
dc.date.accessioned2015-12-04T18:42:57Z
dc.date.created2015-11en_US
dc.date.issued2015-09-23en_US
dc.date.submitted2015en_US
dc.identifier.citationHoloch, Daniel Benjamin. 2015. The Programming and Assembly of a Transcriptional Silencing Complex. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.en_US
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:23845498
dc.description.abstractArgonautes and their small RNA guides form an ancient partnership with diverse roles in controlling gene expression and preserving genome stability. In the fission yeast Schizosaccharomyces pombe, the Argonaute Ago1 acts within the RITS complex to target the repetitive DNA elements that flank each centromere for heterochromatic silencing, which is necessary for faithful chromosome transmission. A separate Ago1-containing complex, termed ARC, is also required for pericentromeric silencing but its precise function, and the mechanisms that regulate the movement of Ago1 between ARC and RITS, have remained unclear. This dissertation investigates both of these questions. By combining distinct approaches, we have defined the role of ARC as that of enabling Ago1 to be programmed with small RNA guides. In an in vitro assay using immunopurified proteins, we found that loading of synthetic double-stranded small RNAs into Ago1 requires the ARC subunit Arb1 but not the RITS subunit Tas3. In parallel, we isolated cellular Ago1- associated small RNAs and, by high-throughput sequencing, observed that deletion of ARC components produced read features indicative of nonspecifically-interacting small RNAs. Together, these data indicate that the small-RNA-loading capability of Ago1 is conferred by ARC. We also discovered using co-immunoprecipitation that the ARC subunits Arb1 and Arb2 are required for the proper association between RITS subunits Ago1 and Tas3, suggesting that small-RNA loading by ARC might license Ago1 for assembly into RITS. Indeed, we went on to show that Ago1 mutants deficient for small-RNA loading universally fail to interact with Tas3, whereas other non-functional Ago1 variants maintain Tas3 association. We conclude that Tas3 distinguishes between loaded and unloaded Ago1, admitting only the former into RITS. Our studies have delineated the mechanisms that control the programming and assembly of the RITS complex. The results illuminate the role of ARC in heterochromatic silencing and identify this complex as the machinery required for loading small RNAs into Argonaute in S. pombe. Furthermore, we have uncovered small-RNA loading as a checkpoint for the entry of Argonaute into RITS, which may reflect a common discriminatory function of GW-repeat proteins such as Tas3 that precludes the formation of inactive and potentially deleterious complexes.en_US
dc.description.sponsorshipMedical Sciencesen_US
dc.format.mimetypeapplication/pdfen_US
dc.language.isoenen_US
dash.licenseLAAen_US
dc.subjectBiology, Geneticsen_US
dc.titleThe Programming and Assembly of a Transcriptional Silencing Complexen_US
dc.typeThesis or Dissertationen_US
dash.depositing.authorHoloch, Daniel Benjaminen_US
dc.date.available2015-12-04T18:42:57Z
thesis.degree.date2015en_US
thesis.degree.grantorGraduate School of Arts & Sciencesen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
dc.contributor.committeeMemberGregory, Richarden_US
dc.contributor.committeeMemberKennedy, Scotten_US
dc.contributor.committeeMemberBartel, Daviden_US
dc.type.materialtexten_US
thesis.degree.departmentMedical Sciencesen_US
dash.identifier.vireohttp://etds.lib.harvard.edu/gsas/admin/view/665en_US
dc.description.keywordsArgonaute; Small RNAs; RITS; ARC; GW proteins; Complex assembly; Schizosaccharomyces pombeen_US
dash.author.emailholoch@gmail.comen_US
dash.identifier.drsurn-3:HUL.DRS.OBJECT:25142751en_US
dash.identifier.orcid0000-0001-6399-0230en_US
dash.contributor.affiliatedHoloch, Daniel Benjamin
dc.identifier.orcid0000-0001-6399-0230


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