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dc.contributor.advisorMarian Walhout, PhD
dc.contributor.authorTamburino, Alex M.
dc.date2022-08-11T08:08:45.000
dc.date.accessioned2022-08-23T16:07:00Z
dc.date.available2022-08-23T16:07:00Z
dc.date.issued2015-08-04
dc.date.submitted2015-11-16
dc.identifier.doi10.13028/M2DK5M
dc.identifier.urihttp://hdl.handle.net/20.500.14038/32160
dc.description.abstractInteractions between 3´ untranslated regions (UTRs) and RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation. Metazoan genomes encode hundreds of RBPs and thousands of 3’ UTRs have been experimentally identified, yet the spectrum of interactions between 3´UTRs and RBPs remains largely unknown. Several methods are available to map these interactions, including protein-centered methods such as RBP immunoprecipitation (RIP) and cross-link immunoprecipitation (CLIP), yeast three-hybrid assays and RNAcompete. However, there is a paucity of RNA-centered approaches for assaying an RNA element of interest against multiple RBPs in a parallel, scalable manner. Here, I present a strategy for delineating protein-RNA interaction networks using a gene centered approach. This approach includes annotating RBPs and identifying physical interactions between an RNA of interest and these RBPs using the Protein-RNA Interaction Mapping Assay (PRIMA). Few RBPs have been experimentally determined in most eukaryotic organisms. Therefore I show that existing RBP annotations can be supplemented using computational predictions of RNA binding domains (RBD) from protein sequences. A single RNA of interest can be tested using PRIMA against a library of RBPs constructed from these annotations. PRIMA utilizes the green fluorescent protein (GFP) in yeast as a reporter. PRIMA is based on reconstitution of the interaction between the 5´ and 3´ ends of an mRNA, which increases mRNA stability and enhances translation. PRIMA recapitulates known and uncovers new interactions involving RBPs from human, Caenorhabditis elegans and bacteriophage with short RNA fragments and full-length 3´UTRs. The development of RBP prey libraries will enable the testing of 3´UTRs against the hundreds of RBPs, which is essential to gain broad insights into post-transcriptional gene regulation at a systems level.
dc.language.isoen_US
dc.publisherUniversity of Massachusetts Medical School
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectDissertations, UMMS
dc.subject3' Untranslated Regions
dc.subjectCaenorhabditis elegans
dc.subjectImmunoprecipitation
dc.subjectRNA-Binding Proteins
dc.subject3' Untranslated Regions
dc.subjectCaenorhabditis elegans
dc.subjectImmunoprecipitation
dc.subjectRNA-Binding Proteins
dc.subjectComputational Biology
dc.subjectGenetics and Genomics
dc.subjectGenomics
dc.subjectMolecular Biology
dc.subjectSystems Biology
dc.titleA Gene-Centered Method For Mapping 3’UTR-RBP Interactions: A Dissertation
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1795&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/793
dc.legacy.embargo2016-08-16T00:00:00-07:00
dc.identifier.contextkey7849480
refterms.dateFOA2022-08-24T04:31:14Z
html.description.abstract<p>Interactions between 3´ untranslated regions (UTRs) and RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation. Metazoan genomes encode hundreds of RBPs and thousands of 3’ UTRs have been experimentally identified, yet the spectrum of interactions between 3´UTRs and RBPs remains largely unknown. Several methods are available to map these interactions, including protein-centered methods such as RBP immunoprecipitation (RIP) and cross-link immunoprecipitation (CLIP), yeast three-hybrid assays and RNAcompete. However, there is a paucity of RNA-centered approaches for assaying an RNA element of interest against multiple RBPs in a parallel, scalable manner.</p> <p>Here, I present a strategy for delineating protein-RNA interaction networks using a gene centered approach. This approach includes annotating RBPs and identifying physical interactions between an RNA of interest and these RBPs using the Protein-RNA Interaction Mapping Assay (PRIMA). Few RBPs have been experimentally determined in most eukaryotic organisms. Therefore I show that existing RBP annotations can be supplemented using computational predictions of RNA binding domains (RBD) from protein sequences. A single RNA of interest can be tested using PRIMA against a library of RBPs constructed from these annotations. PRIMA utilizes the green fluorescent protein (GFP) in yeast as a reporter.</p> <p>PRIMA is based on reconstitution of the interaction between the 5´ and 3´ ends of an mRNA, which increases mRNA stability and enhances translation. PRIMA recapitulates known and uncovers new interactions involving RBPs from human, Caenorhabditis elegans and bacteriophage with short RNA fragments and full-length 3´UTRs. The development of RBP prey libraries will enable the testing of 3´UTRs against the hundreds of RBPs, which is essential to gain broad insights into post-transcriptional gene regulation at a systems level.</p>
dc.identifier.submissionpathgsbs_diss/793
dc.contributor.departmentProgram in Molecular Medicine
dc.description.thesisprogramInterdisciplinary Graduate Program


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