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dc.contributor.advisorDavid P. Bartel.en_US
dc.contributor.authorSubtelny, Alexander O. (Alexander Orest)en_US
dc.contributor.otherMassachusetts Institute of Technology. Department of Biology.en_US
dc.date.accessioned2017-12-05T19:12:18Z
dc.date.available2017-12-05T19:12:18Z
dc.date.issued2014en_US
dc.identifier.urihttp://hdl.handle.net/1721.1/112426
dc.descriptionThesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, June 2014.en_US
dc.descriptionCataloged from PDF version of thesis. "May 2014." Vita.en_US
dc.descriptionIncludes bibliographical references.en_US
dc.description.abstractPoly(A) tails are found at the 3' ends of nearly all eukaryotic messenger RNAs (mRNAs) and long non-coding RNAs. The presence of a poly(A) tail promotes translation and inhibits decay of an mRNA, with both effects mediated through poly(A)-binding protein. However, an understanding of the relationship between the length of a poly(A) tail and these aspects of mRNA metabolism has been limited, primarily because of the lack of a technology that provides high-resolution poly(A)-tail length measurements in a global manner. This dissertation describes a new, high-throughput-sequencing-based method (PAL-seq) that measures the tails of individual mRNA molecules by coupling a fluorescence-based readout of poly(A)-tail length with sequencing of the poly(A)-proximal region. Using PAL-seq, we have found that poly(A)-tail lengths exhibit a notably poor correlation with translational efficiency (as measured by ribosome profiling) across genes in nearly all systems we have examined. In contrast, early zebrafish and Xenopus laevis embryos display a striking correlation (Spearman R > 0.6) that disappears at gastrulation. This developmental uncoupling of tail length and translational efficiency explains the different outcomes of microRNA (miRNA)-mediated poly(A)-tail shortening in zebrafish embryos before and after gastrulation, with translational repression being the predominant effect before and mRNA destabilization after. We have also observed that poly(A)-tail lengths do not correlate positively with mRNA half-lives in mammalian cells, and that miRNAs do not promote any apparent tail shortening in this setting. Since these results could be explained by differences in deadenylation rates, we performed a kinetic analysis in which we captured newly-made mRNAs of different age ranges. The deadenylation rates that we calculated after measuring tails over time correlated strongly with mRNA half-lives (Spearman R < -0.6), reinforcing the notion that tail shortening leads to mRNA downregulation. When we repeated the timecourse with prior overexpression of a miRNA, we found that miRNAmediated tail shortening was generally modest, but of a magnitude not significantly different from that expected given the accompanying decreases in mRNA stability.en_US
dc.description.statementofresponsibilityby Alexander O. Subtelny.en_US
dc.format.extent144 pagesen_US
dc.language.isoengen_US
dc.publisherMassachusetts Institute of Technologyen_US
dc.rightsMIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission.en_US
dc.rights.urihttp://dspace.mit.edu/handle/1721.1/7582en_US
dc.subjectBiology.en_US
dc.titleFunction and regulation of poly(A)-tail lengthen_US
dc.typeThesisen_US
dc.description.degreePh. D.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Biology.en_US
dc.identifier.oclc1008754763en_US


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