{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/112426"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/112426","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Function and regulation of poly(A)-tail length","abstract":"Poly(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.","abstract_html":"Poly(A) tails are found at the 3&#x27; 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 &gt; 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 &lt; -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.","abstract_has_math":false,"creators":["Subtelny, Alexander O. (Alexander Orest)"],"institution":"Massachusetts Institute of Technology","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Department of Biology.","school":null,"contributors":[],"advisors":["David P. Bartel."],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014","date_published":"2014","updated_at":"2026-07-22T22:21:23Z","subjects":["Biology."],"languages":["eng"],"rights":["MIT 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."],"rights_urls":["http://dspace.mit.edu/handle/1721.1/7582"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1721.1/112426","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["David P. Bartel."]},{"key":"dc:contributor.department","label":"Department","values":["Massachusetts Institute of Technology. Department of Biology."]},{"key":"dc:contributor.other","label":"Dc Contributor Other","values":["Massachusetts Institute of Technology. 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They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://dspace.mit.edu/handle/1721.1/7582"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1721.1/112426"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, June 2014.","Cataloged from PDF version of thesis. \"May 2014.\" Vita.","Includes bibliographical references."]},{"key":"dc:description.abstract","label":"Abstract","values":["Poly(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."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph. D."]},{"key":"dc:title","label":"Title","values":["Function and regulation of poly(A)-tail length"]}]}],"canonical_facts":{"dc:contributor.advisor":["David P. Bartel."],"dc:contributor.department":["Massachusetts Institute of Technology. Department of Biology."],"dc:contributor.other":["Massachusetts Institute of Technology. Department of Biology."],"dc:creator":["Subtelny, Alexander O. (Alexander Orest)"],"dc:date.accessioned":["2017-12-05T19:12:18Z"],"dc:date.available":["2017-12-05T19:12:18Z"],"dc:date.issued":["2014"],"dc:description":["Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, June 2014.","Cataloged from PDF version of thesis. \"May 2014.\" Vita.","Includes bibliographical references."],"dc:description.abstract":["Poly(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."],"dc:description.degree":["Ph. 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