2026-09-19T10:44:04Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/1124262026-06-16T18:55:55Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022
David P. Bartel.
Subtelny, Alexander O. (Alexander Orest)
Massachusetts Institute of Technology. Department of Biology.
Massachusetts Institute of Technology. Department of Biology
2017-12-05T19:12:18Z
2017-12-05T19:12:18Z
2014
http://hdl.handle.net/1721.1/112426
1008754763
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, June 2014.
Cataloged from PDF version of thesis. "May 2014." Vita.
Includes bibliographical references.
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.
by Alexander O. Subtelny.
Ph.D.
144 pages
eng
Massachusetts Institute of Technology
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.
http://dspace.mit.edu/handle/1721.1/7582
Biology.
Function and regulation of poly(A)-tail length
Thesis
application/pdf
Publication
unknown
unknown
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<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>
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