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"Finding Intracellular Sites Where mRNAs Containing Premature Termination Codons are Recognized and Degraded"

by
Michael Levandoski
Microbiology and Molecular Genetics Program
B.S. Fairleigh Dickinson University



Thesis Advisor: Sanjay Tyagi, Ph.D.
Associate Professor
Public Health Research Institute

Thursday, May 17, 2012
2:00 P.M., ICPH Auditorium


Abstract

Nonsense-mediated decay (NMD) is a widely conserved process that removes messenger RNAs containing premature termination codons (PTCs) from the cell. In addition to preventing translation of potentially harmful truncated proteins, NMD has also been shown to control expression of many genes by down-regulating their transcripts. Despite extensive research on NMD, the cellular locations where the PTC containing mRNAs are degraded are not clear. Using single-molecule fluorescence in situ hybridization, we show that PTC containing mRNA molecules accumulate in the cytoplasm when NMD is inhibited. We developed a method to visualize the fragments of mRNAs that are believed to be produced by an initial endonucleolytic cleavage near the PTC. When we inhibited the 5-3 exonuclease, Xrn1, the 3 fragment accumulated in the cytoplasm, again suggesting that the sites of decay are distributed throughout the cytoplasm. Additionally, we demonstrate that increasing the length of the 3 un-translated region of an mRNA causes the mRNA to become less stable by making it a substrate of NMD machinery. We showed that such mRNAs are also degraded within the cytoplasm. Presence of a PTC in mRNAs with long un-translated regions did not cause further destabilization. Our single molecule FISH approach provides powerful insights into the intracellular location of NMD.


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