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REGULATION OF DROSOPHILA mRNA STABILITY BY DEADENYLATION ELEMENTS AND miRNAs

Trinh, Tat To

Abstract Details

2015, Doctor of Philosophy, Case Western Reserve University, Biochemistry.
Messenger (mRNA) deadenylation is thought to be rate limiting for and to trigger of mRNA decay, either 5’ to 3’ or 3’ to 5’. In a survey of polyA tail length of specific mRNAs in Drosophila S2 cells, we found several, including reaper and arc-1 mRNAs, that exhibited very short or non-existent polyA tails. Knockdown of deadenylase components revealed that reaper mRNAs were synthesized with normal polyA tails, a result in which indicated that the tail-less mRNAs had been rapidly deadenylated. Maintenance of capped but deadenylated state in the reaper mRNA requires three cis-acting elements in the 3' UTR. Interestingly, arc- 1 transcripts are found to be insensitive to deadenylases depletion, strongly suggesting that they are not ever polyadenylated. More surprisingly, these deadenylated mRNAs were found to be fully associated with actively translating ribosomes. Disaggregation of polysomes under hypertonic conditions followed by recovery in isotonic conditions shows that the deadenylated mRNAs can be efficiently translated. To rule out the possibility of reinitiation of translation required the full polyA tail, we treated S2 cells with harringtonine and showed accumulation of deadenylated species on 80S fraction. For these and possibly other mRNAs, a polyA tail is not required for translation. The mechanism whereby microRNAs (miRNAs) repress protein output from targeted mRNAs remains in question. Substantive evidence indicates that miRNAs function primarily, if not exclusively, by destabilizing their mRNA targets. Whether destabilization is the consequence of inhibition of initiation of translation is still debated. Other evidence has suggested that miRNAs inhibit translation at some step after initiation but mechanistic insight into how this could occur has been difficult to obtain. The notion that miRNAs impact translation post initiation derives largely from many observations that miRNA targets and miRNAs themselves appear to be associated with actively translating ribosomes. The vast majority of studies that had examined mechanism of miRNA-mediated repression have been carried out using transient transfection of both targets and miRNAs; most often with engineered 3' UTRs. Here, we made stable cell lines expressing the well-characterized miRNA-targeted 3' UTRs of Drosophila reaper and hid. Lines expressing two versions of each UTR, one with wild type miRNA recognition sites, and one where the recognition sites were mutated, were studied. All detectable mRNAs from all four lines were associated with actively translating ribosomes as assessed by harringtonine treatment and northern blotting. Half-life measurements showed that mRNAs containing wild type miRNA recognition sites were dramatically destabilized when compared to those with mutant recognition sites. When transcription was arrested, we observed a rapid accumulation of mRNA fragments. These fragments resulted from progressive truncation from the 5' end of the mRNA and were ribosome associated. These data indicate that mRNA-mediated mRNA destabilization and degradation occur while targeted mRNAs are being translated and therefore are cotranslational. These observations may rationalize disparate views of miRNA mechanism. In reports examining subcellular location of targeted mRNAs, half- lives were not determined, and when half-lives were determined, subcellular localization was not.
Timothy Nilsen, Ph.D (Advisor)
Jeffery Coller, Ph.D (Committee Chair)
Richard Padgett, Ph.D (Committee Member)
Donny Licatalosi, Ph.D (Committee Member)
Helen Salz, Ph.D (Committee Member)
155 p.

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Citations

  • Trinh, T. T. (2015). REGULATION OF DROSOPHILA mRNA STABILITY BY DEADENYLATION ELEMENTS AND miRNAs [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1436441986

    APA Style (7th edition)

  • Trinh, Tat. REGULATION OF DROSOPHILA mRNA STABILITY BY DEADENYLATION ELEMENTS AND miRNAs . 2015. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1436441986.

    MLA Style (8th edition)

  • Trinh, Tat. "REGULATION OF DROSOPHILA mRNA STABILITY BY DEADENYLATION ELEMENTS AND miRNAs ." Doctoral dissertation, Case Western Reserve University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1436441986

    Chicago Manual of Style (17th edition)