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Impact of the poly(A) limiting element on mRNA 3' processing efficiency and translation

Peng, Jing
http://rave.ohiolink.edu/etdc/view?acc_num=osu1101745063

Abstract Details

2004, Doctor of Philosophy, Ohio State University, Ohio State Biochemistry Program.
The poly(A)-limiting element (PLE) is a cis-acting sequence whose presence in the terminal exon results in the addition of a short, discrete <20 nt poly(A) tail on reporter mRNA. This study has examined the 3' processing and translation efficiencies of PLE-containing mRNAs with short poly(A) tails. In cells transfected with the human beta-globin reporter genes with or without a PLE, the PLE increases the accumulation of beta-globin mRNA in both nuclear and cytoplasmic RNA fractions. Quantitative RT-PCR and RNase protection assays showed that the PLE increases pre-mRNA 3' cleavage in vivo to the same degree that it increases the amount of beta-globin mRNA. Moreover, in vitro cleavage assays also indicated that the PLE enhances 3' processing efficiency. Thus, in addition to restricting the length of the poly(A) tail to <20 nt, the PLE also acts as an enhancer of pre-mRNA 3' processing. A firefly luciferase reporter gene was used to examine the translation efficiency of PLE-containing mRNAs with short poly(A) tails. In transfected cells, PLE-containing mRNA with a <20 nt poly(A) tail associated with polysomes and was translated as well as the matching control mRNA with a long poly(A) tail. The impact of the PLE and poly(A) tail length on translation in vitro was examined using a cap- and poly(A)-dependent in vitro translation system prepared from HeLa cells. In vitro the presence of a PLE did not overcome the negative effect of a short poly(A) tail on translation. A similar result was observed for capped and polyadenylated mRNAs directly transfected into cells, suggesting that a nuclear processing event facilitates PLE-stimulated translation of short poly(A) mRNA. The addition of the PLE-binding proteins did not selectively enhance the translation of PLE-containing mRNAs with short poly(A) tails in vitro. However, selective PLE-enhanced translation of short poly(A) mRNA was observed in vitro when PABP activity was inhibited. Although the translation of PLE-containing mRNAs was still less efficient than that observed in vivo, these data indicate that the PLE functionally substitutes for bound PABP to stimulate translation of short poly(A) mRNA.
Daniel Schoenberg (Advisor)
201 p.
Chemistry, Biochemistry
PLE; MRNA; POLY; PLE-containing; TRANSLATION; PABP

Recommended Citations

Citations

  • Peng, J. (2004). Impact of the poly(A) limiting element on mRNA 3' processing efficiency and translation [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1101745063

    APA Style (7th edition)

  • Peng, Jing. Impact of the poly(A) limiting element on mRNA 3' processing efficiency and translation. 2004. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1101745063.

    MLA Style (8th edition)

  • Peng, Jing. "Impact of the poly(A) limiting element on mRNA 3' processing efficiency and translation." Doctoral dissertation, Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1101745063

    Chicago Manual of Style (17th edition)

osu1101745063
752
© 2004, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.