Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Cell Biology

Language

English (en)

Date of Award

January 2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Kathleen Hall

Abstract

AU-Rich Elements: ARE) are cis-acting RNA sequences in the 3'UTRs of a wide range of transcripts that function to regulate mRNA stability, localization, and/or translation through interaction with ARE-Binding Proteins. Apobec-1 Complementation Factor: ACF) was originally identified as a co-factor in ApoB mRNA C to U editing, but has recently been implicated in regulation of mRNA stability as an ARE-Binding Protein. Here we have used tissue culture models with RNA turnover assays to show that the stability of reporters cloned upstream of the Interleukin-6: IL-6) 3'UTR or portions of the Cox-2 3'UTR is regulated by levels of ACF expression. Surprisingly, ACF expression results in stabilization of a reporter associated with the IL-6 3'UTR while resulting in destabilization of a reporter associated with the Cox-2 3'UTR. In order to more fully probe this dual ability of ACF, we examined its behavior as an RNA-binding protein. Purified recombinant truncations of ACF were used to probe the affinity and specificity of ACF binding to a panel of RNAs including ApoB mRNA: its canonical target) as well as IL-6 and Cox-2 RNAs, which we have shown are regulated in cellulo by ACF expression The first 380 amino acids of ACF, which contain three RNA recognition motifs: RRM), bind IL-6 and Cox-2 RNAs with higher affinity than ApoB mRNA. This protein is also capable of binding a C/U-rich RNA: GABA Intron), indicating that ACF has a broader target preference than simply AU-Rich RNAs. Furthermore, in vitro binding assays reveal that RRM1 of ACF binds IL-6, Cox-2, and GABA Intron RNA but not ApoB, while RRM3 does not detectably bind any of the RNAs probed. This indicates that RRM1 participates in RNA binding of some targets but not others and RRM3 is not part of the RNA-binding domain. These observations were extended using tryptophan fluorescence to determine that Cox-2 RNA interacts with ACF RRM2, suggesting that RRMs 1 and 2 together bind at least some RNA targets. UV-crosslinking assays identified discrete ACF binding sites within both the IL-6 3'UTR and the Cox-2 3'UTR. On both RNAs, these sites are consistent with regions that confer ACF-dependent mRNA stabilization or destabilization in RNA turnover assays. UV-crosslinking assays also revealed a structural preference in the ACF:Cox-2 interaction. Finally, these observations were examined in the context of ACF structural predictions. While little experimentally determined structural data exists for ACF, homology modeling was used to predict possible secondary and tertiary three-dimensional structures that may account for the physiological and binding activities observed. We propose that ACF binds RNA targets by multiple mechanisms, using one or more of its RRMs, and that the resulting complex displays the RNA to facilitate RNA editing, stabilization, or destabilization. We suggest that the geometry of the complex also impacts ACF interactions with co-factors such as Apobec-1 in mRNA editing or other ARE-Binding proteins that together regulate the stability of common RNA targets.

DOI

https://doi.org/10.7936/K72V2D40

Comments

Permanent URL: http://dx.doi.org/10.7936/K72V2D40

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