Dissertations, Theses, and Capstone Projects

Date of Degree

9-2015

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Kevin Ryan

Committee Members

Serafin Piñol Roma

Zimei Bu

Frida Kleiman

Hualin Zhong

Subject Categories

Biochemistry | Molecular Biology

Keywords

3' cleavage and polyadenylation; ATP analogs; cleavage factor phoshorylation; mRNA 3' end formation; pre-mRNA 3' processing

Abstract

The 3' end cleavage/polyadenylation (3' processing) is important in generating a functional messenger RNA (mRNA) transcript. It is long-known that ATP can significantly stimulate the in vitro cleavage of adenovirus type 2 L3 (Ad2L3) RNA substrate. Here, we used ATP analogs in structure-activity assays to show that the structural features of ATP and its analogs determine in vitro 3' cleavage efficiency. More specifically, we demonstrate that the structure of the nitrogenous base, the nucleotide sugar and the triphosphate group contribute to the efficiency of the nucleotide-stimulated in vitro 3' cleavage reaction.

It was found that AppNp, an analog of ATP whose gamma phosphate cannot be transferred enzymatically or chemically to water (ATPase activity) and cannot serve as a phosphate donor for a protein kinase, is capable of stimulating cleavage. This finding led us to the conclusion that ATP likely functions as an allosteric activator of some protein involved in 3' cleavage activity. The structure-activity relationship studies we carried out also showed that AppCp, another ATP analog whose gamma phosphate cannot be transferred, was an in vitro 3' cleavage reaction inhibitor with an IC50 of approximately 300 μM. Taken together, our experiments provide indirect evidence that ATP (and certain analogs) stimulate 3' cleavage by simply binding to one of the 3' cleavage factors, and that their structures determine the efficiency of the 3' cleavage reaction. Using the in vitro cleavage reaction, site-directed mutagenesis and co-immunoprecipitation (co-IP) we furthermore show that cleavage factors CFIm25, PAP and Clp1 are not likely to be the agencies of ATP action.

From previous research in our lab we know that in vitro dephosphorylation of 3' processing proteins, specifically cleavage factors I and II (denoted as CFm when unseparated), results in the suppression of the 3' cleavage. We carried out experiments aiming to identify the phosphorylated cleavage factor(s) that can restore 3' cleavage activity to dephosphorylated HeLa cell nuclear extract (NxT). It was determined that immunopurified Flag epitope-tagged-CFIm25, -Clp1, -CFIm68, -CFIm59, and tag-independent immunopurified Pcf11, when used in place of CFm, were not capable of restoring 3' cleavage activity to dephosphorylated NxT, and therefore likely do not contain an activating amount of the unidentified phosphorylated cleavage factor.

Download

Share

COinS