Abstract 2338: Functional investigation of the CRD-BP hnRNP-K-Homology domains for C-myc messenger RNA interaction

Abstract

Abstract The coding region determinant binding protein (CRD-BP) is an oncofetal protein, being ubiquitously expressed during embryogenesis, yet highly repressed in adult tissues, except in cancerous tissues [Bell et al, 2013; Mueller-Pillasch et al, 1999]. CRD-BP was inherently identified by its ability to physically associate with c-myc mRNA, and has been shown to protect the transcript from degradation, hence prolonging its mRNA half-life [Prokipcak et al, 1994, Noubissi et al, 2006]. In addition to c-myc mRNA, several other mRNAs bound by CRD-BP have gene products implicated in the development and progression of cancer [Bell et al, 2013]. As an RNA binding protein (RBP), CRD-BP is the Mus musculus variant and homologous to the Homo sapiens IMP-1/Insulin-Like Growth Factor 2 with 99.31% similarity, both categorized into a larger family named VICKZ (Vera/Vg1 RBP, Insulin-Like Growth Factor 1,2,3/IMP-1,2,3, CRD-BP, KOC, and ZBP-1). The VICKZ family of RBPs contains two RNA Recognition Motifs (RRM) and four heterogeneous nuclear ribonucleoprotein [hnRNP]-K-Homology (KH) domains. Deletion analysis had shown that the KH domains, and not the RRM domains, of IMP1 are important for binding RNA substrates. To understand which of the KH domains are critical for interaction with RNA, site-directed mutagenesis was performed to introduce an aspartic acid (D) at the first glycine (G) of the G-X-X-G region in the KH domains of CRD-BP. This D-X-X-G variation was individually introduced to the four KH domains of CRD-BP. These four CRD-BP KH domain variants were expressed, purified and assessed for ability to bind c-myc mRNA using electromobility shift assays (EMSA). In addition, plasmids carrying these variants of FLAG-CRD-BP were transfected into HeLa cells. Immunoprecipitation (IP) using anti-FLAG was performed, followed by RT-qPCR to measure the relative amounts of c-myc mRNA associated with each of the variants as compared to the controls. The in vitro EMSA and IP cell data were all consistent with the notion that all KH domain(s) are critical for CRD-BP-c-myc RNA interaction. Using EMSA, additional binding assessments were carried out in a systematic fashion to determine the smallest region of c-myc mRNA bound by CRD-BP. These results will be presented and discussed, as they provide insight into the role played by the KH domains of CRD-BP in binding RNA, as well as what RNA characteristics are important for binding to CRD-BP. Citation Format: Gerrit van Rensburg, Mark Barnes, Chow Lee. Functional investigation of the CRD-BP hnRNP-K-Homology domains for C-myc messenger RNA interaction. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2338. doi:10.1158/1538-7445.AM2014-2338