Abstract 2133: Investigating the molecular interaction between KRAS mRNA and RNA binding protein CRD-BP

Abstract

Abstract The Coding Region Determinant-Binding Protein (CRD-BP) is an RNA-binding protein which, among other functions, provides a protective effect for select target mRNA transcripts against degradation, effectively increasing their half-life leading to elevated level of gene expression. CRD-BP was first identified due to its inherent ability to bind to a specific coding region of c-myc mRNA. It has further been demonstrated to convey a protective effect to target transcripts, including GLI1, MITF, CD44, and KRAS as well as a host of other mRNAs. CRD-BP is normally under strict spatiotemporal regulation; expressed only during early developmental stages where rapid growth is necessary. However, CRD-BP re-expression has been reported to occur in a number of human cancers, suggesting its roles as an onco-protein. Abnormal expression or function of KRAS, a CRD-BP target, is implicated in some cancers; roughly 90% of pancreatic cancers, 60% of colorectal, and 50% of lung cancers, making KRAS an attractive target for cancer therapies and highlighting the significance of understanding the CRD-BP-KRAS mRNA interaction. To help understand the CRD-BP-RNA interaction, we have previously mutated the first glycine of the G-X-X-G motif in the KH domains of CRD-BP to an aspartate. Using electrophoretic mobility shift assay, we showed that mutation at any two KH domains, except the KH3 and KH4 di-domains, completely abrogated CRD-BP-c-myc and CD44 RNAs interactions. To understand the CRD-BP-KRAS RNA interaction, we used the identical site-directed KH mutants of CRD-BP to assess their binding profiles to a 185-nt KRAS RNA corresponding to a specific coding region of KRAS mRNA. Using EMSA, additional binding assessments were carried out in a systematic fashion to determine the smallest region of KRAS mRNA bound by CRD-BP. In addition, plasmids carrying these variants of FLAG-CRD-BP were transfected into HeLa cells. Immuno-precipitation (IP) using anti-FLAG was performed, followed by RT-qPCR to measure the relative amounts of KRAS 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-KRAS RNA interaction. These experiments and their results will be discussed in further detail upon presentation. Citation Format: Sebastian J. Mackedenski, Chow H. Lee. Investigating the molecular interaction between KRAS mRNA and RNA binding protein CRD-BP. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2133. doi:10.1158/1538-7445.AM2015-2133