Abstract 4478: Characterization of the biochemical interaction of hnRNP A18 to thioredoxin transcript

Abstract

Abstract Introduction: The RNA-binding protein heterogenous ribonucleoprotein A18 (hnRNP A18) is a protein translation regulator found to be elevated in many cancers. Previous work from our lab has shown that under stress conditions, hnRNP A18 is able to bind and regulate the translation of a group of mRNAs, including thioredoxin (TRX), a redox-regulating protein implicated in promoting tumor progression. Additionally, the formation of functional hnRNP A18:TRX complexes increases de novo protein synthesis of TRX through the stabilization of its mRNA transcript. It has been reported that like most RNA-binding proteins, hnRNP A18 is a modular protein and is uniquely composed of a single N-terminal RNA binding domain (RBD) and a C-terminal arginine-glycine rich (RGG) domain. The RBD is able to dictate specificity and affinity of substrate binding, while methylation of the arginine residues in the RGG domain contributes to its RNA binding capability. The observed translational regulation of TRX by hnRNP A18 merits further studies into understanding the physical interaction between the two molecules. Methods/Results: To determine the region which hnRNP A18 interacts with thioredoxin, stable cell lines overexpressing the RBD, RGG or full length (FL) domains of hnRNP A18 were generated in human melanoma cells. Ribonucleoprotein immunoprecipitation assays were performed using specific antibodies to either the RBD or RGG domain epitope. Reverse transcription PCR data reveals the presence of TRX mRNA in the resultant immunoprecipitated materials, indicating that both the RBD and RGG domain of hnRNP A18 protein is able to independently bind TRX mRNA. Maximum binding was observed when both RBD and RGG regions were present. Furthermore, hnRNP A18-GST recombinant proteins were engineered to determine the exact region which hnRNP A18 binds to on its target mRNAs. TRX mRNA was systematically deleted and increasing amounts of hnRNP A18-GST protein was added to determine binding capability. Northwestern analysis reveals that hnRNP A18 binds to its consensus motif in the TRX 3’UTR and to a new 19 nucleotide motif downstream of the consensus site. Conclusion: Heterogenous ribonucleoprotein A18 is a stress-activated RNA-binding protein that functions in the translational regulation of TRX. Although the RBD and RGG domains are each independently able to physically interact with TRX, both regions are necessary for maximal binding. Furthermore, a new 19 nucleotide motif located downstream of the hnRNP A18 consensus motif was also found to be sufficient for binding. The binding interface between RNA-binding proteins and their target mRNAs hold significant importance in understanding how the two molecules interact and will offer new insights into developing novel drug therapeutics that disrupt ribonucleoprotein formation. Citation Format: Elizabeth Tsuying Chang, Palak Parekh, Eun Yong Choi, Ruiqing Yang, France Carrier. Characterization of the biochemical interaction of hnRNP A18 to thioredoxin transcript [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4478. doi:10.1158/1538-7445.AM2017-4478