A 5 ' Utr Rna G-Quadruplex Structure Affects Both The Cap-Dependent And Independent Translation Of Bag-1, An Mrna Involved In Colorectal Cancer

Background: BAG-1 is a gene coding for an anti-apoptotic protein known to be over-expressed in colorectal cancer. This protein is present in three isoforms: BAG-1L, -1M, and -1S (long, medium, and short). The isoforms result from alternative translation initiations at three in-frame start codons and thereby differ only by the length of their N-terminal domains. Translation regulation of this mRNA implies a non-canonical start codon CUG (for -1L isoform) and the presence of an IRES (Internal Ribosome Entry Site) for the -1S isoform. We measured the mRNA and protein expression levels of BAG-1 in tissue samples of colorectal adenomas and tumors stages 1 to 4 compared to their healthy margins. We observed no correlation between mRNA levels and protein expressions, suggesting a post-transcriptional regulation of BAG-1. We predicted the formation of a G-quadruplex structure (G4) at the very beginning of the BAG-1 mRNA 59untranslated region (59 UTR), upstream of all its known translation regulatory motifs. G4s are very stable non-canonical secondary structures made by the stacking of g-tetrads, a co-planar array of 4 guanines linked by Hoogsteen hydrogen bounds and stabilized by potassium. Generally, G4s present in 59 UTR are recognized as translation repression motifs because of their stability impairing ribosome scanning, but some examples are known to be part of IRES structures and favor cap-independent translation. Purposes of the Study: The aim of our study is to confirm the predicted G4 formation and to probe its structural impact on the whole 59 UTR, as well as measuring its effect on both cap-dependent and independent translation of the three BAG-1 protein isoforms. Experimental Procedures and Results: Several in vitro probing analyses, such as In-line probing, Reverse transcriptase stop assay and Selective 29-hydroxyl acylation analyzed by primer extension (SHAPE), confirm the formation of the G4 structure and the global 59 UTR folding. Dual-luciferase reporter assays and RT-qPCR in colorectal HCT116 cells, comparing the WT sequence with a G/A-mutant where G4 formation is abolished, demonstrate that the G4 formation represses the cap-dependent translation of all three isoforms without affecting mRNA levels. On the other hand, bicistronic luciferase reporter assays show that the G4 favors cap-independent translation. With the objective of modulating G4 formation in cellulo, cells were treated with chemical ligands (cPDS, Phen-DC3 and TmPyP4) which are specific G4 stabilizers. The endogenous levels of the BAG-1 protein isoforms is decreased following these treatments as measured by Western blotting. Conclusions : In summary, the G4 folding at the very beginning of the BAG-1 59 UTR affects its whole structure and potentially modulates the IRES structure downstream. The G4 formation has a repression effect on cap-dependent translation and oppositely a favorable effect on cap-independent translation. To our knowledge, this is the first case of a 59 UTR G4 affecting both types of translations on the same transcript. Targeting the structure with ligands shows that G4 modulation can affect protein translation level and thus could be potentially used to modify the phenotype of the colorectal tumor cells. Citation Format: Rachel Jodoin, Jean-Pierre Perreault, Martin Bisaillon. A 59 UTR RNA G-Quadruplex structure affects both the cap-dependent and independent translation of BAG-1, an mRNA involved in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A08.