RNA Group/Groupe ARN, Département de biochimie et de génomique fonctionnelle, Pavillon de Recherche Appliquée au Cancer, Université de Sherbrooke, 3201, Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
RNA Group/Groupe ARN, Département de biochimie et de génomique fonctionnelle, Pavillon de Recherche Appliquée au Cancer, Université de Sherbrooke, 3201, Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
Biochimie. 2020 Sep;176:122-127. doi: 10.1016/j.biochi.2020.06.013. Epub 2020 Jul 4.
The protein "BCL-2-associated athanogene-1" (BAG-1), which exists in multiple isoforms, promotes cancer cell survival and is overexpressed in many different cancers. As a result, BAG-1-targeted therapy appears to be a promising strategy with which to treat cancer. It has previously been shown that the 5'UTR of the BAG-1 mRNA contains a guanine rich region that folds into a G-quadruplex structure which can modulate both its cap-dependent and its cap-independent translation. Accumulating data regarding G-quadruplex binding proteins suggest that these proteins can play a central role in gene expression. Consequently, the identification of the proteins that could potentially bind to the G-quadruplex of the BAG-1 mRNA was undertaken. Label-free RNA pulldown assays were performed using protein extracts from colorectal cancer cells and this leads to the detection of RNA G4 binding proteins by LC-MS/MS. The use of G-quadruplex containing RNA, as well as of a mutated version, ensured that the proteins identified were specific for the RNA G-quadruplex structure and not just general RNA binding proteins. Following confirmation of the interaction, the Small Nuclear Ribonucleoprotein Polypeptide A (SNRPA) was shown to bind directly to the BAG-1 mRNA through the G-quadruplex, and knock down experiments in colorectal cancer cells suggested that it can modulate the expression level of BAG-1.
“BCL-2 相关的抗凋亡基因 1”（BAG-1）蛋白存在多种异构体，可促进癌细胞存活，在许多不同的癌症中过度表达。因此，BAG-1 靶向治疗似乎是一种很有前途的治疗癌症的策略。先前已经表明，BAG-1 mRNA 的 5'UTR 含有一个富含鸟嘌呤的区域，该区域折叠成 G-四链体结构，可调节其帽依赖性和非依赖性翻译。关于 G-四链体结合蛋白的累积数据表明，这些蛋白可能在基因表达中发挥核心作用。因此，进行了鉴定可能与 BAG-1 mRNA 的 G-四链体结合的蛋白的研究。使用来自结直肠癌细胞的蛋白质提取物进行无标记 RNA 下拉测定，通过 LC-MS/MS 检测到 RNA G4 结合蛋白。使用含有 G-四链体的 RNA 以及突变版本，确保鉴定出的蛋白特异性结合 RNA G-四链体结构，而不仅仅是一般的 RNA 结合蛋白。在确认相互作用后，发现小核核糖核蛋白多肽 A（SNRPA）可通过 G-四链体直接与 BAG-1 mRNA 结合，并在结直肠癌细胞中的敲低实验表明，它可以调节 BAG-1 的表达水平。
