Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China.
Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China.
Int J Mol Sci. 2022 Jan 11;23(2):743. doi: 10.3390/ijms23020743.
In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5'-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5'-UTR of vascular endothelial growth factor A () was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of gene by binding to the G4 structure in the IRES.
在真核生物中，mRNA 的翻译主要通过帽依赖性或帽非依赖性方式进行。后者主要在内源核糖体进入位点（IRES）起始于 mRNA 的 5'-UTR。已有报道称，IRES 元件中的 G-四链体（G4）结构可调节 IRES 活性。我们之前曾证实 RBM4（也称为 LARK）是人源 G4 结合蛋白。在这项研究中，为了研究 RBM4 是否通过与 IRES 元件内的 G4 结构结合来参与调节 IRES 活性，我们构建了血管内皮生长因子 A（）5'-UTR 中的 IRES-A 元件到双顺反子报告载体 psiCHECK2，并在 293T 细胞中检测 RBM4 对 IRES 活性的影响。结果表明，IRES 插入显著增加了 FLuc 表达活性，表明该含有 G4 的 IRES 在 293T 细胞中具有活性。当 IRES 中的 G4 结构被碱基突变破坏时，IRES 活性显著降低。用 G4 稳定剂 PDS 处理细胞时，IRES 活性显著增加。EMSA 结果表明，RBM4 特异性结合 IRES 元件中的 G4 结构。RBM4 的敲低显著降低了 IRES 活性，而过表达 RBM4 则增加了 IRES 活性。综合所有结果，我们证明了 RBM4 通过结合 IRES 中的 G4 结构促进了基因的 mRNA 翻译。
