独立于上限,或真核起始因子(eIF)4E非依赖性,真核生物中的翻译起始需要支架蛋白eIF4G或其同源物,死亡相关蛋白5(DAP5)。eIF4G与40S核糖体亚基相关,将核糖体招募到RNA转录物中。RNA转录物的一个子集,如成纤维细胞生长因子9(FGF-9),包含直接绑定DAP5或eIF4GI的5个非翻译区(5个UTR)。内部核糖体进入位点(IRES)样不依赖帽的翻译起始不需要eIF结合的不成对5'端,因为这些eIF在起始密码子处或附近招募40S核糖体。对于病毒mRNA,eIF募集通常利用RNA结构,如假结或茎环,RNA解旋酶eIF4A是DAP5或4G介导的翻译所必需的,这5个UTR是结构化的。然而,对于类似于IRES的蜂窝翻译,尚未鉴定eIF结合的共有RNA结构或序列。FGF-9是在乳腺癌和结肠直肠癌细胞中被帽独立上调的mRNAs子集的成员。可能使用类似IRES的机制。然而,FGF-95'UTR内的DAP5结合位点未知。此外,DAP5绑定到其他,不同的5个UTR,其中一些需要接近未配对的,可访问的5'端刺激独立于上限的翻译。使用SHAPE-seq,我们在体外模拟了186-ntFGF-95'UTRRNA的复杂二级结构。Further,DAP5足迹,Toeprinting,和UV交联实验鉴定DAP5-RNA相互作用。FGF-95'UTR三级结构的建模在预测结构的一面上比对DAP5相互作用的核苷酸。我们认为RNA结构涉及三级折叠,而不是保守序列或二级结构,作为DAP5结合位点。DAP5似乎接触起始密码子附近的核苷酸。我们的发现为寻找与帽无关的翻译增强剂提供了新的视角。结构,而不是序列特异性,eIF结合位点可以作为有吸引力的化疗靶标或作为基于mRNA的治疗的剂量工具。
Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5\' untranslated regions (5\' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5\' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5\' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5\' UTRs, some of which require an unpaired, accessible 5\' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5\'-UTR RNA\'s complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5\'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.