Singha Roy Aditya, Majumder Subhabrata, Saha Partha
Crystallography and Molecular Biology Division, Biophysical Sciences Group, Saha Institute of Nuclear Physics, Kolkata 700064, India.
Homi Bhabha National Institute, Mumbai 400094, India.
Biochemistry. 2024 Feb 9. doi: 10.1021/acs.biochem.3c00521.
RNA G-quadruplex (rG4) structures can influence the fate and functions of mRNAs, especially the translation process. The presence of rG4 structures in 5'-untranslated regions (5'-UTRs) of mRNAs generally represses translation. However, rG4 structures can also promote internal ribosome entry site (IRES)-mediated translation as one of its determinants. Here, we report the identification of an evolutionary conserved rG4-forming sequence motif at the extreme 5'-end of the unusually long 5'-UTR (1.7 kb) in the transcript of human gene encoding the cellular inhibitor of apoptosis protein-1 that promotes cell survival by suppressing apoptosis and is overexpressed in various cancer cells. Expectedly, NMR study, CD spectroscopy, and UV melting assay confirm the formation of a potassium ion-dependent intramolecular and parallel rG4 structure at the sequence stretch. Moreover, the G4-RNA-specific precipitation using biotin-linked biomimetic BioCyTASQ validates the formation of the rG4 structure in the 5'-UTR in cells. Interestingly, disruption of the rG4 structure in the 5'-UTR results in a dramatic reduction in translation of the downstream luciferase reporter in cells, suggesting a translation-promoting effect of the rG4 structure, contrary to many earlier reports. Furthermore, enhancement of translation by the rG4 structure occurs in an IRES-independent manner.
RNA G-四链体（rG4）结构可影响mRNA的命运和功能，尤其是翻译过程。mRNA的5'-非翻译区（5'-UTR）中rG4结构的存在通常会抑制翻译。然而，rG4结构也可作为其决定因素之一促进内部核糖体进入位点（IRES）介导的翻译。在此，我们报告在人类编码细胞凋亡蛋白-1抑制剂基因的转录本中，在异常长的5'-UTR（1.7 kb）的最5'-端鉴定出一个进化保守的形成rG4的序列基序，该蛋白通过抑制细胞凋亡促进细胞存活，且在各种癌细胞中过表达。不出所料，核磁共振研究、圆二色光谱和紫外熔解分析证实了在该序列片段处形成了钾离子依赖性的分子内平行rG4结构。此外，使用生物素连接的仿生BioCyTASQ进行的G4-RNA特异性沉淀验证了细胞中5'-UTR中rG4结构的形成。有趣的是，5'-UTR中rG4结构的破坏导致细胞中下游荧光素酶报告基因的翻译显著减少，这表明rG4结构具有促进翻译的作用，这与许多早期报道相反。此外，rG4结构促进翻译的过程是以不依赖IRES的方式发生的。
