CircHIPK3 and FMRP compete for direct binding to BRCA1 mRNA controlling its protein levels and DNA damage response [RNA_pulldown]

Circular RNAs (circRNAs) are covalently closed RNA molecules widely expressed in eukaryotes and regulated in several pathological processes, including cancer. Many studies point to their mechanism of action as miRNA and protein sponges; however, we propose a new functionality based on circRNA-mRNA interaction to regulate mRNA fate. We show that the widely expressed circHIPK3 directly interacts in vivo with the BRCA1 mRNA through the back-splicing region. This interaction favoured the translation of BRCA1 by competing for the binding of the FMRP1 RNA-binding protein, which acts as a repressor of BRCA1 translation. CircHIPK3 depletion or disruption of the CircRNA-mRNA interaction decreased BRCA1 levels and increased DNA damage, sensitizing several cancer cell lines to DNA-damage-inducing agents and rendering them susceptible to synthetic lethality. Additionally, mimicking circHIPK3 interaction with lock-nucleic acid (LNA) oligonucleotides restores BRCA1 levels in hereditary breast cancer model cells, underscoring circRNA-mRNA interaction’s importance in regulating cell homeostasis and drug response. High-throughput sequencing of total RNA from circHIPK3 AMT-crosslinked pulldown performed in RH4 cells with two sets of biotynilated antisense probes (ODD and EVEN) targeting HIPK3 exon 2 and control biotynilated antisense probes targeting LacZ bacterial mRNA. Two biological replicates.

环状RNA（circular RNAs，circRNAs）是一类共价闭合的RNA分子，广泛表达于真核生物中，并参与包括癌症在内的多种病理过程的调控。既往多项研究认为其作用机制为作为微小RNA（microRNA，miRNA）与蛋白海绵；然而本研究提出一种基于circRNA-mRNA相互作用调控mRNA命运的全新功能机制。本研究证实，广泛表达的circHIPK3可通过反向剪接区域在体内与BRCA1 mRNA直接相互作用。该相互作用通过竞争结合FMRP1 RNA结合蛋白（该蛋白为BRCA1翻译的抑制因子），促进了BRCA1的翻译。敲低circHIPK3或破坏circRNA-mRNA相互作用，会降低BRCA1的表达水平并加重DNA损伤，使多种癌细胞系对DNA损伤诱导剂更为敏感，同时使其易发生合成致死。此外，使用锁核酸（lock-nucleic acid, LNA）寡核苷酸模拟circHIPK3的相互作用，可在遗传性乳腺癌模型细胞中恢复BRCA1的表达水平，这进一步凸显了circRNA-mRNA相互作用在调控细胞稳态与药物反应中的重要性。本研究在RH4细胞中开展circHIPK3 AMT交联下拉实验，使用靶向HIPK3外显子2的两套生物素标记反义探针（ODD与EVEN组），以及靶向大肠杆菌LacZ mRNA的生物素标记对照反义探针，对获取的总RNA进行高通量测序。实验包含两个生物学重复。