Identification of proteins specifically binding to ac4C RNA probes

As a newly identified mRNA modification, the regulation of ac4C remains largely unexplored. RNA-binding proteins (RBPs) that specifically binds to ac4C modification and mediate downstream cellular activities (readers) have not been reported yet. We synthesized acetylated and non-acetylated RNA probes by in vitro transcription. The sequences of the probes were segments of FUS and 18s rRNA, which contain ac4C sites as reported. A biotin-RNA pulldown assay and mass spectrometry were performed with HEK 293T cell lysates.

作为一种新发现的mRNA修饰，ac4C的调控机制仍未得到充分探索。能够特异性结合ac4C修饰并介导下游细胞活动的RNA结合蛋白（RNA-binding proteins, RBPs，即ac4C阅读器），目前尚未有相关报道。我们通过体外转录技术合成了乙酰化与非乙酰化的RNA探针，探针序列为已报道携带ac4C位点的FUS基因片段与18S核糖体RNA（18s rRNA）片段。我们使用HEK 293T细胞裂解液开展了生物素-RNA下拉实验与质谱分析。