RIP-seq using ILF3 antibodies to reveal RNAs that binds to ILF3 protein. RIP-seq using ILF3 antibodies to reveal RNAs that binds to ILF3 protein

RNA pulldown assay showed that lncRNA UBE2CP3 could bind to ILF3 protein. In order to further verify the interaction between ILF3 and UBE2CP3, RNA Immunoprecipitation (RIP) assay was performed to identify the RNAs that binds to ILF3 protein. RIP-seq data verifies the interaction between ILF3 and UBE2CP3. Interestingly, UBE2CP3 could also binds to 3'UTR of IGFBP7 mRNA. Mechanismly, lncRNA UBE2CP3 and 3' UTR of IGFBP7 could forms an double-stranded RNA. Then, the RNA duplex could interact with the Double-Stranded RNA-Binding Protein ILF3. Overall design: The potent RNA was immunoprecipitated by ILF3 antibodies using RIP assay. Then, all the potent ILF3-binding target RNAs were identified by deep sequencing, using Illumina HiSeq 4000.

RNA下拉实验（RNA pulldown assay）结果显示，长链非编码RNA（long non-coding RNA, lncRNA）UBE2CP3可与ILF3蛋白结合。为进一步验证ILF3与UBE2CP3之间的相互作用，本研究开展了RNA免疫沉淀（RNA Immunoprecipitation, RIP）实验，以筛选可结合ILF3蛋白的RNA分子。RNA免疫沉淀测序（RIP-seq）数据证实了ILF3与UBE2CP3的相互作用。值得注意的是，UBE2CP3还可结合胰岛素样生长因子结合蛋白7（insulin-like growth factor binding protein 7, IGFBP7）mRNA的3'非翻译区（3' untranslated region, 3'UTR）。从机制层面分析，lncRNA UBE2CP3与IGFBP7 mRNA的3'UTR可形成双链RNA（double-stranded RNA, dsRNA）；该RNA双链可与双链RNA结合蛋白（double-stranded RNA-binding protein, dsRBP）ILF3发生相互作用。
实验整体设计：通过RNA免疫沉淀实验，利用ILF3抗体免疫富集潜在靶RNA，随后采用Illumina HiSeq 4000平台开展深度测序，以鉴定所有可与ILF3结合的靶RNA分子。