采用MS2 pulldown和质谱分析法检测BT549细胞中的LncRNA SNHG15结合蛋白  

Identification of LncRNA SNHG15 binding proteins in BT549 cells using MS2 pulldown and mass spectrometry

作　　者：陈少英 彭佩 周艳春 顾巍 CHEN Shaoying;PENG Pei;ZHOU Yanchun;GU Wei(Department of Pathophysiology,Shantou University Medical College,Shantou 515041,Guangdong,China)

机构地区：[1]汕头大学医学院病理生理学教研室,广东汕头515041

出　　处：《癌变．畸变．突变》2024年第5期395-398,共4页Carcinogenesis,Teratogenesis & Mutagenesis

摘　　要：目的:鉴定乳腺癌BT549细胞中与长链非编码RNA SNHG15结合的蛋白。方法:用慢病毒感染乳腺癌BT549细胞,构建稳定过表达SNHG15的BT549-SNHG15-MS2细胞株。通过MS2 pulldown和质谱分析法,检测BT549细胞中与SNHG15结合的蛋白。随后采用RNA免疫共沉淀(RIP)和逆转录-定量PCR(RT-qPCR)法,以HEAD框肽5(DEAD box helicases 5,DDX5)为模板验证共沉淀蛋白和SNHG15的结合。结果:经MS2 pulldown和质谱分析筛选出386个潜在的SNHG15结合蛋白。RNA免疫沉淀结合RT-qPCR检测结果证实DDX5与SNHG15的结合。结论:本研究介绍了一种有效分析RNA-蛋白质相互作用的生物学方法,同时为深入研究SNHG15的作用机制提供了新的实验证据。OBJECTIVE:To identify proteins which interacted with long noncoding RNA SNHG15 in breast cancer cells.METHODS:BT549 cells stably overexpressing SNHG15 were established by infection with lentivirus expressing MS2bs-tagged SHNG15.MS2 pulldown and Mass Spectrometry were used to identify proteins associated with SNHG15.Using DDX5(DEAD box helicase 5)as an example,interactions of SHNG15 with identified proteins were further verified by RNA immunoprecipitation(RIP)and RT-qPCR.RESULTS:In total,386 proteins were identified which interacted with SNHG15.DDX5 was truly associated with SNHG15.CONCLUSION:These data indicated an effective method to purify RNA-associated proteins and provided new information for the further study of the function of SNHG15.

关 键 词：长链非编码RNA MS2 pulldown 小核仁RNA宿主基因15 RNA免疫沉淀 RNA结合蛋白 

分 类 号：Q291[生物学—细胞生物学]