Quantitative identification of proteins that influence miRNA biogenesis by RNA pull-down-SILAC mass spectrometry (RP-SMS)

被引：6

作者：

Choudhury, Nila Roy ^{[1
,2
]}

Michlewski, Gracjan ^{[1
,2
,3
]}

机构：

[1] Univ Edinburgh, Wellcome Trust Ctr Cell Biol, Michael Swann Bldg, Edinburgh EH9 3BF, Midlothian, Scotland

[2] Univ Edinburgh, Div Infect & Pathway Med, Chancellors Bldg,49 Little France Crescent, Edinburgh EH16 4SB, Midlothian, Scotland

[3] Zhejiang Univ, Zhejiang Univ Univ Edinburgh Inst, 718 East Haizhou Rd, Haining 314400, Zhejiang, Peoples R China

来源：

METHODS | 2019年 / 152卷

基金：

英国惠康基金;

关键词：

RNA; RNA-binding protein; miRNA; miRNA biogenesis; RNA pull-down; Mass spectrometry; BINDING PROTEINS; MICRORNA BIOGENESIS; PROTEOME; INTERFERENCE; MATURATION; COMPLEX; ATLAS; DICER; KSRP;

D O I：

10.1016/j.ymeth.2018.06.006

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

RNA-binding proteins mediate and control gene expression. As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP-SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using in vitro or chemically synthesized RNAs and protein extracts derived from cultured cells.

引用

页码：12 / 17

页数：6