Optimized RNA gel-shift and UV cross-linking assays for characterization of cytoplasmic RNA-protein interactions.
Considerable interest has recently focused on defining the mechanisms involved in the regulation of gene expression at the level of mRNA stability and translational efficiency. However, the assays used to directly investigate interactions between RNA and cytoplasmic proteins have been difficult to establish, and methods are not widely available. Here, we describe a robust method for RNA electrophoretic mobility shift and UV cross-linking assays that allows rapid detection of cytoplasmic RNA-protein interactions. For added convenience to new investigators, these assays use mini-gels with an electrophoresis time of 15-20 min, enabling a high throughput of samples. The method works successfully with many different probes and cytoplasmic extracts from a variety of cell lines. Furthermore, we provide a system to optimize characterization of the RNA-protein complex and troubleshoot most assay difficulties.
Thomson
A M
AM
Royal Perth Hospital, Australia.
Rogers
J T
JT
Walker
C E
CE
Staton
J M
JM
Leedman
P J
PJ
D000818:Animals; D001943:Breast Neoplasms; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics; D005293:Ferritins / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006493:Heparin / Q000378:metabolism; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis*; D016601:RNA-Binding Proteins / Q000032:analysis*; D006163:Ribonuclease T1 / Q000378:metabolism; D013972:Thyrotropin / Q000235:genetics; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D013972:Thyrotropin; D006493:Heparin; D005293:Ferritins; D066246:ErbB Receptors; D006163:Ribonuclease T1