Characterizing RNA-protein interaction using cross-linking and metabolite supplemented nuclear RNA-immunoprecipitation.
RNA-immunoprecipitation (RNA-IP) is a method used to isolate and identify RNA molecules specifically associated with an RNA-binding protein. Non-coding RNAs are emerging as key regulators of many biological and developmental pathways and RNA-IP has become an important tool in studying their function(s). While RNA-IP is successfully used to determine protein-RNA interaction, specific details regarding the level of this association and the metabolic requirement of this interaction which can influence the success of RNA-IP remain unclear. Here, we investigate the conditions required for efficient nuclear RNA-IP using Arabidopsis AGO4 (Argonaute 4) and siRNA binding as the study model. We showed that formaldehyde cross-linking, but not UV cross-linking, allowed for efficient pull-down of 24-nt siRNAs, suggesting that AGO4-siRNA interaction involves other protein(s). We also showed that, while formaldehyde cross-linking could also be performed on purified nuclei, ATP supplementation to the nuclei isolation buffer was needed to efficiently pull down 24-nt siRNAs. This result indicates that ATP is required for efficient siRNA loading onto AGO4. As most of the known RNA-mediated regulatory processes occur in the nucleus, our findings on cross-linking conditions and metabolite requirement for successful AGO4 nuclear RNA-IP provide a valuable insight and future consideration when studying the function of protein-RNA interactions in plants.
Black Mountain Laboratories, Commonwealth Scientific and Industrial Research Organisation Plant Industry, GPO 1600, Canberra, ACT, 2601, Australia.
Phil Chi Khang
PC
Au
Chris
C
Helliwell
Ming-Bo
MB
Wang
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents; D047468:Immunoprecipitation / Q000379:methods; D011485:Protein Binding; D015138:RNA, Nuclear / Q000378:metabolism*; D018749:RNA, Plant; D016601:RNA-Binding Proteins / Q000378:metabolism*
D029681:Arabidopsis Proteins; D003432:Cross-Linking Reagents; D015138:RNA, Nuclear; D018749:RNA, Plant; D016601:RNA-Binding Proteins