RNase-assisted RNA chromatography.
RNA chromatography combined with mass spectrometry represents a widely used experimental approach to identify RNA-binding proteins that recognize specific RNA targets. An important drawback of most of these protocols is the high background due to direct or indirect nonspecific binding of cellular proteins to the beads. In many cases this can hamper the detection of individual proteins due to their low levels and/or comigration with contaminating proteins. Increasing the salt concentration during washing steps can reduce background, but at the cost of using less physiological salt concentrations and the likely loss of important RNA-binding proteins that are less stringently bound to a given RNA, as well as the disassembly of protein or ribonucleoprotein complexes. Here, we describe an improved RNA chromatography method that relies on the use of a cocktail of RNases in the elution step. This results in the release of proteins specifically associated with the RNA ligand and almost complete elimination of background noise, allowing a more sensitive and thorough detection of RNA-binding proteins recognizing a specific RNA transcript.
Michlewski
Gracjan
G
Medical Research Council Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh EH4 2XU, United Kingdom. Gracjan.Michewski@hgu.mrc.ac.uk
Cáceres
Javier F
JF
D000906:Antibodies / Q000378:metabolism; D002845:Chromatography / Q000379:methods*; D004722:Endoribonucleases / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D011506:Proteins / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism
D000906:Antibodies; D008024:Ligands; D011506:Proteins; D016601:RNA-Binding Proteins; D012313:RNA; D004722:Endoribonucleases; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic