Figure 2

RNA-centric methods for the purification and identification of RNA-binding proteins. (a) Examples of purification schemes for RNA-binding proteins using in vitro and in vivo approaches. For in vitro approaches, a tagged RNA construct is generated and bound to a solid support. In this example the MS2 protein-RNA interaction tagging method is shown with the target RNA (red), MS2-binding motif (purple) and MS2 protein (gray). Cell lysate is prepared and proteins from lysate are captured using the tagged RNA in vitro. For in vivo approaches, the target RNA is crosslinked to specific interacting RNA-binding proteins in living cells using UV, formaldehyde or other cross-linkers. Cells are lysed and the RNA-protein complexes captured from solution. In both scenarios, the complex is washed to remove non-specific interactions (green proteins). Finally the bound proteins are eluted. (b) MS is commonly used to identify the RBPs in a purified sample. In non-quantitative MS approaches, RBPs are purified from unlabeled cell material using either an RNA of interest or a control construct. After separation by one-dimensional gel electrophoresis, specific protein bands from the sample are selected, excised and identified by MS analysis. In quantitative MS approaches, proteins are differentially labeled based on their initial cell populations. Experimental and control purifications are performed on these labeled populations and the purified RBPs are pooled to create a single sample. MS analysis allows direct comparison of labeled peptides, which can then be quantified to determine specific proteins in the sample compared with the control. SILAC, stable isotope labeling by amino acids in cell culture.