Organic Phase Extraction vs Silica-based Purification
Compared to previous strategies, methods built upon consecutive phase extractions are independent of the sequential features of cognate RNAs and suitable to study the RBPome in prokaryotes such asEscherichia coli , Salmonella enterica andStaphylococcus aureus 2,14,15. Moreover, in contrast to titration-based techniques, phase partition methods require the lowest number of input cells for putative RBP identification. However, phenol separation can be technically challenging to perform without residual spill over between fractions, which may result in higher background contrasted with some other protocols if not performed carefully. Furthermore, it has been reported that some stable RNPs can be recovered from interphase fractions even without UV irradiation16. Organic phase extraction protocols are also inherently incapable of detecting RNA-binding activity in molecules with similar characteristics to those of RNPs; namely, glycoproteins17. Nevertheless, a significant advantage of employing organic phase extraction approaches is that they also enrich for cross-linked RNAs, making it possible to globally identify protein-binding sites within RNAs 14,15.
To complement the phase partition protocols, several studies have employed silica-based RNA-centric approaches. Positively charged silica matrices are routinely used to isolate nucleic acids by interacting with the phosphate groups in their backbone. Under stringent conditions, proteins are weakly bound and, consequently, they are easily removed by washes. Importantly, recent work discovered that this charge-based interaction was strong enough to retain RNA-protein adducts in human and yeast lysates 18,19. As ethanol favours RNA adsorption over DNA binding, the complex capture (2C) and total RNA-associated protein purification (TRAPP) procedures have coupled this isolation protocol to LC-MS/MS-based proteomics (Fig. 1) 18,19. Subsequently, these techniques have offered simple and quick alternatives for RBPome profiling in human cell lines, S. cerevisiae , E. coli and S. aureus2,18,19. It would be interesting to combine GRAD-Seq and SEC-seq with RBPome indexing methods as we envision that the fractionation of the cell lysates would further increase the sensitivity of RBPome approaches.