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.