Because of the limitations associated with CARIC/RIC, several groups have explored alternative approaches. Grad-seq established the earliest technical foundation for ribonucleoprotein (RNP) identification in bacteria in vivo 11,12. This method, which does not rely on UV, combines density gradient centrifugation with high-throughput RNA sequencing and mass spectrometry to assemble protein and transcript inventories for each fraction. Subsequent grouping of RNA populations with matching sedimentation patterns identifies likely target cohorts that can be further verified by pull-down assays in a few transcripts of each population. Ongoing methodological advances have expanded on this concept to give rise to SEC-seq 13, which pairs high-resolution size-exclusion chromatography with downstream transcriptomic clustering and bait-based validation. Overall, these unbiased approaches are producing hugely valuable data leading, for example, to the identification of a key bacterial RNA chaperone11. But, since they do not allow recognition of RBPs in direct association with RNA, UV cross-linking based techniques could be highly complementary. In this respect, the first UV-derived snapshots of bacterial RBPomes arrived with the advent of the orthogonal organic phase separation (OOPS) and phenol-toluol extraction (PTex) protocols, which phase-extract molecules depending on their physicochemical properties (Fig. 1) 14,15. Concurrently, the protein-cross-linked RNA extraction (XRNAX) approach employed a similar basis in human cells 16.
OOPS applies identical principles to those of standard acid guanidinium thiocyanate-phenol-chloroform RNA extraction procedure. Briefly, upon detergent and phenol addition to the aqueous phase, denatured proteins and lipids partition to the hydrophobic phenol phase and free RNA molecules migrate to the upper water-rich layer. Due to their intermediate solubility in these conditions, DNA and RNA-protein adducts are predominantly found in the interphase. After trimming the RNA of the RNP complexes in the interphase, downstream phenol extraction rounds enable RBPs recovery from the organic phase. In turn, performing an initial phenol: toluol phase separation at neutral pH, PTex exploits toluol’s higher insolubility to separate DNA and lipids, which shift to a phenol-based organic interphase, from soluble RNA, proteins and covalently linked RNPs in the aqueous phase. Following chaotropic treatment of the aqueous phase, phenol addition confines unfolded proteins and unbound RNA to the organic and aqueous phases respectively and, ultimately, allows direct RBPs precipitation from the interphase fraction.