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.