Defining Microbial RBPomes: Phase-separation vs Silica-based
Strategies
The development of several RBPome profiling methods was inspired by
well-established whole-cell RNA extraction protocols: the
RNA-interactome capture (RIC) procedure employs oligo-dT beads which
hybridised to polyadenylated transcripts 7. Given that
technique specifically detects proteins bound to eukaryotic messenger
RNAs, it is unsuitable to examine RBPs recognising transcripts lacking
(long) poly-A tails (e.g., eukaryotic ribosomal, transfer and small
nucleolar RNAs as well as prokaryotic transcripts in general). Click
chemistry-based methods (click chemistry-assisted RNA-interactome
capture, CARIC; RNA interactome using click chemistry, RICK) have sought
to circumvent this drawback by treating cell with synthetic uridine
analogues. Once incorporated in newly transcribed RNAs, these analogues
can be chemically linked to biotin using click chemistry and
immunoprecipitated by streptavidin-conjugated beads8,9. More recently, a newly identified click
nucleoside analogue was shown to be compatible with metabolic labelling
in many bacteria (2′-deoxy-2′-azidoguanosine (AzG)10), making it possible to adapt CARIC/RICK based
approaches to prokaryotes. Nonetheless, it is important to consider that
these nucleotide analogues can be cytotoxic and alter the transcriptome.