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.