Alternatives to UV irradiation
Although beyond the scope of this article, we feel that it is important highlight recent approaches that have striven to surmount the issues associated with using UV to study protein-RNA interactions. On the one hand, techniques such as TRIBE (targets of RBPs identified by editing) or STAMP (surveying targets by APOBEC-mediated profiling) fuse RBPs of interest to RNA-editing enzymes that introduce nucleotide modifications as a proxy for RNA binding events 41,42. While these strategies minimise handling and input amounts, tagging proteins with modifying enzymes could jeopardise physiological activity and expression of the RBP or induce cytotoxicity by uncontrolled transcriptomic hyperedition. On the other hand, proteins can also be chemically cross-linked to RNA. Traditionally, formaldehyde is used for this purpose 43, but this treatment also links proteins to other proteins and DNA nucleotides located within 2 Å4,43. As a result, several groups have begun to develop more specific chemical cross-linkers that appear to have higher cross-linking efficiencies compared to UV (e.g., NHS-diazirine (SDA) and AMT-NHS 44,45). Yet, a limitation of these reagents is that they only react with lysine residues and, therefore, these chemicals do not fully circumvent the biases associated with UV irradiation. Nevertheless, this work represents a significant advancement in the field, and we hope that it will spur the development of a wide array of chemical cross-linkers.
Unlike UV, which tends to yield RNA-protein duplexes enclosing single-stranded RNA (ssRNA) species, chemical cross-linkers reportedly display uniform adduct formation patterns for single- and double-stranded RNA targets 45. We foresee that this feature will nurture future technical advances to investigate RNA-RNA interactions. To date, even despite its ssRNA bias, UV cross-linking has enabled successful retrieval of RNA-RNA associations from immunoprecipitated RBPs harbouring such interactions. Soon after being identified as a suitable tool to analyse ncRNA-mRNA interactions from yeast RNPs 23, CRAC and other existing CLIP-based protocols were amended to favour intermolecular ligation of complementary RNA species in RNA-RNA base-pairing hubs46. Resulting methods, such as CLASH (cross-linking, ligation and sequencing of hybrids), hiCLIP (RNA hybrid and individual-nucleotide resolution CLIP), and RIL-seq (RNA interaction by ligation and sequencing) 46–48, have used RBPs chaperoning RNA-RNA interactions as bait to map and functionally characterise non-coding transcripts. So far, these procedures have been used in systems including mammalian cells, E. coli , Salmonella and S. aureus 47–55, which unravelled large ncRNA-RNA interactomes in these organisms. Eventually, customising present protocols to replace UV irradiation by chemical cross-linking could verify and expand prevailing knowledge on the microbial and metazoan RNA-RNA interactome.