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.