Mapping the UV Cross-linking Sites in RNA
To be able to sequence the cross-linked RNAs, they need to be converted
into cDNAs. For this purpose, the cross-linked protein is removed using
proteinase K. However, this treatment does not remove amino acids
cross-linked to the RNA, which can significantly impact the processivity
of the reverse transcriptase (RT) during cDNA synthesis. The
cross-linked amino acid residues generally cause the RT enzyme to drop
off 32, yielding truncated cDNAs (Fig.1, iCLIP26). Nevertheless, depending on the reaction
conditions, the reverse transcriptase can introduce mutations in cDNAs
at the site of cross-linking (Fig. 1, CRAC 23),
especially when using highly processive RT enzymes and/or
manganese-supplemented buffers 33. UV cross-linking
sites on the RNA can subsequently be determined at nucleotide resolution
by mapping the nucleotide positions where the RT fell off, or by mining
the data for mutations within reads. Alternatively, photoactivatable
ribonucleoside-enhanced CLIP (PAR-CLIP) uses photoactivable 4-thiouracil
or 6-thioguanine to enhance the cross-linking efficiency and use T-to-C
or G-to-A substitutions as a proxy for RNA cross-linking (Fig. 1)25. Conveniently, the PAR-CLIP protocol has been
modified for some microorganisms, such as the yeasts Saccharomyces
cerevisiae and Schizosaccharomyces pombe34,35, where the technique is now routinely applied.
However, adapting such metabolic labelling methods to prokaryotic model
systems has proved to be significantly more challenging10, which may explain why (at the time of writing)
only a single manuscript describes the application of PAR-CLIP in
bacteria 36. Additionally, prolonged exposure to the
mentioned nucleobase analogues can cause cellular toxicity10,26.