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.