Development of PAR-CLIP to analyze RNA-protein interactions in prokaryotes

The ability to identify RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as “Crosslinking and Immunoprecipitation” (CLIP) has revolutionized the genome-wide discovery of RNA targets. Among the different versions of CLIP developed, the incorporation of photoactivable nucleoside analogs into cellular RNA has proven to be especially valuable, allowing for high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP). Although PAR-CLIP has become an established technique for use in eukaryotes, it has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we first investigated whether 4-thiouridine (4SU), a photoactivable nucleoside, can be incorporated into E. coli RNA. After determining 4SU incorporation into RNA, we developed suitable conditions for crosslinking of proteins in E. coli cells and for the isolation of crosslinked RNA. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA) - messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq. Based on our results, PAR-CLIP represents an improved method to identify the RNAs recognized by RNA-BPs in prokaryotes.