GRIL-Seq: a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base-pairing with partially complementary sequences of targeted transcripts. We present a simple, yet robust method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique (referred to as GRIL-Seq) is based on preferential ligation of sRNAs to the ends of base-paired targets in bacteria expressing the T4 RNA ligase followed by sequencing to identify the ligated chimeras. In addition to the RNA chaperone Hfq, the GRIL-Seq method depends on the activity of the pyrophoshorylase RppH. We used PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, to demonstrate that direct regulatory targets of this sRNA can be readily identified. Therefore, GRIL-Seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but also for better understanding the mechanistic details of their activity during post-transcriptional regulation of gene expression.