title: GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation.
abstract: 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 method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique, referred to as global small non-coding RNA target identification by ligation and sequencing (GRIL-seq), is based on preferential ligation of sRNAs to the ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimaeras. In addition to the RNA chaperone Hfq, the GRIL-seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrated that direct regulatory targets of this sRNA can readily be identified. Therefore, GRIL-seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but also for uncovering novel roles for sRNAs and their targets in complex regulatory networks.
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Kook
authors.initials: K
authors.lastname: Han
authors.affiliation: Computer Science Department, Wellesley College, Wellesley, Massachusetts 02481, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Tjaden
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Lory