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.

Figure 1. T4 RNA ligase-catalyzed in vivo linking of sRNA to mRNAs

(a) Schematic of sRNA-target formation in cells expressing T4 RNA ligase and an sRNA. Also shown is the RT-PCR based strategy for the detection of chimeras between an sRNA and specific target mRNAs. (b) Effect of expression of T4 RNA ligase based on P. aeruginosa cell growth. The T4 RNA ligase was induced in P. aeruginosa (pKH13) when it reached an OD₆₀₀ ~0.5 (indicated by the arrow) and cell viability was monitored for an additional 4 hours. Error bars represent the standard deviation of the average of three biological replicates (c) Determination of optimal induction time for the detection of PrrF1-containing chimeras (white star). Amplicons were detected only, when during the PCR step, primers for the targets (sodB and PA4880, white stars), but not for non-targets (efp and PA3940) were used. PCR amplification of cDNA for a housekeeping gene, rpsL and two non-targets (efp and PA3940) was carried out to ensure the presence of equal amounts of cDNA of two non-target genes in all samples. Results are representative of duplicate experiments. (d) Sites of ligation between PrrF1 and sodB and PA4880. Arrows indicate the location of each junction (relative to the start of translation) based on the sequence of the amplicons indicated by the white stars in 1c.

Figure 2. Factors influencing the ligation of PrrF1 sRNA to the sodB and PA4880 target mRNAs

(a) Effect of mutations in PrrF1, indicated as M1(G83C), M2(G72C) and M3(C64G), predicted to be in its base-pairing region with sodB mRNA. (b) Requirement for a functional T4 RNA ligase in generating sRNA-mRNA chimeras. A mutation in the t4rnl1 gene was engineered, in the codon for lysine (K) position 99 changing it asparagine (N), creating a catalytically inactive T4 RNA ligase. (c) Northern blot analysis of the effects of mutations in the PrrF1 base-pairing region. The M2 or M3 mutations are unable to induce sodB mRNA degradation, while a smaller defect was seen with the M1 mutant of PrrF1. PCR amplification of cDNA for rpsL was carried out to ensure the presence of equal amount of cDNA. (d) Effect of PrrF1 M2 and M3 mutants and catalytically-inactive T4 RNA ligase on ligation of the sRNA to sodB. Ligation between sodB mRNA and PrrF1 (wt, wild type) was monitored by RT-PCR between sodB and wt PrrF1 and M2 and M3 mutants (lanes 2, 4 and 5, respectively) and the inactive (K99N) T4 RNA ligase (lane 3). (e) Requirement for an enzymatically active T4 RNA ligase for the detection of a PA4880 mRNA and PrrF1 chimeric product. (f) Expression levels of target and non-target mRNA in total RNA isolated from in vivo RNA ligated samples in three biological replicates. Error bars represent the standard deviation. Transcript levels were determined by quantitative qRT-PCR after induction of PrrF1 for 20 min when the cultures were at OD₆₀₀ ~1.5. (g) Requirement for the RppH and Hfq proteins for efficient ligation between PrrF1 and sodB (or PA4880), monitored by RT-PCR. All results are representative of at least duplicate experiments.

Figure 3. Overview of the GRIL-Seq method

(a) Ligation of two RNAs is carried out in cells carrying two compatible expression plasmids: pKH6, where the expression of an sRNA (indicated in black) is under the control of the arabinose-inducible P_BAD promoter, while in plasmid pKH13, the expression of the t4rnl1, coding for the T4 RNA ligase (indicated in green) is regulated by IPTG. For optimal ligation, T4 RNA ligase is induced first by addition of 1mM IPTG for 1hr and then the sRNA is expressed by the addition of 0.2% L-arabinose for 20 min. (b) Enrichment for transcripts containing the sRNA chimeras. Magnetic beads with attached oligo dT and poly(A)-tailed sRNA binding oligomer, target (red)-sRNA (black) chimeric RNAs are immobilized. The chimeric RNAs are recovered following DNase treatment. (c) The DNA library for Illumina sequencing is constructed using a commercially available kit (New England BioLab, NEBNext® Ultra^™ RNA Library Prep Kit). (d) The sequences of chimeric target-sRNA are collected and aligned to the reference genome. For the identification of targets and processing sites, coverage of the chimeric RNAs is determined using CLC Genome Workbench (ver. 6.0.1).

Figure 4. Identification of targets of PrrF1 sRNA using GRIL-Seq

(a) Coverage profiling of enriched chimeric PrrF1-target RNAs following mapping to PAO1Δprrf1Δprrf2 genome. Putative targets of PrrF1 sRNA are shown in red color peaks. Peak height represents the value of maximum coverage. Duplicate GRIL-Seq experiments (L1 and L2) are shown. (b) Enrichment of the chimeric RNAs containing sodB and gloA1. The coverage of sequenced chimeric reads corresponding to sodB or gloA1 are shown as red peaks. Data is from the L2 experiment. (c) Sequencing reads corresponding to chimeras between PrrF1 and sodB or gloA1 mRNA. For each sequencing read, the location of the junction between the ligated sRNA and mRNA, at a single nucleotide resolution, was identified. The percentage of each read in the various chimeras was also determined. The sequences shown in red are the most common sodB or gloA1 sequences found in the chimeras, while the PrrF1 sequence is shown as a black rectangle. (d) The predicted base-pairing region between PrrF1 and sodB or gloA1 generated using the IntaRNA algorithm. The Shine-Dalgarno (S/D) sequence and the AUG start codon are indicated as a box and red letters, respectively.

Figure 5. Comparison of the list of genes identified by GRIL-Seq and RNA-seq following overexpression of PrrF1

In the Venn diagram, the blue set represents the top 40 targets of PrrF1 as determined by their coverage following application of the GRIL-Seq method. The red set represents the top 167 differentially expressed genes (q-value < 2.0e-09) based on the relative abundance of transcripts in P. aeruginosa PAO1Δprrf1Δprrf2 and the same strain over-expressing PrrF1 by RNA-seq. Seventeen genes intersect both sets. Results are representative of duplicate experiments.