High-throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites

Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.


Total RNA extraction
Total RNA was extracted from a sample of 1-5 mL of culture. Briefly, 1 mL of room temperature Trizol (Invitrogen) was used to resuspend each cell pellet upon which samples were left at room temperature for 5 min. Next, 200 µL of 24:1 chloroform: isoamyl alcohol were added to each sample and samples were inverted vigorously for 15 seconds prior to centrifugation at 13,000 xg for 10 min at 4ºC. The aqueous phase was then transferred to 2mL DNase/RNase-free microcentrifuge tubes. Equal volumes of 0.8 M sodium citrate and 1.2 M sodium chloride (~ 300 µL) were added to each sample and inverted for 1 min prior to overnight precipitation at -20ºC. The following day, samples were centrifuged at 13,000 xg at 4ºC for 30 min. The liquid was removed carefully as to not disturb the formed RNA pellet. RNA pellets were then washed twice with 75% ethanol (10 min 13,000 xg centrifugations at 4ºC). After the second wash, liquid was completely removed from the RNA pellet and sample was left to dry in a laminar flow hood for 10 min. Once dry, RNA pellets were resuspended in 30 µL nuclease-free water and RNA concentration was measured via spectrophotometry.
Total RNA samples (diluted to 200 ng/µL) were treated with RNAse-free DNase I (PI-90083 Thermo-Fisher Scientific) following manufacturer protocol. After DNase I treatment, 10 μL of GlycoBlue (AM9516 Life Technologies) was added to an equivalent volume solution of 100% isopropanol and DNase-treated RNA (total 55 μL) and precipitated at -20ºC overnight. The following day, samples were pelleted (15 min at 13,000 xg) and liquid was removed carefully. Next, samples were washed with chilled 95% ethanol. After the second wash, liquid was completely removed from the RNA pellet and sample was left to dry in a laminar flow hood for 10 min. Once dry, RNA pellets were resuspended in 50 µL nuclease-free water and stored at -80ºC. Finally, the quality of RNA was evaluated by using a bioanalyzer (Agilent) at the Genomic Sequencing and Analysis Facility (GSAF at UT Austin) to confirm that no significant degradation had occurred.

PCR Amplification and in vitro Transcription (IVT) for EMSA
Primers were designed to amplify sRNAs and mRNAs (Supplementary Table 5) (with an upstream T7 promoter) from previously-designed plasmids or genomic DNA (E. coli BW25113). Specifically, 4 random nucleotides (GACT) were added upstream of the T7 promoter sequence (TAATACGACTCACTATAGGGAGA). PCR product sizes were verified on a 1.5 % agarose gel prior to PCR clean up (GE Illustra GFX PCR). mRNAs were in vitro transcribed using the MEGA Script T7 kit following the manufacturer instructions. Specifically 1-1.5 pmol of DNA template was used per reaction, and each reaction was incubated for 6 hours. Following DNase digestion, one of two reliable RNA recovery methods was performed. If an RNA Clean and Concentrator-5 kit (Zymo Research) was used, 30 µL of nuclease-free water was added to the IVT reaction prior to following manufacturer instruction manual. In the case that RNA was recovered using a chloroform-based method, 115 µL of nuclease-free water and 15 115 µL of Ammonium Acetate Stop Solution (from MEGA Script T7 kit) were added and mixed. Next, two volumes of 25:24:1 phenol: chloroform: isoamyl alcohol were added to each sample and samples were inverted for one minute, then centrifuged at 13,000 xg at 4ºC for 5 min. The top aqueous phase was transferred to two equivalent volumes of 24:1 chloroform: isoamyl alcohol and samples were centrifuged at 13,000 xg at 4ºC for 5 min. The top phase was again transferred, this time to microcentrifuge tubes containing 1 mL isopropyl alcohol and 1.5 µL GlycoBlue each, and left to precipitate overnight at -20ºC.
The following day, samples were centrifuged at 13,000 xg at 4ºC for 15 min. The liquid was removed carefully as to not disturb the formed RNA pellet. One mL of 95% ethanol was added to each sample and the samples were centrifuged at 13,000 xg at 4ºC for 5 min. The liquid was again removed carefully as to not disturb the RNA pellet. One mL of 75% ethanol was added to each sample and the samples were centrifuged for at 13,000 xg at 4ºC for 5 min. The liquid was again removed and samples left to dry in a solvent hood for 2 hours. Once dry, 30 µL of nuclease-free water was added to re-suspend the RNA. RNA concentration was measured via spectrophotometry. To validate transcripts (length and lack of spurious transcripts), 3 µL of each RNA was mixed with 7 µL of nuclease-free water and 5 µL of RNA Loading Buffer II (NEB) and denatured for 5 min at 70ºC. Samples were loaded into an 8% urea gel (SequaGel, National Diagnostics) and run at 100 V for 3 hours. The gel was then stained using Sybr Green II (Thermo-Fisher Scientific) for 30 min and imaged under Sybr Green setting in a gel imager (ChemiDoc XRS +, BioRad).
sRNAs were prepared as were mRNAs with slight modifications to support internal P-32 labeling. Specifically, 0.5-1.5 µL UTP [α-32P] (3000Ci/mmol 10mCi/ml, 250 µCi, PerkinElmer) was added to replace unlabeled UTP from the MEGA Script IVT kit. RNA samples were re-suspended in 20 µL of nuclease-free water and, if RNA was recovered using the chloroform-based method, free NTPs