Rnase t1

The presence or absence of 3′ or 5′ phosphates on RNA cleavage products, and the protection of 3′ phosphates on FANA ligase substrates by formation of phosphorylimidazolides, was assayed by Urea-PAGE gel shift following incubation in rAPid alkaline phosphatase (Roche, Switzerland) or T4 polynucleotide kinase (NEB, USA) in manufacturer’s buffers for 30 min at 37°C. Hydrolysis of cyclic phosphates was achieved by incubation in 10 mM Glycine pH 2.5 for 10mins at room temperature. Partial alkaline hydrolysis of RNA substrates (denoted by ⁻OH) was achieved by incubation at 90°C in 50 mM sodium carbonate buffer (pH 9.2) for 10 mins. Partial RNase T1 digestion was was achieved by incubation at 55°C in 0.1 U/μl RNase T1 (Invitrogen / Life Technologies, USA) in 30 mM sodium acetate (pH 5) for 10 mins, then stopped in 7M Urea 1.5mM EDTA.

Cleavage product length was determined biochemically by comparing the gel migration of AcrVA1-triggered cleavage products with alkaline hydrolysis and RNase T1 digestion ladders of the matched untreated crRNA. Hydrolysis ladders were generated by incubating 15 nM 5’-radiolabeled crRNA at 95°C for 10 min in 1X alkaline hydrolysis buffer (Ambion). Reactions were quenched in 1.5X formamide loading buffer and immediately loaded to a urea-denaturing PAGE (0.5X TBE) gel. For RNase T1 digestion ladders, 15 nM 5’-radiolabeled crRNA were unfolded in 1X RNA sequencing buffer (Ambion) at 65°C for 5 min and cooled to ambient temperature before the addition of 1 U of RNase T1 (Ambion). After incubating at ambient temperature for 15 min, reactions were extracted in phenol-chloroform (pH 8.0) and stored in 1.5X formamide loading buffer before loading to a urea-denaturing PAGE (0.5X TBE) gel. For 3’ end chemistry identification, products from AcrVA1-triggered crRNA truncation reactions were extracted in phenol-chloroform (pH 8.0) before incubation with 10 U of T4 polynucleotide kinase (NEB) in 1X T4 polynucleotide kinase buffer (NEB) for 30 min at 37°C. Reactions were quenched with 1.5X formamide loading buffer for 3 min at 95°C and resolved on a 15% (v/v) urea-denaturing PAGE (0.5X TBE) gel and visualized by phosphoroimaging (Amersham Typhoon, GE Healthcare).

³²P-labeled SdsN₁₃₇ or SdsN₁₇₈ (∼2 nM) was incubated with purified Hfq (or equal volume of buffer) and 1 μg of yeast RNA (Ambion) in 1X RNA structure buffer (Ambion) in a total volume of 8 μl at 37°C for 15 min. Samples were mixed with RNase T1 (0.02 U, Ambion) or an equal volume of buffer and incubated at 37°C for 6 min. Inactivation/Precipitation Buffer (20 μl, Ambion) was added, and samples were placed at −80°C for ∼30 min. RNA pellets were collected by centrifugation, washed with 100 μl of 70% ethanol, air-dried and dissolved in 7 μl Gel Loading Buffer II. For the hydroxide (OH) ladder, 1 μl of ³²P-labeled SdsN₁₃₇ or SdsN₁₇₈ in 9 μl Alkaline Hydrolysis Buffer (Ambion) was incubated 5 min at 90°C. For the RNase T1 ladder, 1 μl of ³²P-labeled SdsN₁₃₇ or SdsN₁₇₈ in 9 μl Sequencing Buffer (Ambion) was denatured by incubating at 95°C for 1 min followed by cooling to 37°C. RNase T1 (0.1 U) was added, and the sample was incubated for 5 min at 37°C. For both ladders, the reactions were stopped by adding 12 μl of Gel Loading Buffer II. Samples (2 μl) were run on a 8% polyacrylamide-7M urea sequencing gel in 1X TBE. The gel was transferred onto Whatman filter paper, dried at 80° for 1 h, and imaged using the STORM 840.