RNA purification and Northern blotting

RNA was isolated from d.mel-2 cells using the SimplyRNA Cells Low Elution Volume kit and Maxwell 16 RSC instrument (Promega). The RNA was quantitated using a NanoDrop spectrophotometer (Thermo Scientific) and its integrity was assessed by gel electrophoresis. For northern blotting, total RNA (5 or 10 μg, as indicated in figure legends) was combined with 0.04 μg/μl Ethidium Bromide in sample buffer (23% formamide, 3% formaldehyde, 4.6 mM MOPS (3-(N-morpholino)propanesulfonic acid) pH 7, 1.1 mM sodium acetate, and 0.2 mM EDTA), and loading dye (2.1% glycerol, 4.2 mM EDTA and 0.01% Bromophenol Blue and Xylene Cyanol) and heated at 75°C for 10 min. RNA was electrophoresed through a 1% denaturing agarose gel containing 1.48% formaldehyde and 1× MOPS buffer (20 mM MOPS pH 7.0, 5 mM sodium acetate, and 1 mM EDTA). The gels were imaged using UV detection with a ChemiDoc (BioRad) prior to transfer to assess integrity, migration of the ribosomal RNA (rRNA) and equivalent loading of lanes. The RNA was then blotted onto Immobilon-Ny+ membrane (Millipore) overnight using capillary transfer in 20× SSC buffer (3 M NaCl and 300 mM sodium citrate), as previously described (25). The blot was then crosslinked with 120 J/cm² UV (λ = 254 nm) using a CL-1000 crosslinker (UVP). The blot was then either probed immediately or stored at 4°C.

Radioactive antisense RNA or DNA probes were used for Northern blot detection. Transcription templates for Nluc and FFluc antisense RNA probes were PCR-amplified using DNA oligonucleotides with a T7 RNA polymerase promoter appended to the antisense strand, described in Supplementary File 1. Using these templates, in vitro transcription was performed for 10 min at 37°C with the T7 MAXIscript transcription kit (Thermo-Fisher) in the supplied 1× Transcription Buffer with 1 μg of DNA template, 0.4 mM final concentration of ATP, CTP, GTP and 8 μM UTP, 2 μl of 800 Ci/mmol 10 mCi/ml 12.5 μM UTP α-³²P (1 μM, 10–20 μCi final) (PerkinElmer), and 30 units T7 RNA polymerase in a 25 μl reaction. Next, 1 μl Turbo DNase (2 U) (Thermo-Fisher) was added to the reactions for 10 min at 37°C and then 1 μl of 250 mM of EDTA and 250 mM EGTA was added to the reaction. The probes were purified using a G25 sephadex (GE Life Sciences) spin-column. To detect 18S rRNA, 1.7 μg of 18S rRNA deoxy-oligonucleotide antisense probe (see Supplementary File 1) was phosphorylated using 2 μl of 6000 Ci/mmol, 150 mCi/ml, 25 μM ATP γ-³²P (2.5 μM final, 25 to 100 μCi) (PerkinElmer) and 40 units of T4 Polynucleotide Kinase (New England Biolabs) in a 20 μl reaction incubated at 37°C for 40 min. The probe was then purified with G25 Sephadex column.

For anti-sense Nluc and FFluc probes, 2.5–7.5 × 10⁶ total cpm was added to the blot that had been pre-hybridized for 45 min at 68°C in 8 ml of ULTRAhyb hybridization buffer (Invitrogen). The blot was then incubated with probe at 68°C overnight, washed two times sequentially with 2 ml each of 2× SSC, 0.1% SDS, and then two more times with 0.1× SSC, 0.1% SDS at 68°C for 15 min each wash. For 18S rRNA probes, 5–6 × 10⁶ cpm was added to the blot that had been pre-hybridized with 8 ml ULTRAhyb-Oligo hybridization buffer (Invitrogen) at 42°C. The blot was incubated with probe overnight and then washed twice with 25 ml 2× SSC containing 0.5% SDS for 30 min each wash at 42°C. Blots were then exposed to phosphor screens and visualized using a Typhoon FLA phosphorimager (GE Life Sciences) and analyzed using ImageQuant TL software (GE Life Sciences). Background signal was subtracted using the ‘Rolling Ball’ method in ImageQuant.

Nluc and FFluc levels were measured in phosphorimager units (PIU). For analysis of steady state reporter mRNA levels, fold change was determined in the same manner as described for the reporter activity measurements, first normalizing Nluc signal to the corresponding FFluc signal in that sample, and then calculating the log₂ fold change relative to the negative control effector/condition. In tethered function assays, MS2-EGFP served as the negative control for normalization of the effectors. In the full-length Pum experiment, the RNA-binding defective mutant, Pum mut R7, served as the negative control effector.

For experiments measuring effect of endogenous Pum and corepressors on reporter mRNA levels, Northern blot data was analyzed in two ways. First, the values of the Pum regulated Nluc 3× PRE were divided by the Nluc ΔPRE reporter to normalize Pum specific activity to global effects on gene expression. From this data, the log₂ fold change was calculated relative to non-targeting control (NTC) negative control RNAi. In the second approach, the RRR of Nluc 3×PRE reporter mRNA was normalized to the RRR of Nluc ΔPRE reporter mRNA within each RNAi condition. From these RRR values, the log₂ fold change in Nluc 3× PRE mRNA was calculated relative to NTC.

To measure RNA decay rates, Nluc signal was normalized to stable 18S rRNA signal for each sample to adjust for potential variation in loading and transfer of RNA in each lane over the time courses. The fraction of reporter mRNA remaining at each time point was plotted relative to time in minutes after Actinomycin D addition. Half-lives and statistical parameters were calculated as described below. Mean mRNA half-lives and 95% credible intervals are reported for each experiment.

High resolution Northern blotting was performed to analyze Nluc 2× MS2 pA and HSL reporter mRNAs. First, 3 μg of total RNA was heated at 70°C with 20 pmol of antisense Nluc cleavage oligo RA 296 (see Supplementary Table S1) in a 30 μl reaction containing 200 mM KCl and 1 mM EDTA. In control reactions that remove the poly(A) tail (the A₀ control) 1.5 μg of oligo deoxythymidine (dT) was included. Reactions were then cooled at room temperature for 20 min. Next, 5 units of RNase H (New England Biolabs) in 20 mM Tris–HCl pH 8.0, 28 mM MgCl₂ and 48 units of RNasin (Promega) were added and reactions were incubated at 37°C for 1 h. Next, EDTA (final 30 mM) was added and reactions were incubated for 15 min at 37°C. The RNA was then purified with Clean and Concentrator–25 kit (Zymo). Next, 1.2 μg of purified RNA was combined equal volume (15 μl) of RNA loading buffer (88% formamide, 0.025% Bromophenol blue, 0.025% xylene cyanol, 10 mM EDTA and 0.025% SDS), heated for 10 min at 75°C. Samples were then electrophoretically separated on a 5% poly-acylamide, 1× Tris–borate–EDTA (TBE), 8 M urea gel (BioRad) that had been pre-run at 20–25 mA, 200 V, in 1× TBE buffer (89 mM Tris, 89 mM Boric acid and 2 mM EDTA). Next, the RNA was transferred onto Immobilon-Ny+ Membrane (Millipore) for 45 min in 0.5× TBE buffer at 60 V at 4°C using a Trans-Blot Cell (BioRad). The blot was crosslinked with 120 J/cm² UV (λ = 254) and probed with a radioactive, antisense 2× MS2 RNA probe (see Supplementary File 1 for primers).