Gel RNA annealing assay
The gel annealing assay protocol was adapted from Doestch et al. (68),
except manganese was used instead of magnesium in the FRET buffer
(69,70). The assay utilizes two short unstructured complementary 21-mer
RNAs, termed J1 and M1, that are labeled with Cy5 and Cy3, respectively,
and are found in Table S3. All RNAs were synthesized by Dharmacon. RNA
annealing reactions were performed in 1× MnCl2 FRET
buffer (50 mM Tris-HCl, pH 7.5, 1 mM MnCl2, 10 mM NaCl,
1 mM DTT) at 37°C. Two controls were included with each annealing assay.
Control 1 (C1) is single stranded M1 RNA (10 nM M1, 1×
MnCl2 FRET Buffer) and control 2 (C2) is preformed J1M1
dsRNA (10 nM M1, 10 nM J1, 1×MnCl2 FRET Buffer). C2
dsRNA was formed by heating the two RNAs (J1 and M1) at 95°C for 2 min
and then slowly cooling to room temperature. A master mix for the
annealing reaction was assembled (10 nM J1 RNA in 1×
MnCl2 FRET buffer) on ice and moved to 37°C to
equilibrate. A time point zero aliquot (10 µl) was taken from the master
mix, added to M1 RNA and 2.5 µl of 5× stop/load buffer (150 mM EDTA, 2%
SDS, 15% (w/v) sucrose, 12.5% (w/v) Ficoll 400, 0.2 mg/ml yeast tRNA
and 3 µM of unlabeled M1 (uM1) and immediately loaded into a running
(100V) 20% native polyacrylamide gel (20% TBE, 12 well, Novex precast
gel). M1 RNA was added to the RNA annealing master mix to start the
annealing reaction. Time points were withdrawn from the annealing
reaction, stopped with 5× stop/load buffer and loaded on the running
gel. To track the progression of the running gel, a lane was loaded with
5× stop/load buffer with the addition of Gel loading buffer II
(Invitrogen). Proteins were added to reactions for a final concentration
of 1.2 µM, which is similar to protein concentrations used by others to
test for non-specific RNA chaperone activity (18,21,68,71). Gels were
run at 100 V at 4°C for 3 h. RNA was visualized using Cy3 and Cy5
channels on an Azure Sapphire using the SmartScan feature and overlays
for each channel were used for gel images. Quantification of band
intensity was measured using the rectangle and gel plot function in
ImageJ. The rate of dsRNA production was quantified by plotting the
amount of dsRNA (J1M1) divided by the total RNA (J1M1, J1uM1) over time,
which was normalized to the zero time point and graphed using GraphPad
Prism with linear regression analysis. The amount of RNA was quantified
using the Cy5 channel. Differences
between the assay endpoints (at 15 min) for each annealing reaction were
determined using Welch’s t -test (GraphPad Prism).