Nuclear Import Activity of Small RNA Molecules Generated In Vitro

<div><p>(A) Nuclear import of YOYO-1–labelled RTCs in permeabilized HeLa cells in the presence of 1× energy-regenerating system and Fr2 (100 ng), the indicated small RNAs generated by in vitro T7 transcription (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-t001" target="_blank">Table 1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-sd001" target="_blank">Protocol S1</a>, and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-st002" target="_blank">Table S2</a>) (140 nM each, corresponding to ~100 ng), human tRNA^Lys1,2 (100 ng), bovine tRNAs (100 ng), or buffer (ctr–). Nuclear import of the G3 RNA molecule in the absence of YOYO-1–labelled RTCs (G3 − RTC) was used as an additional negative control.</p> <p>(B) Quantification of RTC nuclear import as shown in (A). Images acquired by confocal microscopy were analyzed by MetaMorph software version 4.5r4 (Universal Imaging Corp) and the total fluorescence of the nuclei divided by the number of cells per field (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040332#pbio-0040332-sg002" target="_blank">Figure S2</a>). At least 150 cells were counted per experiment. Bars represent the log mean fluorescence per nucleus ± standard deviation of six independent experiments.</p> <p>(C) Quantification of G2/G3 RNA in HSE NA and 60S NA by Northern blot. RNA was separated by 15% denaturing PAGE, transferred onto a nylon membrane, and probed with an oligonucleotide complementary to G2/G3 RNA. There were 100-ng G3 RNA (lane 1), 10-ng G3 RNA (lane 2), 100-ng negative control A9 RNA (tRNA^Gly) (lane 3), 700-ng HSE NA (lane 4), 350-ng HSE NA (lane 5), 175-ng HSE NA (lane 6), and 200-ng 60S NA (lane 7). Upper panel, hybridized membrane visualized by Phosphoimager, lower panel, SYBR Gold staining of denaturing PAGE. Image Quant (Molecular Dynamics) was used to calculate the intensity of the signals.</p> <p>(D) Comparative RTC nuclear import activity of HSE NA (100 ng), 60S NA (100 ng), G3 RNA (100 ng), G3 RNA (10 ng + 90-ng carrier siRNA), buffer (ctr–), or buffer + 100-ng carrier RNA. Nuclear import assays were performed in permeabilized HeLa cells in the presence of labelled RTCs, 1× energy mix, and the indicated RNAs. Bars represent the fold nuclear import increase above background (RTC + energy mix) ± standard deviation of two independent experiments.</p>