p180 Promotes the Ribosome-Independent Localization of a Subset of mRNA to the Endoplasmic Reticulum
Figure 1
Poly(A) transcripts associate with ER independently of ribosomes and translation.
(A) A single digitonin-extracted COS-7 cell co-stained for poly(A) mRNA using poly(dT) FISH probes, and for the ER marker Trapα by immunofluorescence. Note the general co-localization between the mRNA (green) and Trapα (red). (B) The fluorescence intensity (y-axis) of the poly(A) mRNA (green) and Trapα (red) along the arrow (x-axis) in the overlay image in (A). Note the correlation between peaks in intensity of poly(A) mRNA and Trapα (black arrows). (C–D) COS-7 cells were treated with either DMSO, puromycin (“Puro”), homoharringtonine (“HHT”) for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for poly(A) mRNA using poly(dT) FISH probes, and then treated with RNase H (RNase H “+”) or control buffer (“Cont” or RNase H “−”) for 1 h at 37°C. Cells were imaged (C) and the fluorescence intensity of the ER and nucleus were quantified (D). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background normalized to the signal in the ER of DMSO/control treated cells. Note that in cells not treated with RNase H, the amount of mRNA bound to the ER decreased by about half in all the drug-treated cells as compared to DMSO-treated cells. In contrast RNase H treatment eliminated most of the ER fluorescence and the majority of the nuclear signal. All scale bars = 20 µm. (E) COS-7 cells were treated with control medium (DMSO), puromycin, or HHT for 15 min, then incubated in 35S-methionine to label newly synthesized proteins for an additional 15 min. Cell lysates were collected and separated by SDS-PAGE. Total proteins were visualized by Coomassie blue stain, and newly synthesized proteins were detected by autoradiography. Molecular weight markers are indicated on the left (“Ladder”; 188 kD, 62 kD, 49 kD, 38 kD, 28 kD, 18 kD). (F) COS-7 cells were either treated with DMSO (“Cont”) or puromycin for 30 min, then extracted with digitonin (in the absence or presence of 20 mM EDTA). Cytoplasmic (“C”; i.e., non-ER) and ER (“ER”) fractions were separated by SDS-PAGE, then transferred to nitrocellulose, and immunoblotted with antibodies against the small ribosomal protein S6, the ER marker Trapα, and the cytoplasmic marker αtubulin. Note that most of the S6 protein is released from the membrane to the cytoplasmic fraction only after cells are treated with puromycin and then extracted with EDTA. (G) COS-7 and U2OS cells were treated either with cyclohexamide (“control”) and then extracted, or with puromycin for 30 min and then extracted in the presence of 20 mM EDTA. ER and cytoplasmic fractions were isolated as in (F) except that either cyclohexamide, or puromycin and EDTA, was present in all solutions. cDNA was synthesized from each fraction using poly(dT) primers and 32P-dNTPs, and ratio of counts in the ER to total (cytoplasm+ER) were tabulated. Each bar represents the average and standard error of three independent experiments.