Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

Abstract

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions. Video LinkThe video component of this article can be found at

Identification of a Region within the Placental Alkaline Phosphatase mRNA That Mediates p180-dependent Targeting to the Endoplasmic Reticulum

“…Cell Culture, Permeabilization, shRNA Treatment, Transfection, and Microinjection-Cell culture and transfection were carried out as described previously (7,9). Depletion of p180 by lentivirus-mediated delivery of shRNA constructs was carried out in U2OS cells as described previously (7).…”

“…Depletion of p180 by lentivirus-mediated delivery of shRNA constructs was carried out in U2OS cells as described previously (7). 18 -24 h after transfection cells were pretreated with DMEM containing DMSO, 200 M puromycin (Sigma), or 5 m homoharringtonine (HHT) (Tocris Bioscience) for 30 min, permeabilized with digitonin (Sigma), and then fixed as described previously (9). To determine the targeting of newly synthesized transcript, plasmids encoding ALPP, t-ftz, or AP5 were microinjected into U2OS or COS-7 cells pretreated with DMEM containing either DMSO or 5 M HHT for 15 min as described previously (7,14).…”

“…To determine the targeting of newly synthesized transcript, plasmids encoding ALPP, t-ftz, or AP5 were microinjected into U2OS or COS-7 cells pretreated with DMEM containing either DMSO or 5 M HHT for 15 min as described previously (7,14). After injection, cells were incubated with DMEM containing DMSO or HHT for 2 h and then permeabilized with digitonin and fixed as described previously (7,9,14).…”

“…Fluorescent in Situ Hybridization (FISH), Immunofluorescence, and Imaging-FISH, immunofluorescence, and imaging were performed, as described previously (7,9,14). For ALPPand ftz-specific probes, see Ref.…”

“…Importantly, both the calreticulin gene and ALPP require p180 for their translationindependent maintenance at the ER (7). In contrast, certain engineered reporter mRNAs, such as t-ftz, and natural transcripts, such as the insulin-like 3 and cytochrome p450 8B1 (CYP8B1) genes, predominantly use the translation-dependent pathway for ER localization (7,9). In light of this, p180 probably collaborates with supplementary proteins, which have motifdiscriminating RNA-binding domains.…”