mRNPs are blocked at different points of the NPC during various types of mRNA export blocks. (A) U2OS cells stably expressing YFP-MS2–tagged Cerulean-minidystrophin-MS2 mRNPs were induced to transcribe (4 h), either under normal conditions (control) or during mRNA export blocks, after siRNA knockdown of Nup153 or NXF1; transfection with Cerulean-Dbp5-DN; and treatment with WGA (administered together with digitonin for 5 min). Detection of cells that received the siRNA was performed by cotransfection with mCherry-POM121 and mainly by the export defect seen on the tagged mRNPs in the time-lapse videos. Top: Frames from representative movies showing single mRNPs (green dots). Dashed lines show nuclear borders, and arrows show sites of transcription. Middle: Average time projections showing the static mRNPs in each cell (black dots; marked by magenta arrowheads). Green arrows point to sites of transcription, seen in the projection as large black dots. Scale bars, 10 µm. Bottom: Enlarged areas of the average projection showing mRNPs stuck at the nuclear envelope. Scale bar, 1 µm. (B) Numbers of mRNPs anchored at the nuclear envelope (NE) were counted in control and mRNA export blocked cells. Each spot represents the average number of static mRNPs in the nuclear envelope per time point in one cell. The central line represents the average number of static mRNPs in the nuclear envelope per time point in the appropriate condition. Error bars show SD; ***, P < 0.001. See Materials and methods for the number of mRNPs and cells shown. Measurements were performed in at least three independent experiments. (C) Average time projections from Videos 1, 2, 3, and 4, showing the interactions of single mRNPs (green) with POM121-Cherry–tagged NPCs (magenta) under each export blockage condition. Scheme showing the suggested point of blockage is depicted under each treatment. See Materials and methods for the number of mRNPs and cells shown. Scale bar, 0.5 µm.