FIGURE 7:

Perispeckles contain polyA mRNAs and overlap with sites of active Pol II. (A) EJC core factors and polyA RNA colocalize at the perispeckle. HeLa cells were transfected with an empty vector (empty vector, a–f) or a Flag-Magoh construct (magoh, g–l) and then processed for combined immunofluorescence and RNA FISH. Fluorescent Alexa Fluor 488 or Cy3-oligo-d(T) probe were hybridized for polyA RNA detection (green in empty vector, b; red in magoh, g). The EJC core components Magoh and eIF4A3 were stained with anti-Flag (white in magoh, j) and anti-eIF4A3 (white in empty vector, d; green in magoh, h) antibodies, respectively. The splicing factor SC35 is detected by immunofluorescence (red in empty vector, a). (B) Active RNA polymerase II is close to perispeckle. HeLa cells were transfected with Flag-Magoh construct (magoh) and then processed for combined immunofluorescence RNA FISH. A fluorescent Cy3-oligo-d(T) probe was hybridized for polyA RNA detection (red). The EJC core component Magoh and active RNA polymerase II were stained with anti-Flag (white) and anti–Pol II H14 (green) antibodies, respectively. In A and B, nuclei were counterstained with the Hoechst-33258 dye (blue). Insets show a representative speckle with 2.5× magnification. Scale bar, 5 μm. Line-scan graphs were analyzed as described in the legend to Figure 1.