Figure 8.

Inhibition of RNA polymerase II during telophase delays targeting of YFP-SF2/ASF to nuclear speckles. In mitotic cells treated with the specific RNA polymerase II inhibitor α-amanitin, endogenous SF2/ASF (a, arrow) accumulated much more extensively around NORs (labeled with fibrillarin; b and c, arrow) than in untreated cells (Fig. 4). In living mitotic cells treated with α-amanitin (e–h), YFP-SF2/ASF continued to accumulate in NAPs for at least 2 h (h, arrow indicates NAP remnant), ∼100 min longer than it persists in NAPs in untreated cells. Even after 2 h, accumulation of SF2/ASF nuclear speckles was not abundant (h, compare with Fig. 1, j and k). Arrows in a–h indicate NAP position. Confocal images are representative projections from a typical sequence in which z-stacks were collected every 5 min. Immunofluorescence of interphase cells treated with α-amanitin showed that similar to the localization of SR proteins during telophase, a significant amount of YFP-SF2/ASF was redistributed to the periphery of nucleoli (i, arrows). snRNPs remained in rounded, enlarged nuclear speckles and were not targeted to the nucleolar periphery (j, arrowhead). Arrows (i–l) indicate nucleolar periphery. Arrowheads (i–l) indicate rounded-up nuclear speckles. DNA was stained with DAPI (d and l). Bars, 5 μm.