Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei - PubMed (original) (raw)

Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei

Paula A Bubulya et al. J Cell Biol. 2004.

Abstract

Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15-20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis.

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Figures

Figure 1.

Distribution of SR splicing factors during telophase. Endogenous SR splicing factor SF2/ASF (green) and DNA (pseudocolored red) localization during the HeLa cell cycle (a–e). Throughout mitosis, nuclear speckle constituents reside in the cytosol and in MIGs (a–c, arrowheads). At telophase, SF2/ASF enters daughter nuclei and localizes in NAPs (d, arrows) that correspond to DAPI-negative zones (d, inset, arrows) before it is localized in nuclear speckles in G1 (e). YFP-SF2/ASF localization was followed by confocal microscopy during telophase in living cells (f–k), where it initially accumulates in NAPs (f–j, arrows) and later localizes to nuclear speckles (k, arrowheads; see Video 1 for the entire time course, available at

http://www.jcb.org/cgi/content/full/jcb.200404120/DC1

). Images in f–k are 2-μm optical sections. Projections of deconvolved z-stacks (l–o) show that SR splicing factor SC35-CFP (l, arrow, pseudocolored green) colocalizes in NAPs with SF2/ASF (m and n, arrows). The SR protein kinase Clk/STY (q, arrow) colocalizes with endogenous SF2/ASF in NAPs (p and r, arrows). DNA was stained with DAPI to monitor cell cycle phase (a–e, o, and s; arrows in o and s indicate NAP position). Bars, 5 μm.

Figure 2.

SR proteins in NAPs are hypophosphorylated. SC35 antibody (b, arrow), which recognizes hyperphosphorylated SC35, does not recognize the SC35 in NAPs (a, arrow) and is absent from the nucleus (c and d, arrows). 3C5 antibody that recognizes a family of hyperphosphorylated SR proteins (f, arrow) also does not recognize SR proteins in NAPs (e, arrow) and is absent from the nucleus (g and h, arrows). Projections of deconvolved image stacks illustrate the strictly cytoplasmic localization of hyperphosphorylated SR proteins as well as exclusion from nuclei (a–h). SCf11 (j, arrows) colocalizes with YFP-SF2/ASF in NAPs (i and k, arrows) and recognizes SC35 by immunoblot only when cell extract is treated with phosphatase (m, lane 2). Arrows in d, h, and l indicate NAP position. Bar, 5 μm.

Figure 3.

snRNPs and coilin are not enriched in NAPs. snRNPs (b, arrowheads) are enriched in regions of daughter nuclei away from SF2/ASF NAPs (a, arrows). Coilin is also found in regions of daughter nuclei (f, arrowheads; 5P10 antibody), away from SF2/ASF NAPs (e, arrow). The U2snRNP protein B′′ (j, arrowheads) colocalized with coilin (k, arrowheads; R228 antibody; merge shown in m, arrowheads) in regions separate from NAPs, which are indicated by arrows (i–n). The two regions are clearly distinct in a merged image of YFP-SF2/ASF and B′′ (l). DNA was stained with DAPI (d, h, and n). Arrows indicate NAP position (a–n). Arrowheads indicate “polar” localization of U2-B′′ (b–d), coilin (f–h), or both U2-B′′ and coilin (j–n). DNA was stained with DAPI (d, h, and n). Bars, 5 μm.

Figure 4.

NAPs surround transcriptionally active NORs during telophase. During telophase, endogenous SF2/ASF localizes in NAPs (a, arrow) surrounding foci of fibrillarin (b, arrow) in HeLa cells. Endogenous SF2/ASF (d, arrow) is localized in NAPs surrounding foci of fibrillarin (e, arrow) in the nontransformed cell line IMR90. Endogenous SF2/ASF (h, arrow) is localized in NAPs surrounding foci of fibrillarin (i, arrow) in U2OS cells. SF2/ASF is also in MIGs at this stage (a–k, arrowheads). Endogenous SF2/ASF (l, arrow) surrounds foci of the RNA pol I transcription factor upstream binding factor (m, arrow) in HeLa cells. RNA-FISH for ribosomal RNA (q, arrows) showed that SF2/ASF NAPs (p, arrows) surrounded transcriptionally active NORs. RNA-FISH using oligo dT probes demonstrated that polyadenylated RNA (u, arrows) was absent from SF2/ASF NAPs (t, arrows). DNA was stained with DAPI (g, k, o, s, and w). NAP position is indicated by arrows (a–w). Bars, 5 μm.

Figure 5.

NAPs form at established NORs. Dual-color four-dimensional imaging revealed the temporal sequence of nuclear domain establishment (a–l). Cells stably expressing CFP-fibrillarin (a–f) were transiently transfected with a cDNA construct encoding YFP-SF2/ASF (g–l). CFP-fibrillarin (a, arrow) had clearly accumulated in NORs for several minutes before YFP-SF2/ASF first accumulated in NAPs (i, arrows). Images are projections of confocal z-stacks collected every 2 min. Bar, 5 μm.

Figure 6.

Ultrastructure of NAPs and time-lapse of nuclear envelope assembly. Ultrastructure of the NAPs was determined during early telophase (a–c) by using immunoelectron microscopy. Electron-dense material was deposited where SF2/ASF antibody was localized around forming nucleoli (a, arrows) and in MIGs (a, arrowheads). Enlarged images from the top (b) and bottom (c) cells show that SF2/ASF is excluded from the NOR interior. (d–o) Dual four-dimensional imaging of NAPs and nuclear import in living HeLa cells. A functional nuclear envelope was established as indicated by import of IBB-HcRed (e, arrows), before YFP-SF2/ASF began to accumulate in NAPs (m, arrows). Images are projections of confocal z-stacks collected every 2 min. Bars: (a) 1 μm; (d–o) 5 μm.

Figure 7.

Dynamics and quantification of YFP-SF2/ASF in NAPs. FRAP analysis indicates that turnover of YFP-SF2/ASF at NORs is rapid. A representative of 14 NAP photobleaching experiments is shown in panels a–f. Photobleaching of NAPs (a, boxed area) revealed that the average half time of FRAP of YFP-SF2/ASF in NAPs was 1.8 s (± 0.6; recovery curve, f; see also Fig. S3, available at

http://www.jcb.org/cgi/content/full/jcb.200404120/DC1

). High-resolution dual color four-dimensional imaging data set is shown in panels g–v. Intensity sums of fluorescence of YFP-SF2/ASF in different cellular compartments during the early stages of daughter nuclei reformation (g–v) revealed that ∼1.17% of YFP-SF2/ASF is found at NAPs during early telophase in the example shown (m, NAPs; v, right y-axis). Data shown in g–v is representative of three experiments. Bars: (a–e) 2.5 μm; (g–u) 5 μm.

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.

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Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei - PubMed (original) (raw)