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

Bubulya PA, Prasanth KV, Deerinck TJ, Gerlich D, Beaudouin J, Ellisman MH, Ellenberg J, Spector DL - J. Cell Biol. (2004)

Bottom Line: 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.This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.

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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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.
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fig1: 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.

Mentions: IGCs, also called nuclear speckles, are storage, assembly, and/or modification sites for pre-mRNA processing factors in interphase nuclei (for review see Lamond and Spector, 2003). Nuclear speckles disassemble during mitosis, and their constituents are diffusely distributed throughout the cytoplasm, later organizing into cytoplasmic structures called mitotic IGCs (MIGs; Verheijen et al., 1986; Leser et al., 1989; Ferriera et al., 1994). Early studies on the behavior of nuclear speckles through the cell cycle described MIGs as the mitotic equivalent of interphase nuclear speckles based on their similar granular structure and composition (Spector and Smith, 1986; Leser et al., 1989; Thiry, 1995; see Fig. 1, a–c). MIGs disappear concomitant with nuclear entry of pre-mRNA processing factors at telophase (Ferriera et al., 1994; Prasanth et al., 2003), and then nuclear speckles reassemble in G1 and are maintained through interphase.


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

Bubulya PA, Prasanth KV, Deerinck TJ, Gerlich D, Beaudouin J, Ellisman MH, Ellenberg J, Spector DL - J. Cell Biol. (2004)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172523&req=5

fig1: 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.
Mentions: IGCs, also called nuclear speckles, are storage, assembly, and/or modification sites for pre-mRNA processing factors in interphase nuclei (for review see Lamond and Spector, 2003). Nuclear speckles disassemble during mitosis, and their constituents are diffusely distributed throughout the cytoplasm, later organizing into cytoplasmic structures called mitotic IGCs (MIGs; Verheijen et al., 1986; Leser et al., 1989; Ferriera et al., 1994). Early studies on the behavior of nuclear speckles through the cell cycle described MIGs as the mitotic equivalent of interphase nuclear speckles based on their similar granular structure and composition (Spector and Smith, 1986; Leser et al., 1989; Thiry, 1995; see Fig. 1, a–c). MIGs disappear concomitant with nuclear entry of pre-mRNA processing factors at telophase (Ferriera et al., 1994; Prasanth et al., 2003), and then nuclear speckles reassemble in G1 and are maintained through interphase.

Bottom Line: 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.This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.

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.

Show MeSH
Related in: MedlinePlus