Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172523&req=5

fig6: 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.

Mentions: To investigate the ultrastructural relationship between the active NORs and NAPs, immunoelectron microscopy was performed on telophase HeLa cells. The early telophase state of the cell in Fig. 6 a is evident by the abundance of SF2/ASF in cytoplasmic MIGs (Fig. 6 a, arrowheads). Also, although there are several regions of dense heterochromatin throughout the nuclei, there is no indication of granular IGCs, confirming the absence of nuclear speckles at this stage (Fig. 6 a). In the daughter nuclei, endogenous SF2/ASF was localized in several electron-dense granular patches around NORs, and it was clearly excluded from the interior of the NOR (Fig. 6, b and c; enlarged NAPs from the top and bottom nuclei, respectively). Furthermore, the nuclear envelope appeared intact in nuclei where NAPs were observed.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172523&req=5

fig6: 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.
Mentions: To investigate the ultrastructural relationship between the active NORs and NAPs, immunoelectron microscopy was performed on telophase HeLa cells. The early telophase state of the cell in Fig. 6 a is evident by the abundance of SF2/ASF in cytoplasmic MIGs (Fig. 6 a, arrowheads). Also, although there are several regions of dense heterochromatin throughout the nuclei, there is no indication of granular IGCs, confirming the absence of nuclear speckles at this stage (Fig. 6 a). In the daughter nuclei, endogenous SF2/ASF was localized in several electron-dense granular patches around NORs, and it was clearly excluded from the interior of the NOR (Fig. 6, b and c; enlarged NAPs from the top and bottom nuclei, respectively). Furthermore, the nuclear envelope appeared intact in nuclei where NAPs were observed.

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