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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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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.
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fig5: 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.

Mentions: To examine the relationship between the association of fibrillarin with NORs and the assembly of NAPs, both structures were simultaneously examined in living cells. HeLa cells stably expressing CFP-fibrillarin were transiently transfected with YFP-SF2/ASF, and dual color four-dimensional imaging was performed in living mitotic cells (Fig. 5, a–l). The NORs were visible as distinct CFP-fibrillarin foci in early telophase (Fig. 5 a, arrow). YFP-SF2/ASF was not yet detectable in daughter nuclei at this time (Fig. 5 g). Clearly, the NAPs are the first sites where YFP-SF2/ASF is enriched in daughter nuclei (Fig. 5 i), and they appeared a few minutes after CFP-fibrillarin first accumulated in NORs. We conclude that fibrillarin associates with NORs before the accumulation of YFP-SF2/ASF in NAPs.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: 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.
Mentions: To examine the relationship between the association of fibrillarin with NORs and the assembly of NAPs, both structures were simultaneously examined in living cells. HeLa cells stably expressing CFP-fibrillarin were transiently transfected with YFP-SF2/ASF, and dual color four-dimensional imaging was performed in living mitotic cells (Fig. 5, a–l). The NORs were visible as distinct CFP-fibrillarin foci in early telophase (Fig. 5 a, arrow). YFP-SF2/ASF was not yet detectable in daughter nuclei at this time (Fig. 5 g). Clearly, the NAPs are the first sites where YFP-SF2/ASF is enriched in daughter nuclei (Fig. 5 i), and they appeared a few minutes after CFP-fibrillarin first accumulated in NORs. We conclude that fibrillarin associates with NORs before the accumulation of YFP-SF2/ASF in NAPs.

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