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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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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.
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fig2: 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.

Mentions: Immunolocalization studies allowed us to determine the phosphorylation state of SR proteins in NAPs. An antibody that recognizes the phosphorylated form of SC35 (Fu and Maniatis, 1990) labeled cytoplasmic MIGs and did not recognize any nuclear SC35 during telophase (Fig. 2 b). This antibody does not recognize SC35 in NAPs (Fig. 2 b, arrow), which is consistent with the possibility that the SC35 in NAPs must be in a hypophosphorylated state (Prasanth et al., 2003). In fact, hyperphosphorylated SR proteins in general are not found in NAPs, as antibodies 3C5 (Fig. 2 f, arrow; Turner and Franchi, 1987) and mAb104 (Roth et al., 1990), which recognize phosphoepitopes on a subset of SR proteins, also did not label NAPs or recognize nuclear SR proteins until G1. However, antibody SCf11 that preferentially recognizes hypophosphorylated SC35 (Fig. 2 j, arrows; Cavaloc et al., 1999) colocalized with YFP-SF2/ASF in NAPs (Fig. 2 i, arrows). An immunoblot of HeLa extract confirmed that SCf11 recognizes SC35 only in cell extract treated with phosphatase (Fig. 2 m, lane 2), preferentially recognizing hypophosphorylated SC35. Therefore, SR proteins in NAPs are predominantly hypophosphorylated, a characteristic that would promote SR protein–SR protein association 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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172523&req=5

fig2: 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.
Mentions: Immunolocalization studies allowed us to determine the phosphorylation state of SR proteins in NAPs. An antibody that recognizes the phosphorylated form of SC35 (Fu and Maniatis, 1990) labeled cytoplasmic MIGs and did not recognize any nuclear SC35 during telophase (Fig. 2 b). This antibody does not recognize SC35 in NAPs (Fig. 2 b, arrow), which is consistent with the possibility that the SC35 in NAPs must be in a hypophosphorylated state (Prasanth et al., 2003). In fact, hyperphosphorylated SR proteins in general are not found in NAPs, as antibodies 3C5 (Fig. 2 f, arrow; Turner and Franchi, 1987) and mAb104 (Roth et al., 1990), which recognize phosphoepitopes on a subset of SR proteins, also did not label NAPs or recognize nuclear SR proteins until G1. However, antibody SCf11 that preferentially recognizes hypophosphorylated SC35 (Fig. 2 j, arrows; Cavaloc et al., 1999) colocalized with YFP-SF2/ASF in NAPs (Fig. 2 i, arrows). An immunoblot of HeLa extract confirmed that SCf11 recognizes SC35 only in cell extract treated with phosphatase (Fig. 2 m, lane 2), preferentially recognizing hypophosphorylated SC35. Therefore, SR proteins in NAPs are predominantly hypophosphorylated, a characteristic that would promote SR protein–SR protein association 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