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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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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.
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fig3: 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.

Mentions: We expected that all nuclear speckle components would simultaneously reassemble into nuclear speckles upon entry into daughter nuclei, and we initially suspected that NAPs represent the first nuclear speckles in daughter nuclei. However, although SR proteins and snRNPs colocalize in interphase speckles (Huang and Spector, 1992) and in MIGs (Ferriera et al., 1994), NAPs are not enriched in snRNPs as shown by using an antibody against the U2 snRNP protein B′′ (Fig. 3, compare arrows in a and b). Interestingly, the majority of B′′ accumulated in regions of telophase daughter nuclei (Fig. 3 b, arrowheads) distinct from NAPs. DAPI staining indicates that DNA is decondensed or absent in these regions (Fig. 3 d, arrowheads). In addition, an antibody that recognizes the 5′ m3G cap of the snRNAs also showed snRNA accumulation in regions of telophase daughter nuclei distinct from NAPs (unpublished data). Because snRNP maturation is thought to occur in CBs (Sleeman and Lamond, 1999), we examined the localization of the CB protein p80 coilin in telophase cells. Coilin was found in regions of telophase nuclei (Fig. 3 f, arrowheads), distinct from NAPs (Fig. 3 e, arrows). In fact, coilin and U2 snRNP protein B′′ were colocalized in telophase nuclei (Fig. 3, j, k, and m, arrowheads) in areas separate from NAPs (Fig. 3, i–n, arrows). The colocalization of snRNPs and coilin suggests that in addition to NAPs there are regions of daughter nuclei that could be initial sites for snRNP assembly and/or modification (see Discussion).


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)

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.
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fig3: 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.
Mentions: We expected that all nuclear speckle components would simultaneously reassemble into nuclear speckles upon entry into daughter nuclei, and we initially suspected that NAPs represent the first nuclear speckles in daughter nuclei. However, although SR proteins and snRNPs colocalize in interphase speckles (Huang and Spector, 1992) and in MIGs (Ferriera et al., 1994), NAPs are not enriched in snRNPs as shown by using an antibody against the U2 snRNP protein B′′ (Fig. 3, compare arrows in a and b). Interestingly, the majority of B′′ accumulated in regions of telophase daughter nuclei (Fig. 3 b, arrowheads) distinct from NAPs. DAPI staining indicates that DNA is decondensed or absent in these regions (Fig. 3 d, arrowheads). In addition, an antibody that recognizes the 5′ m3G cap of the snRNAs also showed snRNA accumulation in regions of telophase daughter nuclei distinct from NAPs (unpublished data). Because snRNP maturation is thought to occur in CBs (Sleeman and Lamond, 1999), we examined the localization of the CB protein p80 coilin in telophase cells. Coilin was found in regions of telophase nuclei (Fig. 3 f, arrowheads), distinct from NAPs (Fig. 3 e, arrows). In fact, coilin and U2 snRNP protein B′′ were colocalized in telophase nuclei (Fig. 3, j, k, and m, arrowheads) in areas separate from NAPs (Fig. 3, i–n, arrows). The colocalization of snRNPs and coilin suggests that in addition to NAPs there are regions of daughter nuclei that could be initial sites for snRNP assembly and/or modification (see Discussion).

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