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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 surround transcriptionally active NORs during telophase. During telophase, endogenous SF2/ASF localizes in NAPs (a, arrow) surrounding foci of fibrillarin (b, arrow) in HeLa cells. Endogenous SF2/ASF (d, arrow) is localized in NAPs surrounding foci of fibrillarin (e, arrow) in the nontransformed cell line IMR90. Endogenous SF2/ASF (h, arrow) is localized in NAPs surrounding foci of fibrillarin (i, arrow) in U2OS cells. SF2/ASF is also in MIGs at this stage (a–k, arrowheads). Endogenous SF2/ASF (l, arrow) surrounds foci of the RNA pol I transcription factor upstream binding factor (m, arrow) in HeLa cells. RNA-FISH for ribosomal RNA (q, arrows) showed that SF2/ASF NAPs (p, arrows) surrounded transcriptionally active NORs. RNA-FISH using oligo dT probes demonstrated that polyadenylated RNA (u, arrows) was absent from SF2/ASF NAPs (t, arrows). DNA was stained with DAPI (g, k, o, s, and w). NAP position is indicated by arrows (a–w). Bars, 5 μm.
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fig4: NAPs surround transcriptionally active NORs during telophase. During telophase, endogenous SF2/ASF localizes in NAPs (a, arrow) surrounding foci of fibrillarin (b, arrow) in HeLa cells. Endogenous SF2/ASF (d, arrow) is localized in NAPs surrounding foci of fibrillarin (e, arrow) in the nontransformed cell line IMR90. Endogenous SF2/ASF (h, arrow) is localized in NAPs surrounding foci of fibrillarin (i, arrow) in U2OS cells. SF2/ASF is also in MIGs at this stage (a–k, arrowheads). Endogenous SF2/ASF (l, arrow) surrounds foci of the RNA pol I transcription factor upstream binding factor (m, arrow) in HeLa cells. RNA-FISH for ribosomal RNA (q, arrows) showed that SF2/ASF NAPs (p, arrows) surrounded transcriptionally active NORs. RNA-FISH using oligo dT probes demonstrated that polyadenylated RNA (u, arrows) was absent from SF2/ASF NAPs (t, arrows). DNA was stained with DAPI (g, k, o, s, and w). NAP position is indicated by arrows (a–w). Bars, 5 μm.

Mentions: Because NORs are activated in mitosis, we were interested to determine if there is a relationship between NORs and NAPs. Immunofluorescence analysis verified that SR proteins initially surround NORs upon nuclear entry. Endogenous SF2/ASF was localized around foci of fibrillarin in telophase daughter nuclei in HeLa cells (Fig. 4, a–c, arrows), in nontransformed human fibroblast IMR90 cells (Fig. 4, d–g, arrows), and in human osteosarcoma U2OS cells (Fig. 4, h–k, arrows). Some SF2/ASF is still present in MIGs at this stage (Fig. 4, a, d, and h, arrowheads). As there are multiple NORs in each nucleus (see Discussion), multiple NAPs were seen in these cell lines in telophase (Fig. 4, a, d, and h). To determine if the NORs are transcriptionally active when NAPs are formed, we looked for evidence of RNA polymerase I activity in the NORs. Foci of the RNA polymerase I transcription factor upstream binding factor (Fig. 4 m, arrow) that is localized in transcriptionally active NORs (Jordan et al., 1996; Roussel et al., 1996), foci of nascent transcripts (presumably RNA polymerase I transcripts; Fig. S1, i and j), and pre-ribosomal RNAs detected by RNA-FISH (Fig. 4 q, arrows) were surrounded by NAPs (Fig. 4 p, arrows).


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 surround transcriptionally active NORs during telophase. During telophase, endogenous SF2/ASF localizes in NAPs (a, arrow) surrounding foci of fibrillarin (b, arrow) in HeLa cells. Endogenous SF2/ASF (d, arrow) is localized in NAPs surrounding foci of fibrillarin (e, arrow) in the nontransformed cell line IMR90. Endogenous SF2/ASF (h, arrow) is localized in NAPs surrounding foci of fibrillarin (i, arrow) in U2OS cells. SF2/ASF is also in MIGs at this stage (a–k, arrowheads). Endogenous SF2/ASF (l, arrow) surrounds foci of the RNA pol I transcription factor upstream binding factor (m, arrow) in HeLa cells. RNA-FISH for ribosomal RNA (q, arrows) showed that SF2/ASF NAPs (p, arrows) surrounded transcriptionally active NORs. RNA-FISH using oligo dT probes demonstrated that polyadenylated RNA (u, arrows) was absent from SF2/ASF NAPs (t, arrows). DNA was stained with DAPI (g, k, o, s, and w). NAP position is indicated by arrows (a–w). Bars, 5 μm.
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fig4: NAPs surround transcriptionally active NORs during telophase. During telophase, endogenous SF2/ASF localizes in NAPs (a, arrow) surrounding foci of fibrillarin (b, arrow) in HeLa cells. Endogenous SF2/ASF (d, arrow) is localized in NAPs surrounding foci of fibrillarin (e, arrow) in the nontransformed cell line IMR90. Endogenous SF2/ASF (h, arrow) is localized in NAPs surrounding foci of fibrillarin (i, arrow) in U2OS cells. SF2/ASF is also in MIGs at this stage (a–k, arrowheads). Endogenous SF2/ASF (l, arrow) surrounds foci of the RNA pol I transcription factor upstream binding factor (m, arrow) in HeLa cells. RNA-FISH for ribosomal RNA (q, arrows) showed that SF2/ASF NAPs (p, arrows) surrounded transcriptionally active NORs. RNA-FISH using oligo dT probes demonstrated that polyadenylated RNA (u, arrows) was absent from SF2/ASF NAPs (t, arrows). DNA was stained with DAPI (g, k, o, s, and w). NAP position is indicated by arrows (a–w). Bars, 5 μm.
Mentions: Because NORs are activated in mitosis, we were interested to determine if there is a relationship between NORs and NAPs. Immunofluorescence analysis verified that SR proteins initially surround NORs upon nuclear entry. Endogenous SF2/ASF was localized around foci of fibrillarin in telophase daughter nuclei in HeLa cells (Fig. 4, a–c, arrows), in nontransformed human fibroblast IMR90 cells (Fig. 4, d–g, arrows), and in human osteosarcoma U2OS cells (Fig. 4, h–k, arrows). Some SF2/ASF is still present in MIGs at this stage (Fig. 4, a, d, and h, arrowheads). As there are multiple NORs in each nucleus (see Discussion), multiple NAPs were seen in these cell lines in telophase (Fig. 4, a, d, and h). To determine if the NORs are transcriptionally active when NAPs are formed, we looked for evidence of RNA polymerase I activity in the NORs. Foci of the RNA polymerase I transcription factor upstream binding factor (Fig. 4 m, arrow) that is localized in transcriptionally active NORs (Jordan et al., 1996; Roussel et al., 1996), foci of nascent transcripts (presumably RNA polymerase I transcripts; Fig. S1, i and j), and pre-ribosomal RNAs detected by RNA-FISH (Fig. 4 q, arrows) were surrounded by NAPs (Fig. 4 p, arrows).

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