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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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Dynamics and quantification of YFP-SF2/ASF in NAPs. FRAP analysis indicates that turnover of YFP-SF2/ASF at NORs is rapid. A representative of 14 NAP photobleaching experiments is shown in panels a–f. Photobleaching of NAPs (a, boxed area) revealed that the average half time of FRAP of YFP-SF2/ASF in NAPs was 1.8 s (± 0.6; recovery curve, f; see also Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200404120/DC1). High-resolution dual color four-dimensional imaging data set is shown in panels g–v. Intensity sums of fluorescence of YFP-SF2/ASF in different cellular compartments during the early stages of daughter nuclei reformation (g–v) revealed that ∼1.17% of YFP-SF2/ASF is found at NAPs during early telophase in the example shown (m, NAPs; v, right y-axis). Data shown in g–v is representative of three experiments. Bars: (a–e) 2.5 μm; (g–u) 5 μm.
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fig7: Dynamics and quantification of YFP-SF2/ASF in NAPs. FRAP analysis indicates that turnover of YFP-SF2/ASF at NORs is rapid. A representative of 14 NAP photobleaching experiments is shown in panels a–f. Photobleaching of NAPs (a, boxed area) revealed that the average half time of FRAP of YFP-SF2/ASF in NAPs was 1.8 s (± 0.6; recovery curve, f; see also Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200404120/DC1). High-resolution dual color four-dimensional imaging data set is shown in panels g–v. Intensity sums of fluorescence of YFP-SF2/ASF in different cellular compartments during the early stages of daughter nuclei reformation (g–v) revealed that ∼1.17% of YFP-SF2/ASF is found at NAPs during early telophase in the example shown (m, NAPs; v, right y-axis). Data shown in g–v is representative of three experiments. Bars: (a–e) 2.5 μm; (g–u) 5 μm.

Mentions: To determine if SF2/ASF traffics through NAPs, we examined the exchange of SF2/ASF at NAPs by FRAP (Fig. 7, a–f). A representative fluorescence recovery curve is shown in Fig. 7 f for the region bleached in Fig. 7 a (boxed). FRAP analysis of a typical NAP indicated that SF2/ASF had a half time of fluorescence recovery of ∼1.8 s (± 0.6). The average fluorescence recovery curve from 14 experiments is shown in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200404120/DC1). As we also wanted to know how SR protein transit through NAPs compared with transit through the nucleoplasm, half of a nucleus was bleached such that the bleached region included a NAP (Fig. S2). Interestingly, the recovery of YFP-SF2/ASF in the NAPs occurred at the same rate as the YFP-SF2/ASF in the nucleoplasm, suggesting that diffusion is the limiting determinant for recovery.


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)

Dynamics and quantification of YFP-SF2/ASF in NAPs. FRAP analysis indicates that turnover of YFP-SF2/ASF at NORs is rapid. A representative of 14 NAP photobleaching experiments is shown in panels a–f. Photobleaching of NAPs (a, boxed area) revealed that the average half time of FRAP of YFP-SF2/ASF in NAPs was 1.8 s (± 0.6; recovery curve, f; see also Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200404120/DC1). High-resolution dual color four-dimensional imaging data set is shown in panels g–v. Intensity sums of fluorescence of YFP-SF2/ASF in different cellular compartments during the early stages of daughter nuclei reformation (g–v) revealed that ∼1.17% of YFP-SF2/ASF is found at NAPs during early telophase in the example shown (m, NAPs; v, right y-axis). Data shown in g–v is representative of three experiments. Bars: (a–e) 2.5 μm; (g–u) 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172523&req=5

fig7: Dynamics and quantification of YFP-SF2/ASF in NAPs. FRAP analysis indicates that turnover of YFP-SF2/ASF at NORs is rapid. A representative of 14 NAP photobleaching experiments is shown in panels a–f. Photobleaching of NAPs (a, boxed area) revealed that the average half time of FRAP of YFP-SF2/ASF in NAPs was 1.8 s (± 0.6; recovery curve, f; see also Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200404120/DC1). High-resolution dual color four-dimensional imaging data set is shown in panels g–v. Intensity sums of fluorescence of YFP-SF2/ASF in different cellular compartments during the early stages of daughter nuclei reformation (g–v) revealed that ∼1.17% of YFP-SF2/ASF is found at NAPs during early telophase in the example shown (m, NAPs; v, right y-axis). Data shown in g–v is representative of three experiments. Bars: (a–e) 2.5 μm; (g–u) 5 μm.
Mentions: To determine if SF2/ASF traffics through NAPs, we examined the exchange of SF2/ASF at NAPs by FRAP (Fig. 7, a–f). A representative fluorescence recovery curve is shown in Fig. 7 f for the region bleached in Fig. 7 a (boxed). FRAP analysis of a typical NAP indicated that SF2/ASF had a half time of fluorescence recovery of ∼1.8 s (± 0.6). The average fluorescence recovery curve from 14 experiments is shown in Fig. S2 (available at http://www.jcb.org/cgi/content/full/jcb.200404120/DC1). As we also wanted to know how SR protein transit through NAPs compared with transit through the nucleoplasm, half of a nucleus was bleached such that the bleached region included a NAP (Fig. S2). Interestingly, the recovery of YFP-SF2/ASF in the NAPs occurred at the same rate as the YFP-SF2/ASF in the nucleoplasm, suggesting that diffusion is the limiting determinant for recovery.

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