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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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Inhibition of RNA polymerase II during telophase delays targeting of YFP-SF2/ASF to nuclear speckles. In mitotic cells treated with the specific RNA polymerase II inhibitor α-amanitin, endogenous SF2/ASF (a, arrow) accumulated much more extensively around NORs (labeled with fibrillarin; b and c, arrow) than in untreated cells (Fig. 4). In living mitotic cells treated with α-amanitin (e–h), YFP-SF2/ASF continued to accumulate in NAPs for at least 2 h (h, arrow indicates NAP remnant), ∼100 min longer than it persists in NAPs in untreated cells. Even after 2 h, accumulation of SF2/ASF nuclear speckles was not abundant (h, compare with Fig. 1, j and k). Arrows in a–h indicate NAP position. Confocal images are representative projections from a typical sequence in which z-stacks were collected every 5 min. Immunofluorescence of interphase cells treated with α-amanitin showed that similar to the localization of SR proteins during telophase, a significant amount of YFP-SF2/ASF was redistributed to the periphery of nucleoli (i, arrows). snRNPs remained in rounded, enlarged nuclear speckles and were not targeted to the nucleolar periphery (j, arrowhead). Arrows (i–l) indicate nucleolar periphery. Arrowheads (i–l) indicate rounded-up nuclear speckles. DNA was stained with DAPI (d and l). Bars, 5 μm.
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fig8: Inhibition of RNA polymerase II during telophase delays targeting of YFP-SF2/ASF to nuclear speckles. In mitotic cells treated with the specific RNA polymerase II inhibitor α-amanitin, endogenous SF2/ASF (a, arrow) accumulated much more extensively around NORs (labeled with fibrillarin; b and c, arrow) than in untreated cells (Fig. 4). In living mitotic cells treated with α-amanitin (e–h), YFP-SF2/ASF continued to accumulate in NAPs for at least 2 h (h, arrow indicates NAP remnant), ∼100 min longer than it persists in NAPs in untreated cells. Even after 2 h, accumulation of SF2/ASF nuclear speckles was not abundant (h, compare with Fig. 1, j and k). Arrows in a–h indicate NAP position. Confocal images are representative projections from a typical sequence in which z-stacks were collected every 5 min. Immunofluorescence of interphase cells treated with α-amanitin showed that similar to the localization of SR proteins during telophase, a significant amount of YFP-SF2/ASF was redistributed to the periphery of nucleoli (i, arrows). snRNPs remained in rounded, enlarged nuclear speckles and were not targeted to the nucleolar periphery (j, arrowhead). Arrows (i–l) indicate nucleolar periphery. Arrowheads (i–l) indicate rounded-up nuclear speckles. DNA was stained with DAPI (d and l). Bars, 5 μm.

Mentions: Because SR proteins are present in NAPs before entry into nuclear speckles, we were interested in determining the effect of NAP formation and maintenance under conditions where RNA polymerase II transcription was inhibited as cells exited mitosis. Interestingly, inhibition of RNA polymerase II transcription during telophase, by α-amanitin treatment, caused NAPs to become larger and brighter and to persist longer. Endogenous SF2/ASF accumulation at NAPs was exaggerated under these conditions (Fig. 8, a–d), suggesting that SR proteins preferentially target NAPs when transcription sites are absent. This result was confirmed in living cells, as YFP-SF2/ASF accumulation in NAPs continued for at least 2 h (Fig. 8, e–h), during which time the NAPs increased in size over the first 65 min and eventually diminished but were not completely absent by 2 h (Fig. 8 h). This is a dramatically longer association of SF2/ASF with NAPs, compared with SF2/ASF in untreated cells, in which NAPs disappear after ∼20 min (Fig. 1 j). Furthermore, SF2/ASF entry into nuclear speckles was delayed (i.e., speckles were just beginning to accumulate SF2/ASF by 2 h) compared with untreated cells in which nuclear speckles accumulate SF2/ASF immediately (Fig. 1 k). Interestingly, endogenous SF2/ASF (Fig. S4, a–h, available at http://www.jcb.org/cgi/content/full/jcb.200404120/DC1) and YFP-SF2/ASF (Fig. 8 i, arrows) in interphase cells treated with α-amanitin were redistributed from nuclear speckles to the peripheral nucleolar region. In the same cells, snRNPs remained in rounded, enlarged nuclear speckles and were not targeted to the nucleolar periphery (Fig. 8 j, arrowhead), consistent with the segregation of different families of splicing factors at telophase. SC35 tagged with RFP also redistributed from nuclear speckles to the peripheral nucleolar region (Fig. S4, i–p). Identical results were obtained with other RNA polymerase II inhibitors (e.g., actinomycin D and DRB; unpublished data). These results implicate RNA polymerase II transcription in appropriate subnuclear targeting of SR proteins after mitosis, and they demonstrate a previously unrecognized role of NAPs in the pathway of SR splicing factor trafficking and biogenesis of nuclear speckles.


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)

Inhibition of RNA polymerase II during telophase delays targeting of YFP-SF2/ASF to nuclear speckles. In mitotic cells treated with the specific RNA polymerase II inhibitor α-amanitin, endogenous SF2/ASF (a, arrow) accumulated much more extensively around NORs (labeled with fibrillarin; b and c, arrow) than in untreated cells (Fig. 4). In living mitotic cells treated with α-amanitin (e–h), YFP-SF2/ASF continued to accumulate in NAPs for at least 2 h (h, arrow indicates NAP remnant), ∼100 min longer than it persists in NAPs in untreated cells. Even after 2 h, accumulation of SF2/ASF nuclear speckles was not abundant (h, compare with Fig. 1, j and k). Arrows in a–h indicate NAP position. Confocal images are representative projections from a typical sequence in which z-stacks were collected every 5 min. Immunofluorescence of interphase cells treated with α-amanitin showed that similar to the localization of SR proteins during telophase, a significant amount of YFP-SF2/ASF was redistributed to the periphery of nucleoli (i, arrows). snRNPs remained in rounded, enlarged nuclear speckles and were not targeted to the nucleolar periphery (j, arrowhead). Arrows (i–l) indicate nucleolar periphery. Arrowheads (i–l) indicate rounded-up nuclear speckles. DNA was stained with DAPI (d and l). Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172523&req=5

fig8: Inhibition of RNA polymerase II during telophase delays targeting of YFP-SF2/ASF to nuclear speckles. In mitotic cells treated with the specific RNA polymerase II inhibitor α-amanitin, endogenous SF2/ASF (a, arrow) accumulated much more extensively around NORs (labeled with fibrillarin; b and c, arrow) than in untreated cells (Fig. 4). In living mitotic cells treated with α-amanitin (e–h), YFP-SF2/ASF continued to accumulate in NAPs for at least 2 h (h, arrow indicates NAP remnant), ∼100 min longer than it persists in NAPs in untreated cells. Even after 2 h, accumulation of SF2/ASF nuclear speckles was not abundant (h, compare with Fig. 1, j and k). Arrows in a–h indicate NAP position. Confocal images are representative projections from a typical sequence in which z-stacks were collected every 5 min. Immunofluorescence of interphase cells treated with α-amanitin showed that similar to the localization of SR proteins during telophase, a significant amount of YFP-SF2/ASF was redistributed to the periphery of nucleoli (i, arrows). snRNPs remained in rounded, enlarged nuclear speckles and were not targeted to the nucleolar periphery (j, arrowhead). Arrows (i–l) indicate nucleolar periphery. Arrowheads (i–l) indicate rounded-up nuclear speckles. DNA was stained with DAPI (d and l). Bars, 5 μm.
Mentions: Because SR proteins are present in NAPs before entry into nuclear speckles, we were interested in determining the effect of NAP formation and maintenance under conditions where RNA polymerase II transcription was inhibited as cells exited mitosis. Interestingly, inhibition of RNA polymerase II transcription during telophase, by α-amanitin treatment, caused NAPs to become larger and brighter and to persist longer. Endogenous SF2/ASF accumulation at NAPs was exaggerated under these conditions (Fig. 8, a–d), suggesting that SR proteins preferentially target NAPs when transcription sites are absent. This result was confirmed in living cells, as YFP-SF2/ASF accumulation in NAPs continued for at least 2 h (Fig. 8, e–h), during which time the NAPs increased in size over the first 65 min and eventually diminished but were not completely absent by 2 h (Fig. 8 h). This is a dramatically longer association of SF2/ASF with NAPs, compared with SF2/ASF in untreated cells, in which NAPs disappear after ∼20 min (Fig. 1 j). Furthermore, SF2/ASF entry into nuclear speckles was delayed (i.e., speckles were just beginning to accumulate SF2/ASF by 2 h) compared with untreated cells in which nuclear speckles accumulate SF2/ASF immediately (Fig. 1 k). Interestingly, endogenous SF2/ASF (Fig. S4, a–h, available at http://www.jcb.org/cgi/content/full/jcb.200404120/DC1) and YFP-SF2/ASF (Fig. 8 i, arrows) in interphase cells treated with α-amanitin were redistributed from nuclear speckles to the peripheral nucleolar region. In the same cells, snRNPs remained in rounded, enlarged nuclear speckles and were not targeted to the nucleolar periphery (Fig. 8 j, arrowhead), consistent with the segregation of different families of splicing factors at telophase. SC35 tagged with RFP also redistributed from nuclear speckles to the peripheral nucleolar region (Fig. S4, i–p). Identical results were obtained with other RNA polymerase II inhibitors (e.g., actinomycin D and DRB; unpublished data). These results implicate RNA polymerase II transcription in appropriate subnuclear targeting of SR proteins after mitosis, and they demonstrate a previously unrecognized role of NAPs in the pathway of SR splicing factor trafficking and biogenesis of nuclear speckles.

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