We have characterized a transient nuclear domain (NAPs) where SR proteins accumulate during a limited temporal window in telophase. The NAPs form around active NORs after nuclear envelope assembly, but before the onset of nuclear speckle formation. The results of this paper are key to building a time line of nuclear compartment assembly following mitosis. They also bring to light the fact that, in general, constituents of nuclear bodies, including prerRNA processing factors, components of CBs and PML bodies, and pre-mRNA processing factors, do not immediately localize to and/or assemble their resident bodies in daughter nuclei. Instead, there is a lag phase after mitosis as nuclear proteins first enter daughter nuclei and accumulate temporarily with proteins of similar function(s) before nuclear bodies (i.e., nuclear speckles and CBs) are established in G1. These transient associations of subsets of proteins may be necessary for modification/maturation or partial assembly of multimolecular subcomplexes before localization to nuclear bodies in G1. The subsequent organization of nuclear compartments such as nuclear speckles would then provide further modification/maturation and recycling/maintenance of protein complexes during interphase.
We expected that all pre-mRNA processing factors would simultaneously reassemble into speckles in telophase daughter cells. Instead, we found a distinct localization of different families of pre-mRNA processing factors in different regions of telophase nuclei. The U2 snRNP protein B′′, snRNAs (m3
G), and the CB resident protein p80 coilin were all found at polar regions of daughter nuclei. CBs are discrete nuclear compartments that can associate with U1, U2, and U3 snRNA gene loci and histone gene loci (Frey and Matera, 1995
; Smith et al., 1995
; Gao et al., 1997
; Frey et al., 1999
) and have been implicated in snRNP maturation and assembly (Sleeman and Lamond, 1999
; Darzacq et al., 2002
) and in snoRNA posttranscriptional modification and targeting to the nucleolus (Narayanan et al., 1999
; Verheggen et al., 2002
). CBs do not acquire their distinctive morphology until late telophase/G1 (Sleeman and Lamond, 1999
; unpublished data). Therefore, the observed localization of snRNPs and p80 coilin at polar regions of daughter nuclei may be important for assembling the initial nuclear populations of snRNPs before CB formation.
During interphase, snRNPs may follow a nonrandom path as a short pulse of YFP-snRNP protein expression allowed tracking of the accumulation of snRNPs from the CB to the nucleoli, and then to the speckles (Sleeman and Lamond, 1999
). With the entry of snRNPs into the next compartment, there was clear depletion of snRNPs from the previous compartment (Sleeman and Lamond, 1999
). Sleeman and Lamond (1999)
concluded that the snRNPs are directly enriched in “speckles” during telophase without first passing through CBs, suggesting that snRNPs do not require modification/maturation at CBs upon entry into daughter nuclei. However, colocalization with SR proteins was not examined to confirm that these sites of snRNP enrichment are the equivalent of interphase speckles. As we have found a polar distribution of snRNPs in daughter nuclei, what was interpreted as a “speckle” during telophase (Sleeman and Lamond, 1999
) is more likely equivalent to what we have shown here as polar localization of snRNPs. Furthermore, the accumulation of both snRNPs and coilin in the same nuclear region is more suggestive of the early stages of CB organization than of nuclear speckle organization.
The initial localization of SR proteins around NORs in telophase implicates this region in SR protein modification/maturation. The fact that α-amanitin blocks trafficking of SR proteins from the perinucleolar region to transcription sites and/or nuclear speckles during interphase implicates a role for the nucleolar periphery specifically in SR protein modification/maturation during interphase as well. Interestingly, telophase NAPs increased in size and were maintained for a longer period of time when RNA polymerase II transcription was turned off, and SR proteins were left without a pre-mRNA target. Transit through the nucleolar periphery in interphase may be very rapid, similar to the rapid exchange in NAPs observed in FRAP experiments during telophase. Thereby, the steady-state level of SR proteins at the nucleolar periphery may be below the detection limits or indistinguishable from the nuclear speckle pattern (e.g., speckles on the periphery of the nucleoli), and could therefore be observed only upon RNA polymerase II inhibition when a build up occurs due to the depletion of a downstream target. A similar scenario was observed for hnRNP proteins when it was first discovered that they shuttle continuously between the nucleus and the cytoplasm (Pinol-Roma and Dreyfuss, 1993
). The hnRNP proteins are entirely nuclear when observed by immunofluorescence, and no hnRNP is detectable in the cytoplasm. However, upon inhibition of RNA polymerase II transcription, some hnRNP proteins (e.g., A1 and K) accumulated in the cytosol, revealing that these proteins traffic between the nucleus and cytoplasm during interphase (Pinol-Roma and Dreyfuss, 1993
). Here, we have shown interruption in the trafficking pathway of SR proteins, with preferential targeting to the nucleolar periphery under conditions where RNA polymerase II is inhibited, suggesting that this region is important for continuous SR protein modification/maturation/trafficking. This interpretation is strengthened by similar observations made for snRNP trafficking during interphase, as snRNPs were shown to proceed from the cytoplasm to the CBs, and then into nucleoli before accumulation in nuclear speckles (Sleeman and Lamond, 1999
). Interestingly, SR proteins such as SF2/ASF have been identified in proteomic analysis of nucleoli (Leung et al., 2003
). Our results support a model in which SR proteins traffic through the nucleolar periphery in interphase cells, in contrast to snRNPs, which were not detected at the nucleolar periphery, and is consistent with what is seen at mitotic exit as the two families of splicing factors spatially separated in the daughter nuclei. The initial accumulations of SR proteins in NAPs during telophase may reflect the beginning of SR protein modification/maturation/trafficking in the new cell cycle, a time when global transcriptional activity is more reduced than during interphase and much less pre-mRNA target is available for the splicing machinery.
The state of chromatin condensation and nuclear speckle integrity are inversely correlated through the cell cycle (Smith et al., 1985
). In our work, we found that SR proteins did not directly accumulate in nuclear speckles in telophase, but initially accumulated around NORs, which contain repetitive rDNA and are the first extensive regions of chromatin that become highly decondensed and transcribed following mitosis. Human nuclei contain 10 NORs located on the short arms of acrocentric chromosomes 13, 14, 15, 21, and 22 (Kaplan and O'Connor, 1995
), six of which are associated with transcription factors in HeLa cells during mitosis and are the sites where rRNA is synthesized and nucleoli form (Roussel et al., 1996
). We considered the possibility that NORs may be the initial targets for SR proteins because they serve some transient function in nucleolar biogenesis. However, this is unlikely because SF2/ASF is always found in regions directly surrounding NORs, not coincident with NORs. It is also unlikely that the SR proteins are involved in splicing pre-mRNAs originating from transcriptionally active genes that flank NORs or from active chromatin domains in the vicinity of NORs because large regions of heterochromatin isolate the rDNA repeats (Sylvester et al., 1986
; Worton et al., 1988
). Also, during the temporal window that NAPs were observed, polyA+
RNA was absent from nuclei. Furthermore, our data presented here do not support this possibility because the snRNPs, which are enriched elsewhere in the telophase nucleus, would also be expected to accumulate on such transcripts. We also addressed the possibility that SR proteins may be targeted to NAPs because they are directly involved in modulating the condensation state of rDNA. Recent reports indicated that topoisomerase I is found in fibrillar centers of nucleoli and NORs (Christensen et al., 2002
) and that SF2/ASF interacts with topoisomerase I to inhibit its DNA relaxation activity (Andersen et al., 2002
). However, we confirmed that GFP-topoisomerase I is found in fibrillar centers, but does not colocalize with SF2/ASF in NAPs (unpublished data), suggesting that NAPs are not directly involved in these processes.
Perhaps SF2/ASF accumulation in NAPs is necessary for modification of SR proteins required for their targeting to nuclear speckles. Phosphorylation by SR protein kinases regulates the release of SR proteins from nuclear speckles (Colwill et al., 1996b
) as well as their splicing activity (Prasad et al., 1999
). Our earlier studies showed that hyperphosphorylation of SR proteins in vivo does not simply release SR proteins from speckles, but leads to complete disassembly of nuclear speckles (Sacco-Bubulya and Spector, 2002
). On the contrary, hypophosphorylation of SR proteins in vivo causes bright foci to form on the speckle periphery, consistent with inhibition of SR protein release from speckles (Sacco-Bubulya and Spector, 2002
). We concluded that the SR protein–SR protein interactions are the basis for nuclear speckle organization during interphase and would therefore play a critical role in nuclear speckle assembly in daughter nuclei. NAPs provide the only example during the cell cycle in which SR proteins are sequestered from other pre-mRNA processing factors. We have now demonstrated in this paper that SR proteins in NAPs are hypophosphorylated. The sequestration and hypophosphorylated state of the SR proteins at NAPs would favor RS domain–RS domain interactions for establishing NAPs. As the recovery time of SR proteins in NAPs is rapid, a simple mechanism such as this for the establishment of NAPs is the most logical. The presence of Clk/STY at NAPs could be a result of the RS repeats found in the amino terminus of the kinase; however, it also suggests phosphorylation as a means for SR protein activation for subsequent participation in pre-mRNA splicing. Alternatively, the function of Clk/STY at NAPs could be to release SR proteins from NAPs, or both activities could result from the same phosphorylation event. Although it would potentially be informative to examine the localization of SR protein deletion mutants and substitutions with regard to their localization to NAPs during telophase, this is not possible due to the delayed entry of such proteins into daughter nuclei (unpublished data).
If SR protein interactions are the basis for nuclear speckle organization, nuclear speckles could be assembled/disassembled during the cell cycle simply by regulating the level of phosphorylation of RS domains. Phosphorylation of different serine residues within the RS domain may confer different activities or functions upon SR proteins or interactions with different partners and/or subnuclear compartments. This possibility is also supported by the finding that Schizosaccharomyces pombe
contains only two SR proteins and nuclear speckles have not been observed in these cells (Potashkin et al., 1990
), and no SR proteins are encoded in the Saccharomyces cerevisiae
genome and once again no speckles have been identified in these cells (for review see Graveley, 2000
). In mammalian cells, it is not clear how pre-mRNA processing factors are activated to a splicing-competent state in newly forming nuclei that have not yet assembled nuclear speckles. Our current observations suggest that regions surrounding NORs are important sites for initially segregating SR proteins away from other splicing factors, to modify them or to allow them to interact before association with pre-mRNA transcripts or nuclear speckles. This finding raises questions for future study regarding how SR proteins and snRNPs follow two separate pathways to eventually become targeted to the same speckle regions within daughter nuclei. It will be important to determine if SR proteins and snRNPs first meet at transcription sites before recycling of complexes through nuclear speckles. It also raises questions about how different nuclear compartments assemble/disassemble and communicate with each other as nuclear domains are established after mitosis.