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Article |
Address correspondence to M. Dundr, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892. Tel.: (301) 402-0303. Fax: (301) 402-0055. email: dundrm{at}mail.nih.gov; or A.G. Matera, Case Western Reserve University School of Medicine, Cleveland, OH 44106. Tel.: (216) 368-4922. Fax: (216) 368-1257. email: a.matera{at}case.edu
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Abstract |
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Key Words: Cajal body; gems; coilin; SMN; iFRAP
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Introduction |
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CBs are molecularly defined by the presence of the marker protein coilin (Raska et al., 1991). In most cell types and tissues, CBs also contain the survival of motor neurons (SMN) protein (Matera and Frey, 1998; Carvalho et al., 1999; Young et al., 2000). SMN is part of a multiprotein complex that plays an essential role in the assembly of spliceosomal snRNPs (Meister et al., 2002; Paushkin et al., 2002). In the absence of a functional SMN complex, snRNPs are improperly assembled, resulting in defects in pre-mRNA splicing (Fischer et al., 1997). In some cell lines and certain fetal tissues (Liu and Dreyfuss, 1996; Sleeman and Lamond, 1999b; Young et al., 2001), the SMN complex localizes in separate nuclear structures, called gems (Gemini of CBs). The localization of SMN to CBs is regulated by the methylation status of the RG box in coilin (Boisvert et al., 2002; Hebert et al., 2002).
Using time-lapse fluorescence microscopy in living cells, several groups have shown that CBs are mobile within the nucleoplasm (Boudonck et al., 1999; Platani et al., 2000, 2002). Furthermore, Handwerger et al. (2003) demonstrated that GFP fusions of both coilin and TATA-binding protein are dynamically exchanged between the CB and the nucleoplasm in isolated Xenopus oocyte nuclei, although the exchange is slow in comparison to the nucleoplasmic mobility of these proteins (Handwerger et al., 2003). Furthermore, FRAP experiments in mammalian cells suggest rapid exchange of fibrillarin, SMN, and coilin from CBs (Snaar et al., 2000; Sleeman et al., 2003). These observations suggest that CBs are dynamic structures.
In this work, we have systematically investigated the dynamic properties of various CB components. To this end, we have analyzed the kinetics of 14 proteins belonging to several functionally distinct groups of CB components, including factors involved in pre-mRNA splicing, pre-rRNA processing, and snoRNA biogenesis. Using a modified photobleaching technique termed inverse fluorescence recovery after photobleaching (iFRAP), we find three kinetically distinct groups of components within CBs. Furthermore, fluorescence resonance energy transfer (FRET) experiments provide evidence for coilinSMN, coilincoilin and SMNSMN interaction within the CB. The residence times of CB components that localize to residual CBs were not dependent on coilin, and the residence times of coilin and SMN in CBs were independent of their physical interaction. The sum of our observations provides a kinetic framework for CB components in vivo and has implications for the dynamic functional interplay of nuclear subcompartments.
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Results |
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Coilin dynamics
First, we tested the dissociation kinetics of the CB marker protein coilin. To this end, a previously characterized HeLa cell line (Sleeman et al., 1998; Platani et al., 2000) stably expressing GFP-coilin was used (Fig. 1 B). When iFRAP was applied to these cells, a small fraction of the fluorescence signal was lost within the first 5 s after the bleaching event, followed by a slow decline of the CB signal. After 200 s, a plateau representing the dissociation/association equilibrium of the unbleached population of molecules was reached (Fig. 1 J). Although the rapid initial loss likely reflects of a fraction of loosely bound or diffusing GFP-coilin, the slow loss of the majority of protein indicates that GFP-coilin is temporarily retained in the CB. The
200-s plateau indicates that coilin resides in mammalian CBs on the order of 23 min (Fig. 1 J). Typical measurement errors in all experiments were
15%. Equivalent results were obtained by transient transfection of full-length GFP-coilin (Fig. 1 A; unpublished data). These results are consistent with the reported dynamic exchange of coilin from CBs in isolated Xenopus oocyte nuclei, although Xenopus coilin exhibited significantly longer residence times (Handwerger et al., 2003).
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Kinetics of splicing and RNP assembly factors
In addition to coilin, the CB contains a large number of components involved in pre-mRNA splicing. To determine the dynamic properties of splicing factors in the CB, we tested the dissociation kinetics of human snRNP core proteins GFP-SmB (Fig. 2 A), GFP-SmD1 (Fig. 2 B), and the U4/U6 snRNP assembly factor GFP-SART3 (Fig. 2 C) in transiently transfected HeLa cells. GFP-tagged versions of these proteins have previously been demonstrated to be incorporated into snRNPs or to interact with them (Sleeman and Lamond, 1999a; Stanek et al., 2003; unpublished data), suggesting that these proteins are functional. Consistent with their presence in a discrete complex, the fluorescence decay curves for the Sm proteins were superimposable, and these proteins displayed dissociation kinetics that were significantly faster than GFP-coilin (Fig. 2 D; P < 0.001). More than 50% of the initial fluorescence signal was lost within 30 s, whereas in comparison, 50% reduction for GFP-coilin was only reached after 100 s (Fig. 2 D). The U4/U6 snRNP assembly factor SART3 showed even more rapid dissociation from CBs (Fig. 2 D; P < 0.001), consistent with biochemical data suggesting a transient association of SART3 with the U4/U6 snRNP (Bell et al., 2002; Stanek et al., 2003). Together, these observations suggest that pre-mRNA splicing factors have significantly shorter residence times in CBs than coilin.
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Coilin and SMN interact in the CB in vivo
Because coilin and SMN have similar CB retention dynamics (Fig. 6), we were interested to test whether coilin physically interacts with SMN within the CBs of intact living nuclei. Previous work has shown that SMN and coilin form complexes in vivo (Hebert et al., 2001), and that methylation of the coilin RG box motif is required for recruitment of SMN to CBs (Hebert et al., 2002). However, it was not clear from the biochemical data whether SMN and coilin interact in the nucleoplasm, the CB, or in both places. To test whether these proteins interact in CBs, we performed FRET by acceptor photobleaching in living cells (Karpova et al., 2003; see Materials and methods for details). In acceptor photobleaching FRET, energy transfer between the donor and the acceptor is reduced or eliminated when the acceptor is irreversibly bleached, resulting in an increase in donor fluorescence as an indicator of physical interaction between the test proteins (Karpova et al., 2003). CFP-coilin and YFP-SMN were coexpressed in HeLa cells, the YFP-SMN acceptor was bleached, and the fluorescent signal of CFP-coilin was monitored in living cells (Fig. 7 A). Upon bleaching, an 6.5 ± 1.2% increase in donor CFP-coilin fluorescence was observed, indicating an in vivo interaction of coilin and SMN in CBs (Fig. 7 B). Reversal of the tags to CFP-SMN and YFP-coilin gave a similar increase in donor CFP-SMN fluorescence of
6.29 ± 1.3% (unpublished data). A fusion protein between CFP and YFP served as a positive control and yielded a 14 ± 1.3% increase in fluorescence signal (Fig. 7 B). As negative controls, an increase of <0.5 ± 0.2% was observed for coexpressed CFP and YFP, and a loss of fluorescence signal was detected in a CB in the same nucleus to which no acceptor bleaching had been applied (Fig. 7 B). To demonstrate that the positive FRET signal was not merely due to the accumulation of the two proteins in the CB, we performed FRET between fibrillarin-CFP and YFP-coilin or fibrillarin-CFP and YFP-SART3, which are all concentrated in the CB but have not been found to physically interact. FRET signals for these pairs were similar to negative controls and were 0.75 ± 0.54 for fibrillarin/SART3 and 1.19 ± 0.61 for fibrillarin/coilin (Fig. 7, E and F). Interaction of coilin and SMN in the nucleoplasm could not be accurately assessed due to the low nucleoplasmic signal of SMN (unpublished data). Furthermore, when purified, recombinant proteins are incubated in vitro, coilin and SMN each have the ability to self-oligomerize (Lorson et al., 1998; Hebert and Matera, 2000). To test whether these proteins self-interact in vivo in CBs, we performed acceptor FRET between CFP-coilin and YFP-coilin or CFP-SMN and YFP-SMN (Fig. 7, C and D). An
10.26 ± 1.7% increase of CB donor fluorescence was detected for CFP-coilin, and an
5.23 ± 0.6% increase of CB donor fluorescence was detected for CFP-SMN (Fig. 7, C and D). We conclude that coilin interacts with SMN and that both proteins self-interact in the CB in vivo.
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Discussion |
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Kinetically distinct CB components
In comparing the dissociation kinetics of 14 CB proteins using iFRAP, we found three distinct kinetic classes. The group of CB proteins with the longest residence times includes coilin, SMN, Gemin3, and Tgs1. These proteins typically reside in CBs on the order of minutes. Consistent with previous biochemical and subcellular localization analyses (Carvalho et al., 1999; Hebert et al., 2001, 2002), we provide direct evidence for an interaction of SMN and coilin within CBs by FRET analysis. We cannot directly determine whether these complexes are stable within the nucleoplasm or if they exchange from CBs as intact complexes. However, the comparable retention dynamics of SMN-associated proteins are consistent with this latter possibility. Thus, the SMN complex might not only exist during early steps of Sm assembly in the cytoplasm, but might also remain associated with the assembled snRNPs after their import into the nucleus, perhaps functioning in later steps of snRNP maturation in CBs (Narayanan et al., 2002; Jady et al., 2003; Sleeman et al., 2003). This interpretation is consistent with heterokaryon assays showing that newly imported GFP-Sm proteins are targeted only into CBs that contain SMN (Sleeman et al., 2001). This scenario is also supported by our observation of similar dissociation kinetics of GFP-Tgs1 and GFP-SMN and by the recent finding that Tgs1 physically associates with the SMN complex (Mouaikel et al., 2003). Furthermore, the observation that 3'-extended U3 snoRNA precursors with a monomethylated m7G-cap were detected in CBs also suggests that 5'-cap trimethylation of U3 snoRNA occurs in CBs (Verheggen et al., 2002).
The second kinetic group of CB proteins contains nucleolar snoRNP components and the spliceosomal snRNP core proteins. These proteins have significantly faster dissociation kinetics than coilin and the SMN-associated proteins. We find that fibrillarin-GFP, a box C/D snoRNP factor, and the box H/ACA snoRNP component GAR1 have similar dissociation kinetics from CBs, suggesting that the two snoRNP classes might have analogous mechanisms of retention in CBs. This latter scenario is supported by the fact that GFP-Nopp140, which is associated with both classes of snoRNPs, exhibited comparable dissociation kinetics from CBs as both snoRNP classes. Because 3'-extended precursor U3 snoRNA is only associated with NHPX/15.5k but not with fibrillarin, our data are consistent with findings that the majority of U3 snoRNA detected in CBs are mature U3 snoRNPs (Verheggen et al., 2002). The dissociation kinetics of these proteins was significantly slower than that of the third (and fastest) kinetic group containing the U4/U6 assembly factor SART3 (Bell et al., 2002) and RNA polymerase I transcription factor UBF1. GFP-SART3 exhibited dissociation kinetics, indicating that its association with CBs lasts only a few seconds. This kinetic behavior is in agreement with the observation that SART3 is only transiently associated with snRNPs, and supports a model in which SART3 delivers U6 to the CB where U4/U6 snRNP (re)assembly may occur (Bell et al., 2002; Stanek et al., 2003).
The rapid exchange dynamics of CB components, in the range of seconds to minutes, indicates that all measured proteins are dynamic and are continuously exchanged between the nucleoplasm and CBs. Proteins are likely retained in CBs via transient interaction with less mobile CB components. The dynamic nature of CB association also suggests that any putative modification steps taking place within the CB are relatively rapid and occur on the order of seconds. Furthermore, our observations are consistent with the possibility that many proteins may enter CBs nondiscriminatorily and that collisions with interacting partners lead to their retention. This scenario might explain why certain CB components are retained in CBs only when their partner protein is present, for example in a cell cycledependent manner such as cyclin E and p220NPAT, a substrate of the CB component cyclin E/cdk2 (Liu et al., 2000; Wei et al., 2003).
The observed dynamic nature of CB components is in agreement with the demonstration of dynamic exchange of proteins from several other nuclear compartments, including nucleoli and splicing factor compartments (Phair and Misteli, 2000; Snaar et al., 2000; Chen and Huang, 2001; Dundr et al., 2002; Handwerger et al., 2003; Sleeman et al., 2003). In contrast, at least some components of promyelocytic leukemia bodies appear to be more stably associated (Boisvert et al., 2001). Dynamic exchange of CB components has also been demonstrated for coilin, TATA-binding protein, and U7 snRNA from CBs in Xenopus germinal vesicles, although in this system coilin resides in CBs for up to 30 min (Handwerger et al., 2003). The reason for the significantly longer residence time of coilin in CBs from Xenopus is unclear, but might be due to lower levels of rRNA and snRNA metabolism. In agreement with our observations, a rapid exchange of fibrillarin and coilin from CBs in mammalian cells has previously been reported (Snaar et al., 2000; Sleeman et al., 2003). In contrast to our iFRAP experiments, FRAP analysis has suggested slower exchange of SMN compared with coilin (Sleeman et al., 2003). The apparent discrepancy is likely explained by different measurement technique and the smaller nucleoplasmic pool of SMN compared with coilin (Sleeman et al., 2003). Because FRAP recovery kinetics (but not iFRAP loss kinetics) are sensitive to the size of the unbleached pool, the smaller unbleached nucleoplasmic pool of GFP-SMN compared with GFP-coilin may result in underestimation of exchange kinetics when using FRAP.
Compartment-specific retention mechanisms
Comparison of the dissociation kinetics of nucleolar components that also localize in CBs revealed that nucleolar CB components reside in CBs for a significantly shorter time than they do in nucleoli, indicating the existence of compartment-specific retention mechanisms. These kinetic differences between CBs and nucleoli might be due to interactions of the nucleolar pool of the processing components with pre-rRNA during modification and maturation steps. Because CBs do not contain any pre-rRNA transcripts, retention is reduced in CBs, resulting in faster dissociation. This interpretation is supported by the fact that truncated mutants of fibrillarin-GFP lacking the RNA-binding domain exhibit significantly faster kinetics in nucleoli than wild-type fibrillarin (Snaar et al., 2000). Similarly, when nucleolar rRNA transcription is selectively inhibited by actinomycin D, pre-rRNA processing components nucleolin, fibrillarin, B23, and Rpp29 exhibit faster kinetics in the nucleoli (Chen and Huang, 2001). In contrast, the inhibition of transcription of all three RNA polymerases by actinomycin D does not affect the dissociation kinetics of coilin from CBs, although the structures are relocated to the nucleolar periphery (unpublished data). The even slower dissociation kinetics of GFP-B23 from the nucleolus in comparison to the snoRNP components might reflect its function not only as a processing enzyme but also as a protein chaperone in the assembly of preribosomal subunits (Szebeni et al., 2003), which have longer residence time in nucleoli than the relatively short-lived pre-rRNA processing intermediates (Lazdins et al., 1997; Chen and Huang, 2001).
Kinetic independence of CBs and gems
The fact that coilin has one of the longest residence times of all CB components might suggest that it is a structural element. Consistent with this model, coilin is a multi-interactive protein that, apart from interacting with itself (Hebert and Matera, 2000; this paper), associates with SMN, SmB/B' (Hebert et al., 2001), and Nopp140 (Isaac et al., 1998). However, loss of coilin in mice does not result in complete disappearance of CBs, but rather causes relocalization of various CB components into at least three distinct types of residual nuclear bodies, suggesting that coilin is not essential for the maintenance of nuclear compartments that contain many of the CB components (Tucker et al., 2001; Jady et al., 2003). Nevertheless, coilin does clearly play a critical role in recruitment of CB components to a combined CB/gem structure. Similarly, the SMN protein interacts with a multitude of partners including spliceosomal snRNPs and nucleolar snoRNPs, fibrillarin, GAR1, nucleolin, and B23 (Friesen and Dreyfuss, 2000; Jones et al., 2001; Pellizzoni et al., 2001; Lefebvre et al., 2002). However, in contrast to coilin, whose self-interaction domain targets it to CBs, SMN requires the presence of coilin for its recruitment into CBs (Hebert et al., 2001; Tucker et al., 2001). Despite the physical association and the direct interaction of coilin with SMN, CBs and gems are kinetically autonomous compartments because the dissociation kinetics of several respective components remain unchanged upon separation of the two structures. The basis for this kinetic autonomy may be the ability of SMN and coilin to maintain self-interactions within the separated nuclear bodies. These observations paint a picture in which two independent nuclear structures, the CB and gem, are spatially superimposed due to the dynamic interaction of their marker proteins.
One possible advantage of fusing the two structures into one spatially overlapping domain might be that the concentration of substrates (e.g., snRNPs) and enzymes (e.g., SMN complexes) increases the efficiency of RNA metabolism. Conceivably, SMNsnRNP complexes might localize to the CB after passage through nuclear pores; coilin could then dissociate this complex by competing for SMN's Sm-binding sites (Hebert et al., 2001). This would allow for modification of snRNAs by CB components such as the scaRNPs (Darzacq et al., 2002; Jady et al., 2003). However, such a pathway cannot be an obligate one, as coilin is not an essential protein. Another possibility is that gems represent a storage depot for excess nuclear SMN complexes, forming in response to the methylation status of coilin and/or Sm proteins. Clearly, uncovering the complex links between CBs and gems will require further investigation of the structural, functional, and dynamic relationships between these two nuclear subcompartments.
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Materials and methods |
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Cell culture and transfection
HeLa wild-type, HeLa-PV, and MEFs were grown in DME (Invitrogen) supplemented with 10% FCS (Invitrogen), 1% glutamine, and penicillin and streptomycin at 37°C in 5% CO2. The cells were electroporated with an electroporator (model ECM 830; BTX) using 5 µg plasmid DNA and 15 µg sheared salmon sperm carrier DNA in a 2-mm gap cuvette at 200 V, 1-ms pulse, 4 pulses, and 0.5-s intervals. After electroporation, the cells were plated in Lab-Tek® II chambers (Nalgene), and after 6 h the medium was changed to DME with 25 mM Hepes without phenol red (Invitrogen). In cotransfection experiments, the ratio of
RG-coilin to CFP-coilin (SMN) to YFP-SMN (coilin) was 3:1:1.
Photobleaching
iFRAP experiments were performed on a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.) with a 100x/1.3 N.A. Plan Apochromat oil objective and 3x zoom. GFP was excited with the 488-nm line of argon laser, and GFP emission was monitored above 505 nm as described previously (Dundr et al., 2002). Cells were maintained at 37°C with an Air Stream incubator (ASI 400; Nevtek). The whole nuclear area of transfected cells was bleached except for a small region of one CB using the 488-nm laser line at 100% laser power. Cells were monitored in 0.5-s intervals for 195 s. To minimize the effect of photobleaching due to imaging, images were collected at 0.1% laser intensity. For quantification, the loss of total fluorescent intensity in the unbleached region of interest was measured using software from Carl Zeiss MicroImaging, Inc. Background fluorescence was measured in a random field outside of the cells. For each time point, the relative loss of fluorescent intensity in the unbleached region of interest was calculated as: Irel = (I(t) - BG)/(Io - BG)*(T(t) - BG), where Io is the background-corrected average intensity of the region of interest during prebleach, and T(t) is the background-corrected total fluorescence intensity of a neighboring control cell. Typical measurement errors in all experiments were 15%. For statistical comparison of iFRAP data, t tests on t50 (defined as time after bleaching when 50% of the initial fluorescence signal was lost) were performed.
FRET
FRET experiments were performed on a confocal microscope (LSM 510; Carl Zeiss MicroImaging, Inc.) with a 100x/1.3 N.A. Plan Apochromat oil objective and 3x zoom. FRET was measured using the acceptor photobleaching method (Karpova et al., 2003). In the presence of FRET, bleaching of the acceptor (YFP) results in a significant increase in fluorescence of the donor (CFP). A single CB was bleached for 500 ms in the YFP channel using the 514 argon laser line at 100% intensity. Before and after the bleach, CFP images were collected to assess changes in donor fluorescence. To minimize the effect of photobleaching due to imaging, images were collected at 0.1% laser intensity. To ensure that bleaching due to imaging was minimal, the level of bleaching in each experiment was monitored by collecting five CFP/YFP prebleach and postbleach image pairs. Each image was collected first in the CFP channel, and then in the YFP channel. The gain of the photomultiplier tubes was adjusted to obtain the best possible dynamic range. The FRET efficiency as a percentage of EF was calculated as: EF = (Ipost - Ipre) x 100/Ipost, where Ipre is the prebleach CFP intensity in the last prebleach image, and Ipost is the postbleach CFP intensity in the bleached region of the first post-bleach image. As a negative control, an unbleached CB in the same cell was measured, or FRET between monomeric CFP and YFP was measured in the nucleoplasm. As a positive control, a CFP-YFP fusion protein was used.
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Acknowledgments |
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This work was supported by National Institutes of Health grants GM53034 and NS41617 (to A.G. Matera).
Submitted: 21 November 2003
Accepted: 5 February 2004
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References |
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---|
Bell, M., S. Schreiner, A. Damianov, R. Reddy, and A. Bindereif. 2002. p110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor. EMBO J. 21:27242735.
Boisvert, F.M., M.J. Kruhlak, A.K. Box, M.J. Hendzel, and D.P. Bazett-Jones. 2001. The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body. J. Cell Biol. 152:10991106.
Boisvert, F.M., J. Cote, M.C. Boulanger, P. Cleroux, F. Bachand, C. Autexier, and S. Richard. 2002. Symmetrical dimethylarginine methylation is required for the localization of SMN in Cajal bodies and pre-mRNA splicing. J. Cell Biol. 159:957969.
Boudonck, K., L. Dolan, and P.J. Shaw. 1999. The movement of coiled bodies visualized in living plant cells by the green fluorescent protein. Mol. Biol. Cell. 10:22972307.
Carmo-Fonseca, M. 2002. New clues to the function of the Cajal body. EMBO Rep. 3:726727.
Carvalho, T., F. Almeida, A. Calapez, M. Lafarga, M.T. Berciano, and M. Carmo-Fonseca. 1999. The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body. J. Cell Biol. 147:715728.
Chen, D., and S. Huang. 2001. Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells. J. Cell Biol. 153:169176.
Darzacq, X., B.E. Jady, C. Verheggen, A.M. Kiss, E. Bertrand, and T. Kiss. 2002. Cajal body-specific small nuclear RNAs: a novel class of 2'-O-methylation and pseudouridylation guide RNAs. EMBO J. 21:27462756.
Dundr, M., T. Misteli, and M.O. Olson. 2000. The dynamics of postmitotic reassembly of the nucleolus. J. Cell Biol. 150:433446.
Dundr, M., U. Hoffmann-Rohrer, Q. Hu, I. Grummt, L.I. Rothblum, R.D. Phair, and T. Misteli. 2002. A kinetic framework for a mammalian RNA polymerase in vivo. Science. 298:16231626.
Fischer, U., Q. Liu, and G. Dreyfuss. 1997. The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis. Cell. 90:10231029.[Medline]
Friesen, W.J., and G. Dreyfuss. 2000. Specific sequences of the Sm and Sm-like (Lsm) proteins mediate their interaction with the spinal muscular atrophy disease gene product (SMN). J. Biol. Chem. 275:2637026375.
Gall, J.G. 2000. Cajal bodies: the first 100 years. Annu. Rev. Cell Dev. Biol. 16:273300.[CrossRef][Medline]
Handwerger, K.E., C. Murphy, and J.G. Gall. 2003. Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle. J. Cell Biol. 160:495504.
Hebert, M.D., and A.G. Matera. 2000. Self-association of coilin reveals a common theme in nuclear body localization. Mol. Biol. Cell. 11:41594171.
Hebert, M.D., P.W. Szymczyk, K.B. Shpargel, and A.G. Matera. 2001. Coilin forms the bridge between Cajal bodies and SMN, the spinal muscular atrophy protein. Genes Dev. 15:27202729.
Hebert, M.D., K.B. Shpargel, J.K. Ospina, K.E. Tucker, and A.G. Matera. 2002. Coilin methylation regulates nuclear body formation. Dev. Cell. 3:329337.[Medline]
Isaac, C., Y. Yang, and U.T. Meier. 1998. Nopp140 functions as a molecular link between the nucleolus and the coiled bodies. J. Cell Biol. 142:319329.
Jady, B.E., X. Darzacq, K.E. Tucker, A.G. Matera, E. Bertrand, and T. Kiss. 2003. Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm. EMBO J. 22:18781888.
Jones, K.W., K. Gorzynski, C.M. Hales, U. Fischer, F. Badbanchi, R.M. Terns, and M.P. Terns. 2001. Direct interaction of the spinal muscular atrophy disease protein SMN with the small nucleolar RNA-associated protein fibrillarin. J. Biol. Chem. 276:3864538651.
Karpova, T.S., C.T. Baumann, L. He, X. Wu, A. Grammer, P. Lipsky, G.L. Hager, and J.G. McNally. 2003. Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J. Microsc. 209:5670.[CrossRef][Medline]
Lazdins, I.B., M. Delannoy, and B. Sollner-Webb. 1997. Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization. Chromosoma. 105:481495.[CrossRef][Medline]
Lefebvre, S., P. Burlet, L. Viollet, S. Bertrandy, C. Huber, C. Belser, and A. Munnich. 2002. A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy. Hum. Mol. Genet. 11:10171027.
Leung, A.K., and A.I. Lamond. 2002. In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles. J. Cell Biol. 157:615629.
Lorson, C.L., J. Strasswimmer, J.M. Yao, J.D. Baleja, E. Hahnen, B. Wirth, T. Le, A.H. Burghes, and E.J. Androphy. 1998. SMN oligomerization defect correlates with spinal muscular atrophy severity. Nat. Genet. 19:6366.[Medline]
Liu, Q., and G. Dreyfuss. 1996. A novel nuclear structure containing the survival of motor neurons protein. EMBO J. 15:35553565.[Abstract]
Liu, J., M.D. Hebert, Y. Ye, D.J. Templeton, H. Kung, and A.G. Matera. 2000. Cell cycle-dependent localization of the CDK2-cyclin E complex in Cajal (coiled) bodies. J. Cell. Sci. 113:15431552.
Matera, A.G. 1999. Nuclear bodies: multifaceted subdomains of the interchromatin space. Trends Cell Biol. 9:302309.[CrossRef][Medline]
Matera, A.G., and M.R. Frey. 1998. Coiled bodies and gems: Janus or gemini? Am. J. Hum. Genet. 63:317321.[CrossRef][Medline]
Meister, G., C. Eggert, and U. Fischer. 2002. SMN-mediated assembly of RNPs: a complex story. Trends Cell Biol. 12:472478.[CrossRef][Medline]
Mouaikel, J., U. Narayanan, C. Verheggen, A.G. Matera, E. Bertrand, J. Tazi, and R. Bordonne. 2003. Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron. EMBO Rep. 4:616622.
Narayanan, U., J.K. Ospina, M.R. Frey, M.D. Hebert, and A.G. Matera. 2002. SMN, the spinal muscular atrophy protein, forms a pre-import snRNP complex with snurportin1 and importin beta. Hum. Mol. Genet. 11:17851795.
Ochs, R.L., T.W.J. Stein, and E.M. Tan. 1994. Coiled bodies in the nucleolus of breast cancer cells. J. Cell Sci. 107:385399.
Ogg, S.C., and A.I. Lamond. 2002. Cajal bodies and coilinmoving towards function. J. Cell Biol. 159:1721.
Ou, Q., J.F. Mouillet, X. Yan, C. Dorn, P.A. Crawford, and Y. Sadovsky. 2001. The DEAD box protein DP103 is a regulator of steroidogenic factor-1. Mol. Endocrinol. 15:6979.
Paushkin, S., A.K. Gubitz, S. Massenet, and G. Dreyfuss. 2002. The SMN complex, an assemblyosome of ribonucleoproteins. Curr. Opin. Cell Biol. 14:305312.[CrossRef][Medline]
Pellizzoni, L., J. Baccon, B. Charroux, and G. Dreyfuss. 2001. The survival of motor neurons (SMN) protein interacts with the snoRNP proteins fibrillarin and GAR1. Curr. Biol. 11:10791088.[CrossRef][Medline]
Phair, R.D., and T. Misteli. 2000. High mobility of proteins in the mammalian cell nucleus. Nature. 404:604609.[CrossRef][Medline]
Phair, R.D., S.A. Gorski, and T. Misteli. 2004. Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy. Methods Enzymol. 375:393414.[Medline]
Platani, M., I. Goldberg, J.R. Swedlow, and A.I. Lamond. 2000. In vivo analysis of Cajal body movement, separation, and joining in live human cells. J. Cell Biol. 151:15611574.
Platani, M., I. Goldberg, A.I. Lamond, and J.R. Swedlow. 2002. Cajal body dynamics and association with chromatin are ATP-dependent. Nat. Cell Biol. 4:502508.[CrossRef][Medline]
Pluk, H., J. Soffner, R. Luhrmann, and W. J. van Venrooij. 1998. cDNA cloning and characterization of the human U3 small nucleolar ribonucleoprotein complex-associated 55-kilodalton protein. Mol. Cell. Biol. 18:488498.
Pogacic, V., F. Dragon, and W. Filipowicz. 2000. Human H/ACA small nucleolar RNPs and telomerase share evolutionarily conserved proteins NHP2 and NOP10. Mol. Cell. Biol. 20:90289040.
Raska, I., L.E. Andrade, R.L. Ochs, E.K. Chan, C.M. Chang, G. Roos, and E.M. Tan. 1991. Immunological and ultrastructural studies of the nuclear coiled body with autoimmune antibodies. Exp. Cell Res. 195:2737.[Medline]
Shpargel, K.B., J.K. Ospina, K.E. Tucker, A.G. Matera, and M.D. Hebert. 2003. Control of Cajal body number is mediated by the coilin C-terminus. J. Cell Sci. 116:303312.
Sleeman, J.E., and A.I. Lamond. 1999a. Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway. Curr. Biol. 9:10651074.[CrossRef][Medline]
Sleeman, J.E., and A.I. Lamond. 1999b. Nuclear organization of pre-mRNA splicing factors. Curr. Opin. Cell Biol. 11:372377.[CrossRef][Medline]
Sleeman, J., C.E. Lyon, M. Platani, J.P. Kreivi, and A.I. Lamond. 1998. Dynamic interactions between splicing snRNPs, coiled bodies and nucleoli revealed using snRNP protein fusions to the green fluorescent protein. Exp. Cell Res. 243:290304.[CrossRef][Medline]
Sleeman, J.E., P. Ajuh, and A.I. Lamond. 2001. snRNP protein expression enhances the formation of Cajal bodies containing p80-coilin and SMN. J. Cell Sci. 114:44074419.[Medline]
Sleeman, J.E., L. Trinkle-Mulcahy, A.R. Prescott, S.C. Ogg, and A.I. Lamond. 2003. Cajal body proteins SMN and Coilin show differential dynamic behaviour in vivo. J. Cell Sci. 116:20392050.
Snaar, S., K. Wiesmeijer, A.G. Jochemsen, H.J. Tanke, and R.W. Dirks. 2000. Mutational analysis of fibrillarin and its mobility in living human cells. J. Cell Biol. 151:653662.
Stanek, D., S.D. Rader, M. Klingauf, and K.M. Neugebauer. 2003. Targeting of U4/U6 small nuclear RNP assembly factor SART3/p110 to Cajal bodies. J. Cell Biol. 160:505516.
Szebeni, A., K. Hingorani, S. Negi, and M.O. Olson. 2003. Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23. J. Biol. Chem. 278:91079115.
Tucker, K.E., M.T. Berciano, E.Y. Jacobs, D.F. LePage, K.B. Shpargel, J.J. Rossire, E.K. Chan, M. Lafarga, R.A. Conlon, and A.G. Matera. 2001. Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product. J. Cell Biol. 154:293307.
Verheggen, C., D.L. Lafontaine, D. Samarsky, J. Mouaikel, J.M. Blanchard, R. Bordonne, and E. Bertrand. 2002. Mammalian and yeast U3 snoRNPs are matured in specific and related nuclear compartments. EMBO J. 21:27362745.
Wei, Y., J. Jin, and J.W. Harper. 2003. The cyclin E/Cdk2 substrate and Cajal body component p220(NPAT) activates histone transcription through a novel LisH-like domain. Mol. Cell. Biol. 23:36693680.
Young, P.J., T.T. Le, N. thi Man, A.H. Burghes, and G.E. Morris. 2000. The relationship between SMN, the spinal muscular atrophy protein, and nuclear coiled bodies in differentiated tissues and cultured cells. Exp. Cell Res. 256:365374.[CrossRef][Medline]
Young, P.J., T.T. Le, M. Dunckley, T.M. Nguyen, A.H. Burghes, and G.E. Morris. 2001. Nuclear gems and Cajal (coiled) bodies in fetal tissues: nucleolar distribution of the spinal muscular atrophy protein, SMN. Exp. Cell Res. 265:252261.[CrossRef][Medline]
Zhu, Y., R.L. Tomlinson, A.A. Lukowiak, R.M. Terns, and M.P. Terns. 2004. Telomerase RNA accumulates in Cajal bodies in human cancer cells. Mol. Biol. Cell. 15:8190.