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© The Rockefeller University Press,
0021-9525/1999//715 $5.00
The Journal of Cell Biology, Volume 147, Number 4,
, 1999 715-728
Original Article |
The Spinal Muscular Atrophy Disease Gene Product, Smn
: A Link between Snrnp Biogenesis and the Cajal (Coiled) Body
b Department of Anatomy and Cell Biology, Faculty of Medicine, University of Cantabria, 39011 Santander, Spain
Institute of Histology and Embryology, Faculty of Medicine, Av. Prof. Egas Moniz, 1649-028 Lisboa Codex, Portugal.+351 1 7951780+351 1 7934340
hcarmo{at}correio.fm.ul.pt
The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway.
Key Words: Cajal (coiled) body • leptomycin B • nucleocytoplasmic transport • SMN protein • spliceosomal small nuclear ribonucleoproteins
© 1999 The Rockefeller University Press
SMALL nuclear ribonucleoproteins (snRNPs)1 are essential mediators of RNA processing events. In particular, the so-called spliceosomal snRNPs play a key role in splicing of pre-mRNA (reviewed by Burge et al. 1999). This class includes the U1, U2, U4, U5, and U6 snRNPs. The five spliceosomal snRNAs are synthesized in the nucleus and, with the single exception of U6 ( Vankan et al. 1990), they are rapidly exported to the cytoplasm ( Lührmann 1988; Mattaj 1988). The U1, U2, U4, and U5 snRNAs contain an evolutionarily conserved structural element composed of a single-stranded, uridylic acid–rich region that is typically flanked by two hairpin loops ( Branlant et al. 1982). This motif binds a complex of eight proteins (B, B', D1, D2, D3, E, F, and G), collectively known as the Sm or core proteins ( Lührmann et al. 1990). After assembly of the Sm core domain, the 5'-monomethylated guanosine cap structure of the snRNAs is hypermethylated to yield 2,2,7-trimethylguanosine (m3G) ( Mattaj 1986). In addition, varying numbers of nucleotides are trimmed from the 3' end of several of the snRNAs and many internal nucleotides are modified ( Reddy and Busch 1988). Assembly of the Sm core, cap hypermethylation, and 3' end processing occur in the cytoplasm and are crucial for the subsequent nuclear import of the snRNP particles ( Mattaj 1988; Izaurralde and Mattaj 1992). The final step in the biogenesis of these particles involves the association of more than 30 snRNP-specific proteins that are found in only one type of snRNP. It remains unclear whether the assembly of these proteins takes place just before or after nuclear import. When and where the internal snRNA modifications are introduced is also uncertain, but a recent study by Yu et al. 1998 indicates that 2'-O-methylation and pseudouridylation of U2 snRNA take place in the nucleus and are required for assembly of snRNP-specific proteins.
Transport of snRNP particles from the cytoplasm to the nucleus involves a receptor- and energy-dependent mechanism that is not completely understood. Both the m3G-cap and a nuclear localization signal located within the Sm core have been implicated, but not all spliceosomal snRNPs have the same m3G-cap requirements ( Fischer et al. 1991; Michaud and Goldfarb 1992). Differential m3G-cap requirements are also detected between Xenopus oocytes and mammalian somatic cells, presumably due to differences in soluble cytosolic factors ( Marshallsay and Lührmann 1994). Competition experiments indicate that snRNPs are imported by specific receptors not shared by other classes of nuclear proteins ( Fischer et al. 1991; Michaud and Goldfarb 1992; Izaurralde et al. 1997), and the first nuclear import receptor for snRNPs, termed snurportin1, was recently identified ( Huber et al. 1998).
In the course of studies on heterogeneous nuclear ribonucleoproteins (hnRNPs), two novel proteins involved in snRNP biogenesis, SMN and SIP1, were discovered ( Liu and Dreyfuss 1996; Liu et al. 1997). The SMN protein is encoded by the Survival Motor Neurons gene, which is deleted or mutated in >98% of patients with Spinal Muscular Atrophy (SMA), and is therefore considered the SMA disease gene ( Lefebvre et al. 1998). The SMN gene is duplicated in humans ( Lefebvre et al. 1995), but is an essential, single-copy locus in mice ( Schrank et al. 1997). SIP1 is a novel protein that forms a stable heteromeric complex with SMN in vivo and in vitro ( Liu et al. 1997). In addition to SIP1, SMN binds to RNA ( Lorson and Androphy 1998; Bertrandy et al. 1999) and interacts directly with several snRNP Sm core proteins forming a large SMN-SIP1–snRNP complex ( Liu et al. 1997). Importantly, microinjection of anti–SIP1 antibodies in the cytoplasm of Xenopus oocytes completely inhibited the Sm core assembly on U1 and U5 snRNAs and, consequently, blocked their nuclear import ( Fischer et al. 1997). In addition to playing an essential role in snRNP biogenesis, more recent data suggest that SMN may also be involved in recycling of the nuclear spliceosomal snRNPs after each round of splicing ( Pellizzoni et al. 1998).
The SMN-SIP1–snRNP complexes are biochemically detected in both the cytoplasm and the nucleus of somatic cells, and immunolocalization studies revealed SMN and SIP1 proteins concentrated in novel nuclear structures that were called "gemini of coiled bodies" or "gems" ( Liu and Dreyfuss 1996; Liu et al. 1997) because they are frequently located in close proximity to coiled bodies, a subnuclear structure that is highly enriched in snRNPs (for recent reviews see Lamond and Earnshaw 1998; Matera 1999).
Coiled bodies were first described as accessory bodies of the nucleolus ( Ramón y Cajal 1903), and later rediscovered by electron microscopists ( Hardin et al. 1969; Monneron and Bernhard 1969; Hervás et al. 1980). The name "coiled body" was introduced because when this subnuclear structure is viewed with the electron microscope it resembles a tangle of coiled threads ( Monneron and Bernhard 1969). At present, coiled bodies have been renamed Cajal bodies ( Gall et al. 1999) and are known to contain three major classes of small nuclear ribonucleoproteins. These are the spliceosomal U1, U2, U4, U5, and U6 snRNPs ( Carmo-Fonseca et al. 1991, Carmo-Fonseca et al. 1992; Huang and Spector 1992; Matera and Ward 1993), the U7 snRNA required for 3' end processing of histone mRNA ( Wu and Gall 1993; Frey and Matera 1995; Wu et al. 1996), and the U3 and U8 small nucleolar RNAs (snoRNAs) involved in processing of pre–rRNA ( Wu et al. 1993; Jiménez-Garcia et al. 1994; Bauer et al. 1994). Although the function of the Cajal body is still unknown, some of these structures are located near the replicative histone gene cluster ( Gall et al. 1981; Frey and Matera 1995), where they may recruit histone processing factors to the sites of histone pre–mRNA transcription ( Bellini and Gall 1998; Abbott et al. 1999). In addition, Cajal bodies associate with the human U3 snoRNA genes ( Gao et al. 1997), and with the tandemly repeated human U1 and U2 snRNA genes ( Frey and Matera 1995; Smith et al. 1995). Remarkably, the association with U2 genes requires transcriptionally competent loci, indicating that targeting of the body to this chromosomal site is mediated by nascent snRNAs ( Frey et al. 1999).
The discovery that SMN and SIP1, two proteins essential for snRNP biogenesis, are located in close proximity to the Cajal body represented a major breakthrough in unraveling the function of this structure. However, an intriguing question was how SMN plays a direct role in nuclear snRNP metabolism when it is concentrated in gems, which do not contain snRNPs under physiologic conditions (see Dietz 1998). In the present work, we demonstrate that SMN and SIP1 are normally located in the Cajal body, whereas gems are only detected in a variable but small proportion of rapidly proliferating cells in culture. The Cajal body is shown to represent a unique intranuclear site specifically enriched in snRNPs, SMN, and SIP1, thus suggesting a direct involvement of this structure in the snRNP biogenesis pathway.
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Immunofluorescence and Immunoelectron Microscopy of Cultured Cells
For indirect immunofluorescence cells were grown on 10 x 10-mm glass coverslips. The cells were washed twice in PBS, fixed with 3.7% formaldehyde (freshly prepared from paraformaldehyde) in PBS for 10 min at room temperature, and subsequently permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature. Alternatively, cells were first permeabilized with 0.5% Triton X-100 in CSK buffer (100 mM NaCl, 300 mM sucrose, 10 mM PIPES, 3 mM MgCl2, 1 mM EGTA, pH 6.8; Fey et al. 1986) containing 0.1 mM PMSF for 1 min on ice, and subsequently fixed with 3.7% formaldehyde in CSK buffer, for 10 min at room temperature. After fixation and permeabilization, the cells were rinsed in PBS containing 0.05% Tween-20 (PBS-Tw), incubated for 30 min with primary antibodies diluted in PBS, washed in PBS-Tw, and incubated for 30 min with the appropriate secondary antibodies conjugated to fluorescein (FITC), Texas red, or indodicarbocyanine (Cy5) (Jackson ImmunoResearch Laboratories, Inc.). Finally, the coverslips were mounted in VectaShield (Vector Laboratories) and sealed with nail polish.
For immunoelectron microscopy, cells were fixed with 3.7% formaldehyde in PBS, 
20 min. First, the medium was washed twice with PBS and the fixative was added to the culture dishes for 10 min; then, the cells were scraped using a rubber policeman and centrifuged at 7,000 g for 10 min. The cell pellets were sequentially dehydrated with continuous low-speed stirring, in 30% methanol at 4°C for 5 min, 50% methanol at 4°C for 5 min, 75% methanol at –20°C for 5 min, and 90% methanol at –20°C for 30 min. This was followed by mixtures of 90% methanol/Lowicryl K4M (1:1 and 1:2 vol/vol, for 1 h each at –20°C). Finally, the cell pellets were embedded in Lowicryl K4M for 24 h at –20°C and transferred to gelatine capsules. Polymerization was induced by UV irradiation for 5 d at –20°C, followed by one more day at room temperature. Ultrathin sections on gold grids were sequentially incubated with 0.1 M glycine in PBS (15 min), 5% BSA in PBS (15 min), and finally the primary antibody was diluted in 1% BSA, 0.1 M glycine in PBS (1 h at room temperature). After washing, the sections were incubated with appropriate secondary antibody conjugated to either 5 or 10 nm gold particles diluted 1:25 in 0.1% BSA in PBS (45 min at room temperature). Finally, the sections were washed and stained with 10% aqueous uranyl acetate. As controls, sections were treated as described, but omitting the primary antibody.
Immunocytochemistry of Rat Neurons
Male rats of the Sprague-Dawley strain were kept on a 12-h day/night lighting regime with lights out at 8:00. All animals were killed by overdose of pentobarbital. For immunofluorescence, the rats were perfused through the ascending aorta with 3.7% formaldehyde (freshly prepared from paraformaldehyde) in PBS, pH 7.4, for 15 min at room temperature. Supraoptic nuclei were dissected out of 500-µm-thick hypothalamic slices and trigeminal ganglia were removed and cut into small fragments. These tissue fragments were washed in PBS for 1 h and individually transferred to a drop of PBS on a siliconized slide. Then, a coverslip was applied on top of the slide and the tissue was squashed by percussion with a histologic needle. The preparation was then frozen in dry ice and the coverslip was removed using a razor blade. The slides with adhered neurons were sequentially dehydrated in 96% and 70% ethanol at 4°C for 10 min and rinsed in PBS. Before immunostaining, the samples were sequentially incubated with 0.5% Triton X-100 in PBS for 10 min, 0.1 M glycine in PBS for 30 min, and 0.01% Tween 20 in PBS for 5 min.
For immunoelectron microscopy, animals were perfused with 4% formaldehyde (freshly prepared from paraformaldehyde) in 0.12 M phosphate buffer, pH 7.4. The hypothalamic supraoptic nucleus was dissected out from brain slices and left in the same fixative for 3 h at 4°C, dehydrated in increasing concentrations of methanol at –20°C, and embedded in Lowicryl K4M at –20°C. Ultrathin sections were immunostained as described above.
Microscopy and Image Processing
Confocal microscopy was performed with either a Zeiss laser scanning microscope LSM 410 or a BioRad MRC-1024, using excitation wavelengths of 488 nm (for FITC), 543 nm (for Texas red), and 633 nm (for Cy5). Each channel was recorded independently and pseudocolor images were generated and superimposed. Ultrathin sections were examined with a JEOL CXII electron microscope operated at 80 Kv.
For quantitative analysis, images were obtained from untreated and leptomycin B–treated cells, and double labeled with probes for U2 snRNA (green staining) and coilin (red staining). Each image was decomposed in RGB (yR = red, yG = green) and segmented according to Fwu and Djuriç 1996 into four classes: C0 = extracellular background, C1 = intracellular background (i.e., cytoplasm and nucleoli), C2 = nucleoplasm, C3 = Cajal bodies. The average intensities for yG 
C3 (m2) and yG C2 (m1) were estimated. Finally, the ratio m2/m1 was determined.
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Since colocalization studies based on computer-superimposed images of fluorochrome-coupled antibody labeling are constrained by the resolution limits of light microscopy, immunoelectron microscopy was also performed. Cells were fixed in formaldehyde, embedded in Lowicryl K4M at –20°C, and ultrathin sections were incubated with anti–SMN and anti–coilin antibodies. Fig. 2 depicts thin sections through the nucleus of Huh7 cells. In Fig. 2 A, the section was incubated with anticoilin antibody and appropriate secondary antibody conjugated to 10-nm gold particles. The gold particles decorate the characteristic entangled threads of a Cajal body. A similar labeling pattern (i.e., concentration of gold particles over a Cajal body) is observed in B, which depicts a thin section incubated with anti–SMN antibody. Fig. 2C and Fig. D, shows Cajal bodies simultaneously labeled by anticoilin and anti–SMN antibodies (detected with 5- and 10-nm gold particles, respectively). This demonstrates that SMN is present in the Cajal body of cultured cell lines.
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10–15% of the nuclei contained SMN in gems ( Fig. 3H and Fig. I). In conclusion, these results show that SMN may either be present in discrete focal structures that are not enriched in snRNPs (i.e., gems) or colocalize with snRNPs in the Cajal body. The association of SMN with Cajal bodies is observed in both cultured cell lines and primary neurons, whereas gems are only detected in a small proportion (<15%) of rapidly proliferating cells in culture. Although gems tend to be more frequently detected during the G1 stage of the cell cycle (T. Carvalho, unpublished data), the overall proportion of nuclei containing gems is variable depending on cell type and passage number.
SMN Is Targeted to Preformed Cajal Bodies
In agreement with data from earlier studies ( Ferreira et al. 1994), remnants of Cajal bodies are visible in the cytoplasm of mitotic HeLa cells ( Fig. 4). We have previously reported that these mitotic Cajal bodies are labeled with antibodies directed against both snRNP proteins and the m3G cap, and by snRNA-specific antisense probes, indicating that they contain assembled snRNP particles ( Ferreira et al. 1994). Here we show that these structures are additionally labeled by anti–SMN antibodies ( Fig. 4, A–C), indicating that the association of SMN and snRNPs with Cajal bodies persists during mitosis. As cells progress from telophase to G1, Cajal bodies reappear in the nucleus ( Fig. 4 D, arrows), but SMN remains exclusively in the cytoplasm ( Fig. 4 D, arrowheads). The presence of SMN in Cajal bodies is detected later in G1, indicating that there is a lag period between assembly of the Cajal body and localization of SMN therein. This suggests that SMN is targeted to preformed Cajal bodies.
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Taken together, these results suggest that Cajal bodies provide a structural framework for the colocalization of SMN and snRNPs in the nucleus.
SIP1 Colocalizes with SMN and snRNPs in the Cajal Body
Next, we examined the distribution of SIP1, a novel protein that forms a complex with SMN ( Liu et al. 1997). Immunolocalization studies were performed using anti–SIP1 monoclonal antibody 2E17 ( Liu et al. 1997), anti–Sm, and anticoilin antibodies. As depicted in Fig. 6, both primary neurons and HeLa cells show a precise colocalization of SIP1 and snRNPs in the Cajal body. The presence of SIP1 in Cajal bodies was further confirmed at the electron microscopical level (data not shown). Thus, Cajal bodies represent unique intranuclear sites where snRNPs colocalize with SMN and SIP1.
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HeLa cells were incubated with 10, 20, or 30 nM leptomycin B for 1–10 h. Although these drug concentrations produced similar effects, at the lower doses the time of response was longer and more variable. We therefore decided to work with a concentration of 30 nM. For snRNP detection, the cells were labeled with antibodies directed against Sm proteins (Y12; Lerner et al. 1981), the U2 snRNP-specific B'' protein (4G3; Habets et al. 1989), and the m3G cap of snRNAs (R1131; Reuter et al. 1984); in addition, in situ hybridization was performed with a U2 snRNA-specific antisense probe ( Carmo-Fonseca et al. 1991). In nontreated cells, snRNPs are highly enriched in Cajal bodies (foci in Fig. 7), whereas after 3 h of drug treatment, snRNPs are no longer concentrated in the Cajal body ( Fig. 7). In fact, the U2 snRNP is normally approximately threefold more concentrated in Cajal bodies than in the nucleoplasm, but after leptomycin B treatment the average intensity of U2 snRNP labeling in the Cajal body is similar to that in the nucleoplasm ( Fig. 8).
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The SMN and SIP1 proteins were previously immunolocalized in HeLa cells to discrete nuclear foci termed gemini of coiled bodies or gems ( Liu and Dreyfuss 1996). The data presented here indicate that gems are observed in a variable (but small) proportion of cells in culture. In the vast majority of cells studied, including primary neurons directly removed from organisms, SMN and SIP1 are present exclusively in the Cajal body and gems were never identified. Notably, the presence of SMN in Cajal bodies was also detected by other groups (see Matera and Frey 1998; Matera 1999; Sleeman and Lamond 1999a).
A major difference between Cajal bodies and gems concerns the presence or absence of snRNPs. The SMN and SIP1 proteins colocalize with snRNPs in the Cajal body, whereas gems do not concentrate snRNPs. This makes it likely that Cajal bodies contain the SMN-SIP1–snRNP complexes detected in the nuclear fraction of somatic cells ( Liu et al. 1997). As SMN and SIP1 associate with snRNPs in the cytoplasm, one possibility is that the SMN-SIP1–snRNP complex enters the nucleus and is targeted to the Cajal body. The Cajal body may then represent an assembly platform where snRNPs in transit from the cytoplasm accumulate transiently, until an as yet unidentified stage of their maturation process is completed. Taking into account that 2'-O-methylation and pseudouridylation of U2 snRNA take place in the nucleus and are required for the final assembly of snRNP-specific proteins ( Yu et al. 1998), it will be important to clarify whether snRNP complexes enriched in the Cajal body contain modified snRNAs. In this regard, it is important to note that the U2-specific proteins collectively termed SF3a/SF3b ( Krämer 1996), which assemble into the splicing-competent U2 snRNP 17S particle, are not detected in Cajal bodies (D. Nesic and A. Krämer, personal communication). This observation further supports the view that snRNPs concentrated in the Cajal body are not yet fully mature.
Assuming that both snRNPs and SMN complexes are constantly in motion throughout the cell and accumulate transiently in either Cajal bodies or gems, differences in their trafficking dynamics could account for the differences observed in their steady state nuclear distribution. In fact, expression of a dominant-negative amino-terminal deletion mutant of SMN (SMN
N27), which is thought to block nuclear snRNP recycling, causes an accumulation of SMN in extremely enlarged Cajal bodies ( Pellizzoni et al. 1998). Moreover, SMN contains a self-oligomerization domain that overlaps with the Sm-interacting domain ( Lorson et al. 1998). This implies that SMN may either form a complex with snRNPs (most probably localized in the Cajal body) or self-oligomerize, giving rise to gems. A prediction from this model is that gems should become more prominent after depletion of snRNPs from the Cajal body, as indeed was observed after treatment of cells with leptomycin B.
The observed depletion of snRNPs from Cajal bodies caused by leptomycin B strongly suggests that spliceosomal snRNPs newly imported from the cytoplasm travel through the Cajal body. Leptomycin B is a cytotoxin that interacts directly with the nuclear export receptor CRM1, inhibiting its binding to nuclear export signals and Ran-GTP ( Fornerod et al. 1997). As a consequence, this drug blocks the export of substrates recognized by CRM1, including Rev ( Wolff et al. 1997) and proteins containing Rev-like leucine-rich nuclear export signals ( Fornerod et al. 1997; Fukuda et al. 1997; Ossareh-Nazari et al. 1997). Although CRM1 mutations in yeast interfere with mRNA export ( Stade et al. 1997), to date leptomycin B has been found to inhibit export of only one class of RNA in vertebrates, the U snRNAs ( Fornerod et al. 1997). As shown in Fig. 7 and Fig. 8, Fig. 3 h after drug treatment, Cajal bodies are still present but are no longer brightly stained by antibodies directed against Sm proteins, the U2 snRNP-specific B'' protein, and the m3G cap of snRNAs, or by an antisense probe specific for the U2 snRNA. Thus, after exposure to leptomycin B the Cajal body fails to concentrate snRNPs. Since leptomycin B inhibits nuclear export of U snRNAs, the assembly of new snRNP particles in the cytoplasm and their subsequent transport to the nucleus is also blocked. The observed redistribution of snRNPs in the nucleus can therefore be explained if mature snRNPs present in the Cajal body leave the structure while new particles are prevented from reaching it. Direct visual evidence that snRNPs flow through the Cajal body has been recently obtained by microinjection of fluorescently tagged U snRNAs into the cytoplasm of Xenopus oocytes ( Gall et al. 1999; Narayanan et al. 1999) and expression of green fluorescent protein–tagged Sm proteins in HeLa cells ( Sleeman and Lamond 1999b). These studies revealed that both snRNAs and snRNP proteins are first detected in Cajal bodies before they start to accumulate at other nuclear locations.
In addition to interfering with the concentration of snRNPs in the Cajal body, treatment of cells with leptomycin B also affects the distribution of coilin in the nucleus. Shortly after exposure to the drug, coilin is detected inside the nucleolus while the number of Cajal bodies decreases. Within the nucleolus, coilin colocalizes with fibrillarin in foci that correspond to the dense fibrillar component of the nucleolus. A similar staining pattern was previously observed in HeLa cells labeled with an anticoilin monoclonal antibody (mAb-
) ( Almeida et al. 1998). Remarkably, this antibody reacts with an epitope adjacent to a critical serine residue (serine 202) that, when mutated to aspartate, induces the formation of Cajal body-like structures inside the nucleolus ( Lyon et al. 1997). The presence of Cajal bodies inside the nucleolus has also been reported in certain rare cases such as breast carcinoma cells, brown adipocytes, and hepatocytes of hibernating dormice ( Malatesta et al. 1994; Ochs et al. 1994). Much more frequently, Cajal bodies are located in the vicinity of nucleoli, even physically attached to them, seemingly emerging from the dense fibrillar component ( Ferreira and Carmo-Fonseca 1995; Lafarga et al. 1998). Supporting this morphological evidence, several molecules are shared by both the Cajal body and the dense fibrillar component of the nucleolus, namely, fibrillarin ( Raska et al. 1990, Raska et al. 1991), NAP57 and Nopp140 ( Meier and Blobel 1994). In particular, Nopp140 interacts with coilin and has been proposed to chaperone the transport of molecules from the nucleolus to the Cajal body ( Isaac et al. 1998). It therefore appears that leptomycin B inhibits the flow of molecules between the nucleolus and the Cajal body, causing a retention of coilin in the dense fibrillar component and, consequently, a decrease in the number of Cajal bodies.
In summary, the major conclusions from this study are, first, that two proteins essential for snRNP biogenesis, SMN and SIP1, are localized in the Cajal body, and, second, that the nuclear export inhibitor leptomycin B causes a depletion of snRNPs from the Cajal body. The first observation provides a link between Cajal bodies and snRNP biogenesis, whereas the second indicates that the accumulation of snRNPs in the Cajal body is transient. Taken together, these results suggest that snRNPs flow through the Cajal body along their biogenesis pathway.
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Acknowledgments |
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This study was supported by grants from Fundação para a Ciência e Tecnologia/PRAXIS XXI (SAU/1310/95, Portugal), Programa de Promoción General del Conocimiento (PM-960036, Spain), and from the European Union (ERBCHRXCT-930251 and Biomed2 PL 963147).
Submitted: 26 August 1999
Revised: 23 September 1999
Accepted: 30 September 1999
1.used in this paper: m3G, 2,2,7-trimethylguanosine cap; SIP1, SMN interacting protein 1; SMN, survival motor neurons; snRNP, small nuclear ribonucleoprotein
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