Human Molecular Genetics, 2000, Vol. 9, No. 13 1977-1986
© 2000 Oxford University Press
Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins
Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany
Received 6 April 2000; Revised and Accepted 16 June 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinal muscular atrophy (SMA) is a neurodegenerative disease of motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. Cytoplasmic SMN directly interacts with spliceosomal Sm proteins and facilitates their assembly onto U snRNAs. Nuclear SMN, in contrast, mediates recycling of pre-mRNA splicing factors. In this study, we have addressed the function of SMN in the nucleus. We show that a monoclonal antibody directed against SMN inhibits pre-mRNA splicing. Interestingly, the mode of inhibition suggests a novel role for SMN in splicing that occurs prior to, or in addition to, its role in recycling. Using biochemical fractionation and anti-SMN immunoaffinity chromatography, we identified two distinct nuclear SMN complexes termed NSC1 and NSC2. The biochemical properties and protein composition of NSC1 were determined in detail. NSC1 migrates in sucrose gradients as a U snRNA-free 20S complex containing at least 10 proteins. In addition to SMN, these include the SMN-interacting protein 1 (SIP-1), the putative helicase dp103/Gemin3, the novel dp103/Gemin3-interacting protein GIP1/Gemin4 and three additional proteins with apparent masses of 43, 33 and 18 kDa, respectively. Most surprisingly, NSC1 also contains a specific subset of spliceosomal Sm proteins. This shows that the SMN–Sm protein interaction is not restricted to the cytoplasm. Our data imply that nuclear SMN affects splicing by modulating the Sm protein composition of U snRNPs.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The motor neuron disease spinal muscular atrophy (SMA) is one of the most prevalent causes of infant mortality, affecting ~1 in 10 000 live births (1). The disease is characterized by a progressive loss of
motor neurons in the anterior horn of the spinal cord, leading to muscle weakness of the trunk and limbs (2–4). The clinical types of the disease (types I–III) are distinguished by the severity of the symptoms. Patients suffering from the most severe form of SMA (type I) display dramatic muscle weakness and a life expectancy of typically <3 years. Milder forms of SMA (SMA II and III) are characterized by a later onset of the disease and a less pronounced muscle weakness. Genetic approaches have been used to show that mutations in the survival of motor neurons (SMN) gene are the major cause of SMA (5). The SMN gene is duplicated in an inverted repeat in chromosome 5. The telomeric copy of SMN (SMN1) is homozygously deleted or mutated in >90% of all SMA patients, whereas the second copy (SMN2, or centromeric SMN) remains unaffected. These SMN genes differ in that the full-length protein is almost exclusively produced from the SMN1 gene. In contrast, the primary transcript of SMN2 undergoes alternative splicing of exon 7, which leads to the predominant expression of an unstable and C-terminally truncated SMN protein (5,6). At the cellular level, this leads to apoptotic cell death of motor neurons in the anterior horn of the spinal cord, and consequently to SMA (7).
In contrast to humans, mice harbour only one copy of the SMN gene, which is equivalent to human SMN1. Gene targeting studies in mice revealed that SMN is essential for viability, i.e. mice that are null for the SMN gene die prior to implantation at the blastocyst stage (8). Interestingly, the human SMN2 copy can rescue the early embryonic lethal phenotype in SMN–/– mice. These SMN–/–SMN2 mice develop postnatal symptoms very similar to those found in SMA patients (9–11).
The SMN gene encodes a ubiquitously expressed protein of 294 amino acids which is predominantly found in the cytoplasm. However, SMN also localizes to the nucleus, where it is concentrated in a subnuclear structure of unknown function, termed gemini of coiled bodies (gem) (12). It is thought that SMN is incorporated in large complexes, since it can be co-immunoprecipitated with several recently identified proteins, namely the SMN-interacting protein 1 (SIP1), the putative helicase Gemin3 and spliceosomal U snRNP-proteins of the Sm class (13,14). Moreover, it has been shown that SMN transiently associates with spliceosomal snRNAs U1, U4 and U5 in the cytoplasm of Xenopus laevis oocytes (15).
The association with spliceosomal components suggested two cellular functions of SMN. A critical role for SMN and SIP1 in the cytoplasmic assembly of spliceosomal snRNPs U1, U2, U4 and U5 could be deduced from studies in X.laevis oocytes. In this process, the U snRNAs are bound in a step-wise and ordered manner by the Sm hetero-oligomeric complexes of B/B'.D3, D1.D2 and E.F.G. The U snRNPs are thereafter imported into the nucleus, where they function in pre-mRNA splicing (16–18). Antibodies directed against SIP1 and SMN strongly interfere with binding of the Sm proteins onto the U snRNA, indicating that both proteins are essential assembly factors for U snRNPs (15,19).
Since U snRNP assembly is a cytosolic process, it is likely to be mediated by one or several cytoplasmic SMN complexes. In contrast to cytoplasmic SMN, it has recently been proposed that the nuclear pool of SMN contributes directly to nuclear pre-mRNA splicing (20). Pellizzoni et al. (20) observed inhibition of splicing in vitro after pre-incubation of HeLa nuclear extract with monoclonal antibody 2B1 directed against SMN. Similarly, pre-incubation of nuclear extract with a deletion mutant of the SMN protein that lacks the first 27 amino acids inhibited splicing by blocking the formation of mature spliceosomes. Hence, it was suggested that SMN recycles splicing factors after pre-mRNA processing (20). The putative RNA-helicase dp103/Gemin3 might confer the enzymatic activity required for this recycling process (14). However, the precise mode of action of SMN and possible other components in this process remains elusive.
Nuclear SMN may also have a function related to gene regulation at the transcriptional level. This was concluded from the observation that the papillomavirus nuclear transcription activator, E2, interacts with SMN in vitro and in vivo and that SMN enhances the E2-dependent transcriptional activation of genes (21). Furthermore, the SMN-interacting protein dp103/Gemin3 was originally identified as a factor that interacts with viral and cellular transcription factors (22,23). Thus, SMN may also regulate gene expression, although the mechanism by which this is achieved is not yet understood in detail.
In this study, we have used a different monoclonal anti-SMN antibody to analyse SMN function in the nucleus. We provide evidence for a novel role of SMN in the first step of pre-mRNA splicing. Using a biochemical strategy, we report on the isolation of two distinct nuclear SMN complexes, termed NSC1 and NSC2, from HeLa nuclear extract. NSC1 migrates in sucrose gradients as a U snRNA-free 20S complex containing at least 10 proteins. These include SIP1, dp103/Gemin3, a novel dp103/Gemin3-interacting protein and three additional proteins with apparent masses of 43, 33 and 18 kDa, respectively. Interestingly, NSC1 also contains a specific subset of spliceosomal Sm-proteins. Our data support a model in which nuclear SMN can modulate the Sm protein composition of U snRNPs and hence affect splicing.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A monoclonal antibody directed against the N-terminus of SMN inhibits the first step of pre-mRNA splicing
To obtain a monoclonal antibody against SMN, we injected mice with bacterially expressed human SMN protein. A selected hybridoma cell line, termed 7B10, produced antibody that specifically detected SMN in HeLa whole-cell extract and recombinant SMN on western blots (Fig. 1A). To map the epitope of 7B10, we expressed glutathione S-transferase (GST) fusions of SMN fragments and tested whether these fragments are recognized by 7B10. As shown in Figure 1B, any fragment that harboured the N-terminal 30 amino acids of human SMN was readily detected by the antibody (Fig. 1B, lanes 5–7), whereas a fragment that lacked the first 30 amino acids was not (Fig. 1B, lane 8). Thus, 7B10 specifically recognizes an epitope at the N-terminus of SMN.
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Next, we tested whether 7B10 antibody can bind to SMN in solution. 7B10 or a control antibody against FLAG-peptide was coupled to Protein G–Sepharose and incubated with 35S-labelled SMN, generated by translation in vitro. After extensive washes, the bound SMN protein was eluted, separated on SDS–PAGE and detected by fluorography. As shown in Figure 1C (lanes 2 and 3), antibody 7B10, but not the anti-FLAG control, efficiently immunoprecipitated SMN. Finally, we tested 7B10 for immunoprecipitation of SMN from HeLa cell extracts. To test this, antibodies 7B10 and anti-FLAG were bound to Protein G–Sepharose and incubated with HeLa whole-cell extract. Immunoprecipitates were subsequently separated by SDS–PAGE and tested in western blots for the presence of SMN (Fig. 1D). SMN was detected in 7B10 immunoprecipitates (Fig. 1D, lane 3), but not in the anti-FLAG antibody control (lane 2). Further characterization by immunofluorescence showed that 7B10 stains the cytoplasm and nuclear gems in somatic cells (data not shown). Taken together, 7B10 is a specific monoclonal anti-SMN antibody that recognizes SMN on western blots and in solution.
To gain more insight into the nuclear function of SMN, we tested whether the monoclonal antibody 7B10 inhibits the proposed function of SMN as a recycling factor for splicing (Fig. 2). Increasing amounts of 7B10 antibody were added to a splicing assay and preincubated for 30 min at 30°C before splicing was monitored by the addition of radiolabelled pre-mRNA (Fig. 2A, lanes 1 and 5–7). The preincubation period should allow for the consumption of endogenous pre-mRNA and force the reaction into recycling of spliceosomes (20). Under these conditions, the 7B10 antibody exhibited a strong inhibitory effect on the first step of splicing, as indicated by the lack of products from either chemical step of the splicing reaction (Fig. 2A, lanes 5–7). An affinity-purified control antibody did not affect splicing (Fig. 2A, lanes 2–4). Significantly, a closer inspection of the requirements for inhibition revealed that the 7B10 anti-SMN antibody could block splicing regardless of preincubation temperature (4 or 30°C) (Fig. 2A, lanes 8 and 9). In fact, the inhibitory effects were identical with and without preincubation (Fig. 2A, lanes 10–11).
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To test whether the antibody inhibited spliceosome formation, splicing reactions were subjected to native gel electrophoresis at several time points after the addition of radiolabelled pre-mRNA, either in the absence of antibody, or in the presence of 7B10 or a control antibody (Fig. 2B). In the absence of an inhibitory antibody, spliceosome formation proceeds from early heteronuclear RNA–protein complexes (complex H) to complex A, which harbours U1 and U2 snRNPs and a number of non-snRNP protein factors. Mature spliceosomes, as indicated by integration of the [U4/U6.U5] tri-snRNP, could be detected prior to (complex B) and after the first step of splicing (complex C) (Fig. 2B, lanes 1–6) (24,25). Interestingly, only complexes H and A were detected at significant levels in the presence of the 7B10 antibody, whereas complexes B and C were seen only in the controls (Fig. 2B, compare lanes 1–6 with lanes 7–9). Moreover, complex H, which disappears at later time points (30–60 min) in the controls (Fig. 2B, lanes 2 and 3, and 5 and 6), is still evident in the presence of 7B10 (lanes 8 and 9). Thus, 7B10 blocks spliceosomes from proceeding to stable, mature complexes B and C. This suggests that nuclear SMN engages in the formation of spliceosomes prior to, or in addition to, their reassembly.
Two distinct nuclear SMN complexes
As a first step in characterizing components that interact with nuclear SMN, and hence may be involved in its function in pre-mRNA splicing, we performed a gradient centrifugation of HeLa nuclear extract. The fractions of the gradient were separated by SDS–PAGE and tested for the presence of SMN by western blotting using antibody 7B10. As shown in Figure 3A, SMN was mainly detected in fractions that corresponded to a sedimentation value of 20S. Only minor fractions of SMN (<5%) migrate at the top and bottom of the gradient. This suggested that soluble SMN is predominantly associated with 20S complex(es) in the nucleus.
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To test whether SMN sedimenting at 20S is part of more than one complex, we fractionated HeLa nuclear extract using a CM–Sepharose column. The flow-through and fractions eluted at 75 mM, 300 mM and 1 M NaCl were then separated by SDS–PAGE and blotted with the anti-SMN antibody, 7B10. Surprisingly, only ~20% of nuclear SMN bound to CM–Sepharose and was eluted with 300 mM salt. The remaining SMN pool did not bind to the column matrix even after repeated loading on the column (Fig. 3B and data not shown). This indicates that two biochemically distinct SMN complexes co-sediment in the 20S range in sucrose gradients as shown in Figure 3A. To test this directly, the flow-through fraction and the CM300-eluate were separately analysed on a high resolution sucrose gradient. Indeed, we were able to distinguish two complexes under these conditions. These complexes, termed NSC1 and NSC2, can be distinguished by their slightly different sedimentation at ~20S and 18S, respectively (Fig. 3C). Thus, HeLa nuclear extract contains at least two distinct SMN complexes.
To gain insight into the protein composition of NSC1, HeLa nuclear extract from 109 cells was fractionated on a CM–Sepharose column as described above. CM flow-through fractions were then subjected to immunoaffinity chomatography using 7B10. After extensive washing of the column, the bound proteins were eluted by pH-shock and analysed by SDS–PAGE. Eight proteins could readily be detected in a Coomassie stained gel and designated according to their apparent molecular mass: p105, p99, p43, p38, p34, p33, p15 and p14, respectively (Fig. 4A, lane 4). None of these bands was detected in control eluates from either anti-FLAG or anti-GST columns (Fig. 4A, lanes 2 and 3). Preliminary experiments indicate that some of the proteins found in NSC1, such as p38 and p34, are also associated with NSC2. Otherwise, however, the protein composition differs between both complexes (G. Meister and U. Fischer, unpublished data).
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The NSC1 proteins that eluted from the 7B10 column were analysed by a combination of western blotting with specific antibodies, as well as by mass spectroscopy. To control the specificity of the affinity-purification, we first tested whether SMN and its known interactor SIP1 could be detected in the 7B10 eluate (Fig. 4B). Indeed, proteins p38 and p34 reacted readily with anti-SMN and anti-SIP1 antibody, respectively (Fig. 4B, lanes 1 and 3), whereas none of these proteins was detected in the eluate of the anti-FLAG control (lanes 2 and 4). This shows that the elution from the 7B10 column was highly specific.
p105 encodes a novel interactor of dp103/Gemin3
To identify proteins p105 and p99, bands were excised from the gel, digested with trypsin and the peptide fragments analysed by mass spectroscopy. Using this approach, p105 was identified as the putative DEAD-box helicase Gemin3 previously shown to associate with SMN (data not shown) (14). Tryptic digestion of p99 yielded 14 fragments that matched a partial sequence of a hypothetical protein found in the database (GenBank accession no. Al080150). Since this protein has not previously been identified as a component that interacts with SMN in vivo, it was characterized further. Primers complementary to the known sequence of the 3' part of p99 were used to amplify a partial cDNA from a human fetal brain cDNA library. Subsequently, the correct 5' terminus of the coding sequence was determined by 5' rapid amplification of cDNA ends. The isolated full-length gene codes for a protein with a size of 1058 amino acids (Fig. 5A). Inspection of the sequence of p99 shows no significant homology to any known protein in the database other than a putative leucine zipper (Fig. 5A, underlined sequence).
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The specific elution of p99 from our anti-SMN column, together with known interactors of SMN, strongly implied that it is a constituent of the nuclear SMN complex. We therefore tested whether p99 binds to other proteins of this complex (Fig. 5B). To this end, SMN and dp103/Gemin3 were expressed as fusions with GST. SIP1, which could not be stably expressed as a single GST fusion protein, was co-expressed in a complex with GST–SMN1/160 and tested in binding studies. GST–SMN, GST–dp103/Gemin3, GST–SMN1/160–His–SIP and GST alone were immobilized on glutathione–Sepharose and tested in pull-down assays for interaction with in vitro translated 35S-labelled untagged p99. As shown in Figure 5B, only dp103/Gemin3 bound p99 efficiently (lane 1), whereas SMN, SIP1 and the GST control did not (lanes 2–4). These data indicate that p99 is incorporated in a nuclear SMN complex via the interaction with the SMN-interacting protein dp103/Gemin3. We hence propose to term p99 dp103/Gemin3-interacting protein 1 (GIP1). While this work was in progress, Charroux et al. (26) reported on a novel dp103/Gemin3-interacting protein termed Gemin4 that co-immunoprecipitates with SMN from total cellular extracts (26). Comparison between Gemin4 and GIP1 revealed that both proteins are identical.
A specific subset of Sm proteins in NSC1
A hallmark of SMN is its direct interaction with Sm proteins. It has been shown that this interaction occurs in the cytoplasm and is required for the assembly of the Sm core domain in vivo (13,15,19). It was therefore of interest to test whether Sm proteins are also part of the SMN complexes found in the nucleus. NSC1 proteins p14 and p15 co-migrate with SmD1 and SmD2 from affinity-purified U snRNPs and hence were analysed first. Indeed, p15 and p14 were identified by western blotting or direct sequencing as SmD2 and SmD1, respectively (Fig. 6B, lane 1, and data not shown). Since the small Sm proteins SmE, SmF and SmG are often difficult to visualize by Coomassie staining, we separated purified NSC1 and a U snRNP protein marker on SDS–PAGE and silver-stained the eluted proteins. Figure 6A shows a magnification of the 5–35 kDa region of the gel in which these Sm-proteins migrate. This procedure revealed that not only SmD1 and SmD2, but also SmF, form part of NSC1. An additional 18 kDa protein which does not co-migrate with any of the U snRNP protein markers could also be detected under these conditions (Fig. 6A, band indicated with a star in lane 3). However, none of the other Sm proteins (SmB, SmD3, SmE or SmG) were detected in this preparation either by silver staining (Fig. 6A) or by western blotting (Fig. 6B and data not shown). Thus, NSC1 contains a specific subset of spliceosomal Sm proteins.
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The presence of Sm proteins in NSC1 led us to test whether U snRNAs are also present in the nuclear SMN complex studied here. To this end, nuclear extract and eluates from the 7B10 and anti-FLAG columns were radiolabelled with [32P]pCp using T4 RNA ligase. As shown in Figure 6C, no RNA that could be labelled by [32P]pCp was specifically eluted from either column (Fig. 6C, lanes 2 and 4) whereas U snRNAs and tRNAs were readily detected in the nuclear extract used for the affinity purification (Fig. 6C, lanes 1 and 3). We conclude that NSC1 is not stably associated with U snRNAs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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It has recently been shown that SMN is involved in nuclear pre-mRNA splicing, most likely in recycling of spliceosomal factors after splicing (20). Assuming that this function is mediated, at least in part, by SMN found in the nucleus, we have taken a biochemical approach to identify nuclear components that associate with, and hence may be functionally connected to, SMN.
In accordance with earlier reports (20,27), we provide evidence that SMN is an essential pre-mRNA splicing factor. However, inhibition of splicing by 7B10 does not depend on pre-incubation with nuclear extract. This is somewhat surprising in the light of recent data from Pellizzoni et al. (20) who, with the use of a different SMN-specific monoclonal antibody or a trans-dominant SMN mutant (SMN
27), showed a strict requirement for preincubation. A likely interpretation of these discrepancies is that the 7B10 antibody tested in our study and the 2B1 antibody tested by others (20) do not recognize the same epitope, and thus exhibit different effects. This indicates that the function of nuclear SMN may not be restricted to its proposed role in the recycling of spliceosomes.
We provide evidence that nuclear SMN is part of two distinct complexes. Although the biochemical composition of NSC2 remains to be elucidated, we were able to purify NSC1 to homogeneity and identify 10 associated proteins including GIP1. Recently, Charroux et al. (26) reported on the identification of SMN-interacting proteins by anti-SMN immunoprecipitation. Among the proteins identified were dp103/Gemin3, SIP1, Sm proteins and a novel protein termed Gemin4. Comparison of the Gemin4 sequence revealed that it is identical to GIP1 identified in this study. Otherwise, however, the protein composition differs. These differences may be explained by the fact that a whole-cell extract was used in the study by Charroux et al. (26) which contains several different SMN complexes, including those studied here.
Although purified by anti-SMN affinity chromatography, Gemin4/GIP1 does not directly bind to SMN in vitro, but can interact with dp103/Gemin3. Since it has previously been shown that dp103/Gemin3 interacts with SMN (14), it is likely that Gemin4/GIP1 is incorporated into an SMN complex via the interaction with dp103/Gemin3. Very similar results have been obtained independently by Charroux et al. (26). Importantly, the putative helicase dp103/Gemin3 does not exhibit either RNA-dependent ATPase activity or helicase activity when recombinant protein was analysed (14,23). However, immunoprecipitates of dp103/Gemin3 were shown to contain ATPase activity. Hence it was suggested that the putative enzymatic activity of dp103/Gemin3 may be modulated by cofactors (23). Our data suggest that Gemin4/GIP1 might be such a cofactor that influences the helicase and/or ATPase activity of dp103/Gemin3 and thereby confer enzymatic properties to the SMN complex.
The data presented here suggest a model of how a core structure of an SMN complex could be organized (Fig. 7). In this model, an SMN dimer/oligomer interacts with at least three components: SIP1, Sm proteins and dp103/Gemin3. These components interact with SMN via the N-terminus (15,19), the central (tudor) domain (19) and the C-terminus (14), respectively. Gemin4/GIP1 appears to be incorporated in this complex via the interaction with dp103/Gemin3. Recent data also suggested that Sm proteins bind to dp103/Gemin3 and/or Gemin4/GIP1. Hence, it cannot be excluded that Sm proteins are incorporated via dp103/Gemin3 into the nuclear SMN complex.
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Although it is known that SMN interacts with Sm proteins, it has been thought that this interaction occurs exclusively in the cytoplasm (13,15,19). This assumption was based on the previous finding that SMN mediates cytosolic assembly of Sm proteins onto the Sm site of U snRNA (15,19). Surprisingly, however, we report here that stochiometric amounts of a subset of the Sm proteins specifically associate with a nuclear SMN complex. Earlier findings suggested that Sm proteins are stored in the cytoplasm and enter the nucleus only after they have assembled onto U snRNAs (18,28). The presence in the nucleus of specific Sm proteins that are not bound to U snRNA was therefore unexpected. Currently, we can only speculate as to what the functional consequences of the SMN–Sm interaction might be. One possible scenario is that SMN dissociates some Sm proteins from assembled U snRNPs in the nucleus. This would imply that SMN can alter the biochemical composition of spliceosomal U snRNPs and may therefore directly regulate splicing. Hence, this hypothesis is consistent with the proposed role of nuclear SMN in recycling of splicing factors (20,27). Alternatively, the nuclear SMN complex may recycle U snRNPs that are no longer needed in the nucleus, i.e. those which are defective or functionally inactive. In this scenario, SMN would promote disassembly and hence act in an opposite manner as has been described for cytosolic SMN. Compartment-specific factors may regulate these opposed functions. A third possibility is that SMN can transport Sm proteins that are not assembled onto U snRNAs from the cytoplasm to the nucleus. This would imply that assembly of U snRNPs in somatic cells is not restricted to the cytoplasmic compartment as has been shown for the X.laevis oocyte system (15,18). Given the proposed role of SMN in transcription (21,22) we cannot exclude the possibility that the nuclear SMN complexes described in this work also have a function in gene regulation. Clearly, a detailed knowledge of all components that interact with SMN in the nucleus will be required to analyse the cellular process that is facilitated by SMN. To this end, the preparative purification and molecular characterization of NSC1 and NSC2 as described in this study will be a prerequisite for further functional studies both in vivo and in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmid constructs
cDNAs for full-length SMN, SIP1, dp103/Gemin3 and Gemin4/GIP1 were amplified by polymerase chain reaction (PCR) using primers that introduced EcoRI and XhoI sites to the 5' and 3' ends, respectively, and cloned into pET21a and pET28a (Novagen, Madison, WI). For the production of GST fusions, fragments corresponding to the different regions of SMN, full-length dp103/Gemin3 and Gemin4/GIP1 were obtained by PCR and subcloned into pGEX 5X-1 (Amersham-Pharmacia, Freiburg, Germany).
In vitro translation and production of recombinant proteins
For production of 35S-labelled proteins, plasmids containing SMN or Gemin4/GIP1 were transcribed and translated in vitro using a TNT T7/T3-coupled reticulocyte lysate systems kit (Promega, Madison, WI). For recombinant protein expression and purification expression, plasmid pGEX5X-1 containing SMN and deletion fragments thereof, or dp103/Gemin3 were transformed into the Escherichia coli strain BL21(DE3). For co-expression of GST–SMN1/160 with His-tagged SIP1, plasmid pGEX5X-1:SMN1/160 was cotransformed with pET28a:SIP1 and simultaneously selected by kanamycine and ampicillin. After induction with 1 mM isopropyl-ß-D-thiogalactoside for 4 h at 22°C, cells were pelleted, resuspended in lysis buffer (500 mM NaCl, 50 mM Tris–HCl pH 7.4, 5 mM MgCl2) and sonicated. Lysates were centrifuged at 20 000 g for 1 h and purified over glutathione–Sepharose 4B beads (for GST-tagged proteins; Amersham-Pharmacia) or Ni-NTA-agarose (for His-tagged proteins; Qiagen, Hilden, Germany). Bound proteins were eluted with lysis buffer containing 10 mM glutathione or 150 mM imidazole, respectively, and dialysed against binding buffer (300 mM NaCl, 50 mM Tris–HCl pH 7.4, 5 mM MgCl2).
Monoclonal antibodies
Monoclonal antibodies were obtained by repeated immunization of BALB/c mice with bacterially expressed His-tagged human SMN dissolved in 50% Freund’s adjuvant. Spleen cells were fused with PAIB3Ag81 myeloma cells, and clones that produced anti-SMN antibodies were selected by western blotting against recombinant SMN protein. One clone (termed 7B10) recognized full-length GST–SMN in western blots and was characterized as described in the Results section. To affinity-purify 7B10 hybridoma supernatants were passed over a column containing a GST–SMN fragment comprising the first 30 amino acids coupled to NHS-activated Sepharose (Amersham-Pharmacia). After washing with phosphate-buffered saline (PBS) (pH 7.4) and 10 mM Tris–HCl (pH 8.0), bound antibodies were eluted with 100 mM glycin pH 2.3 and dialysed against PBS pH 7.4.
Western blot analysis
Approximately 0.1 µg of GST–SMN fragments or 10 µg of HeLa cell lysates were separated on a 12% SDS–polyacrylamide gel and transferred to a Hybond C nitrocellulose filter (Amersham-Pharmacia) using a standard semi-dry blot apparatus. HeLa whole-cell lysates were obtained by boiling of cells in SDS–PAGE sample buffer and brief sonification. Filters were incubated in blocking solution [Tris-buffered saline (TBS), 5% non-fat milk] for 1 h at room temperature and then incubated with primary antibodies for 1 h at room temperature. After extensive washing with TBS containing 0.1% Tween 20, filters were incubated with horseradish peroxidase-coupled anti-mouse or anti-rabbit IgG (Sigma, St Louis, MO) for 1 h at room temperature and subsequently washed three times in TBS containing 0.1% Tween 20. Protein bands were visualized with an ECL detection kit (Amersham-Pharmacia).
HeLa cell extracts
HeLa cell extracts were prepared according to a protocol by Dignam et al. (29). Briefly, 5 x 109 cells from HeLa suspension cultures were pelleted and washed in PBS (pH 7.4). Cells were again pelleted, resuspended in two pellet volumes of 10 mM KCl, 10 mM HEPES–KOH pH 7.9, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM PMSF and homogenized by douncing. Subsequently, cytoplasm and nuclei were separated by centrifugation of the suspension in a swingout rotor (1000 g, 10 min, 4°C). Nuclei were resuspended in 420 mM KCl, 20 mM HEPES–KOH pH 7.9, 1.5 mM MgCl2, 0.5 mM PMSF, 0.2 mM EDTA, 5% glycerol (3 ml/109 cells), homogenized by douncing and stirred for 30 min on ice. Nuclear extracts were then centrifuged (30 000 g, 30 min, 4°C) and the supernatant frozen in liquid nitrogen. For further fractionation, the nuclear extract was passed over a CM–Sepharose Fast Flow column (Amersham-Pharmacia) and eluted with wash buffer (50 mM Tris–HCl pH 7.4, 5 mM MgCl2) containing 75 mM, 300 mM or 1 M NaCl. Flow-through and CM300 fractions were then used for further characterization of NSC1 and NSC2. For preparation of whole-cell extracts, HeLa cells from suspension cultures were harvested and lysed in PBS (pH 7.4) containing 0.2% Triton X-100. After centrifugation (15 000 g, 10 min, 4°C), supernatants were used for immunoprecipitation experiments.
Immunoprecipitation and immunoaffinity purification
For antibody characterization, 1 µg of affinity-purified monoclonal antibody 7B10 was coupled to 20 µl of Protein G–Sepharose (Amersham-Pharmacia), washed with binding buffer (300 mM NaCl, 50 mM Tris–HCl pH 7.4, 5 mM MgCl2) and incubated with either HeLa whole-cell extract or in vitro translated [35S]SMN. After extensive washing with binding buffer bound proteins were eluted with SDS–PAGE sample buffer, separated by SDS–PAGE and visualized by either western blotting or fluorography. For identification of SMN-interacting proteins, 50 µg of affinity-purified monoclonal antibody 7B10 was coupled to 250 µl of Protein G–Sepharose and incubated with CM300 eluates or CM flow-through fractions for 3 h at 4°C. After extensive washing with binding buffer, bound proteins were eluted with 1 ml of 100 mM glycin (pH 2.3) and precipitated with trichloroacetic acid. Protein pellets were resuspended with SDS–PAGE sample buffer and analysed by SDS–PAGE.
Protein–protein interaction assays
Approximately 50 µl of glutathione–Sepharose was incubated for 1 h at 4°C with 1 µg of purified GST fusion protein. Resin was then washed three times in binding buffer. For protein binding, 3 µl of 35S-labelled protein was incubated with the appropriate resin for 1 h at 4°C, and resin was subsequently washed extensively with binding buffer. Bound proteins were eluted with SDS sample buffer separated by SDS–PAGE and visualized either by staining with Coomassie or by autoradiography using Amplify (Amersham-Pharmacia).
3'-end-labelling of RNA
For 3'-end-labelling of RNA, nuclear extract or eluates from columns (elution conditions: 1.5 M MgCl2) were phenol-extracted and ethanol-precipitated. The RNA was dissolved in water and assayed in a buffer containing 50 mM HEPES–KOH pH 7.9, 18 mM MgCl2, 3 mM dithioerythrol, 1 µg/ml bovine serum albumin, 1 U/µl T4 RNA ligase (Gibco BRL, Karlsruhe, Germany) and 60 µCi [32P]pCp (3000 Ci/mmol) (Amersham-Pharmacia). The assay mixture was then incubated at 4°C for 5 h and RNA was subsequently precipitated with ethanol. The RNA was dissolved in RNA-loading dye and separated on denaturing 5% polyacrylamide gels containing 7.5 M urea. Bands were visualized by autoradiography.
In vitro splicing assays
HeLa nuclear extracts were prepared as described by Dignam et al. (29). 32P-labelled pre-mRNA substrate was obtained by run-off transcription from a MINX minimal intron construct as described by Zillmann et al. (30) in the presence of m7G(5')ppp(5')G cap analogue and [32P]UTP (Amersham-Pharmacia). The sequence of MINX is based on the Adenovirus 2 major late transcription unit and contains duplicates of exon 2 (59 and 41 nucleotides) interspersed by a truncated intron (100 nucleotides). In vitro splicing reactions were essentially set up as described by Ségault et al. (31). Reactions contained 30% (v/v) nuclear extract, 2 x 104 c.p.m. of 32P-labelled pre-mRNA, 3 mM MgCl2, 40 mM KCl, 2 mM ATP and 10 mM creatine phosphate in a total volume of 12.5 µl. Pre-incubation was carried out for 30 min at 4 or 30°C, respectively, with affinity-purified monoclonal antibodies directed against SMN (clone 7B10) or, as a negative control, anti-Rac1 monoclonal antibody (kindly provided by J. Faix, Madison, WI). After pre-incubation, the reaction was started by the addition of radiolabelled pre-mRNA and incubated for 1 h at 30°C. Reactions were stopped by 200 µl of PK buffer [0.1 M Tris–HCl pH 7.4, 12.5 mM EDTA, 150 mM NaCl, 1% (w/v) SDS]. RNA was extracted with 1 vol of phenol–chloroform–isoamylalcohol (25:24:1), precipitated from the aqueous phase with 2.5 vol of ethanol and analysed on 14% polyacrylamide/ 8 M urea gels.
For the analysis of spliceosomal complexes, 2 µl 86% (w/v) glycerol and 2 µl heparin (5 mg/ml) were added to splicing reactions following incubation and loaded on native composite gels containing 0.5% (w/v) agarose, 3.5% polyacrylamide [acrylamide/N,N'-methylene bisacrylamide, 80:1 (w/w)], 10% (w/v) glycerol and 0.3x TBE (32). Gels (29 cm x 19 cm x 1 mm) were run in 0.3x TBE at 25 mA for 5 h and complexes were visualized by autoradiography.
Sedimentation experiments
Nuclear extract (500 µl, ~1–2 mg total protein) was layered on a 15–45% sucrose (w/v) gradient containing 150 mM NaCl, 50 mM Tris–HCl pH 7.4 and 5 mM MgCl2. After centrifugation at 40 000 r.p.m. for 21 h in a Beckman SW41 rotor, 0.5 ml fractions were collected and proteins precipitated. Proteins were separated by SDS–PAGE and analysed by western blotting using the 7B10 antibody. Svedberg values were determined by a gradient to which the marker proteins cytochrome C (2S), immunoglobulin (7S), catalase (11S) and ß-galactosidase (19S) had been applied. For high resolution gradients, we used 10–35% sucrose gradients under the same conditions as described above.
Protein identification by mass spectroscopy
Protein bands were excised and cleaved directly in gel with endoproteinase LysC according to Eckerskorn and Lottspeich (33). Peptide mass fingerprinting was performed using a Bruker reflex III MALDI–TOF mass spectrometer (Bruker-Franzen, Bremen, Germany) equipped with a 337 nm nitrogen laser. One microlitre of the eluted peptide mixture was applied onto the sample target. After drying at room temperature, 0.7 µl of matrix solution (5 mg of -cyano-4-hydroxy-cinnamic acid dissolved in 1 ml of acetonitrile/water/TFA (50:50:0.1 v/v/v) was overlaid and dried again at room temperature. Mass analyses were performed using positive reflector mode with delayed extraction, accelerating voltage 20 kV and reflector voltage 22.8 kV with a deflection cut-off mass of ~400. Typically, 100–150 shots were accumulated. The peptide masses found were used in a database search using the program MSFIT (http://prospector.ucsf.edu ).
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ACKNOWLEDGEMENTS |
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We thank R. Lührmann, C. Will and M. Sendtner for helpful suggestions and reagents. This work is part of the PhD thesis programmes of G.M. and D.B. D.B. is funded by Boehringer Ingelheim Fonds. B.L. is the recipient of a Liebig-Fellowship from the Verband der Chemischen Industrie. U.F. received a grant from the German Science Foundation (Hess-Programm) and support from the Max-Planck Society.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +49 89 8578 2475; Fax: +49 89 8578 3810; Email: ufischer@biochem.mpg.de
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A. K. Gubitz, Z. Mourelatos, L. Abel, J. Rappsilber, M. Mann, and G. Dreyfuss Gemin5, a Novel WD Repeat Protein Component of the SMN Complex That Binds Sm Proteins J. Biol. Chem., February 8, 2002; 277(7): 5631 - 5636. [Abstract] [Full Text] [PDF]
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L. Pellizzoni, J. Baccon, J. Rappsilber, M. Mann, and G. Dreyfuss Purification of Native Survival of Motor Neurons Complexes and Identification of Gemin6 as a Novel Component J. Biol. Chem., February 22, 2002; 277(9): 7540 - 7545. [Abstract] [Full Text] [PDF]
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