| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy
Introduction
Results
Sm proteins directly interact with the tudor domain of SMN
SMN harbouring an SMA-causing point mutation within the tudor domain fails to interact with Sm proteins
Antibodies directed against the tudor domain interfere with U snRNP assembly in vivo
Discussion
Materials And Methods
Plasmid constructs
Generation of antiserum and affinity-purification of specific antibodies
Recombinant proteins
Western blot analysis
Preparation of RNA-free, native snRNP TPs
Binding assays
Microinjection in X.laevis oocytes
Acknowledgements
References
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Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy
Received August 17, 1999; Revised and Accepted October 4, 1999
Spinal muscular atrophy (SMA) is a neurodegenerative disease of spinal motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. SMN is part of a macromolecular complex that contains the SMN-interacting protein 1 (SIP1) and spliceosomal Sm proteins. Although it is clear that SIP1 as a component of this complex is essential for spliceosomal uridine-rich small ribonucleoprotein (U snRNP) assembly, the role of SMN and its functional interactions with SIP1 and Sm proteins are poorly understood. Here we show that the central region of SMN comprising a tudor domain facilitates direct binding to Sm proteins. Strikingly, the SMA-causing missense mutation E134K within the tudor domain severely reduced the ability of SMN to interact with Sm proteins. Moreover, antibodies directed against the tudor domain prevent Sm protein binding to SMN and abolish assembly of U snRNPs in vivo. Thus, our data show that SMN is an essential U snRNP assembly factor and establish a direct correlation between defects in the biogenesis of U snRNPs and SMA.
INTRODUCTION
Spinal muscular atrophy (SMA) is a common, often fatal autosomal recessive disorder characterized by degeneration of anterior horn cells of the spinal cord (1). This typically leads to progressive paralysis of the trunk and limb associated with muscular atrophy. SMA is caused by mutations in the survival of motor neurons (SMN) gene (2). Two copies of the SMN gene, termed SMN1 and SMN2, are located in a 500 kb inverted repeat at chromosome 5p13. SMN1 and SMN2 differ in that full length protein is predominantly produced from SMN1. The primary transcript of SMN2, in contrast, undergoes alternative splicing of exon 7 which leads to the predominant expression of C-terminally truncated SMN. In >95% of all SMA patients SMN1 is deleted or mutated whereas the expression of SMN2 is unaffected. This leads to a reduced level of functional full length SMN protein in the cell and consequently to muscular atrophy (3,4).
SMN localizes both to the cytoplasm and the nucleus where it is highly concentrated in structures termed gems (gemini of coiled bodies) (5). The nuclear pool of SMN has been shown to be involved in splicing (6). However, the majority of SMN protein is cytoplasmic, where it is part of a large protein complex that contains the SMN-interacting protein 1 (SIP1) and Sm proteins of spliceosomal uridine-rich small ribonucleoproteins (U snRNPs) (7). Previous experiments have implicated SIP1 to be involved in the cytoplasmic assembly of Sm core U snRNPs (8). In this process, U snRNAs are bound in a step-wise and ordered manner by the Sm hetero-oligomeric complexes of B/B[prime].D3, D1.D2 and E.F.G (9,10). Formation of the Sm core is a prerequisite for several further processing steps of the U snRNPs, including their 5[prime] cap hypermethylation and import into the nucleus, where pre-mRNA splicing takes place (11-14). The involvement of SIP1 in U snRNP assembly has previously been inferred from injection studies in Xenopus laevis oocytes (8). In these studies anti-SIP1 antibodies were shown to strongly interfere with the formation of the Sm core in vivo (8). The role of SMN in this process, however, is less well understood. Anti-SMN antibodies injected into oocytes did not inhibit, but rather slightly stimulated, the assembly process (8). In another study, overexpression of SMN mutant in somatic cells caused Sm protein aggregation in the nucleus and the cytoplasm (6). Although these studies have provided a first link between SMN and U snRNP assembly, they could not provide evidence for a direct and active involvement of SMN in this process.
SMN appears to consist of several functionally important regions (Fig. 1C): the N-terminal part (amino acids 1-91) has been implicated in SIP1 binding (7) and association with RNA (15), whereas the C-terminus (amino acids 242-278) was shown to be important for SMN oligomerization (16) and Sm protein binding (7). In addition, the C-terminus has recently been shown to interact with the nuclear transcription activator E2 of papillomavirus (17). The central region of SMN (amino acids 90-160) constitutes a so-called tudor domain (18,19). Tudor domains, originally described for the Drosophila tudor protein, are evolutionarily conserved sequence motifs of unknown function found in many eukaryotic proteins (18).
Figure 1. Regions of SMN responsible for binding to Sm proteins. (A) Binding of in vitro translated 35S-labeled SmB to zz-tagged SMN mutants immobilized on IgG-Sepharose. Bound proteins were detected by autoradiography. Input lanes represent 20% of translated protein added to assays. (B) Direct binding of native HeLa Sm proteins to the tudor domain of SMN. Sm and U1- and U2-specific proteins (TPs) were incubated with GST-SMN fragments (lanes 1 and 2) or GST alone (lane 3) immobilized on GST-Sepharose beads. After GST pull-down, bound proteins were analyzed by electrophoresis and silver-staining. Input lanes represent 100% of protein added to assays. (C) Schematic representation of binding domains within SMN. An amino acid substitution found in one SMA patient is indicated.
We have performed a detailed study of the interactions between SMN and Sm proteins, to determine whether these interactions are important for the formation of the Sm core domain of U snRNPs in vivo. We present evidence that the tudor domain of SMN is necessary and sufficient for binding of Sm proteins. Moreover, a point mutation within the tudor domain found in one patient with SMA strongly reduces the affinity of SMN for Sm proteins. Finally we show that antibodies directed against the tudor domain of SMN interfere with Sm protein binding and strongly inhibit U snRNP assembly in vivo. Our results provide direct evidence for a function of SMN as an essential assembly factor for U snRNPs and suggest that defective U snRNP assembly might significantly contribute to the pathogenesis of SMA.
RESULTS
Sm proteins directly interact with the tudor domain of SMN
We initially chose to investigate the mode of interaction between SMN and Sm proteins in more detail based on the assumption that this interaction might be crucial for the formation of the Sm core domain of U snRNPs. For this, bacterially expressed SMN fragments fused to two IgG-binding domains of protein A (zz-tag) were immobilized on IgG-Sepharose and tested in pull-down assays for interaction with in vitro translated 35S-labeled SmB protein. SmB bound strongly to full-length human SMN and also to the SMN construct encompassing the N-terminal 160 amino acids (Fig. 1A, lanes 1 and 7). However, no binding to SmB was observed with the C-terminal fragment SMN159-294 (Fig. 1A, lane 12) or to SMN249-294 (data not shown), and only weak binding was observed with fragments of the first 105 amino acids of SMN (lanes 3, 4, 5 and 9). In contrast, all SMN constructs containing the tudor domain strongly bound to SmB (Fig. 1A, lanes 2, 7 and 8). Indeed, the smallest fragment necessary and sufficient for SmB binding is the C-terminal half of the tudor domain (amino acids 120-160; Fig. 1A, lane 13), whereas the N-terminal part showed only weak binding to SmB (lane 6). Similar results were also obtained for SmD1, SmD2, SmD3 and SmE which were shown previously to interact with SMN (data not shown). Thus, the tudor domain is crucial for Sm protein binding by SMN. To exclude the possibility that the observed SMN-Sm protein interactions are mediated by proteins in the reticulocyte lysate used in the binding assay, we tested whether recombinant glutathione (GST)-tagged SMN can directly bind to native HeLa Sm proteins (Fig. 1B). HeLa Sm proteins were obtained from highly purified U snRNPs in a total protein (TP) mixture that also contained a subset of the U1 and U2 snRNP-specific proteins (20). Since GST-fusion proteins containing solely the tudor domain could not be effectively expressed, we tested binding of Sm proteins to GST-SMN constructs containing amino acids 36-160 or 30-105, or, as a control, to the GST protein alone. As shown in Fig. 1B, SMN36-160 bound strongly to Sm proteins D1 and D2, moderately to SmB/B[prime] and D3, and weakly to SmE, -F and -G (lane 1). In contrast, Sm proteins did not bind to either SMN30-105 or GST protein (lanes 2 and 3), and none of the snRNP-specific proteins bound to any of the GST constructs. This strongly indicates that all Sm proteins specifically and directly interact with SMN via its tudor domain. We repeatedly observed a specificity in the binding of recombinant SMN to in vitro translated Sm proteins B/B[prime], D1, D2, D3 and E, while SmF and -G were bound to a much lesser extent (7). However, when TP fractions were used, SMN bound to all Sm proteins including F and G albeit with different affinities (Fig. 1B). This may indicate that Sm hetero-oligomers such as B/B[prime].D3, D1.D2 and E.F.G, which have been shown to form efficiently in TP fractions (10), bind to SMN. Previous peptide competition studies have implicated the C-terminus of SMN in Sm protein binding (7); the discrepancy with our results cannot yet be explained (see also Discussion).
We further investigated which residues within the tudor domain are important for interactions between SMN and Sm proteins. The sequence GYGNREE (position 129-135) within the tudor domain is highly conserved and thus may be functionally important. We therefore substituted this region to VFVDRQQ (Mu1) and tested whether this substitution would affect Sm protein binding (Fig. 2A). In vitro translated 35S-labeled B and D1 proteins interacted with wild-type GST-SMN (Fig. 2A, lane 1). However, binding of both Sm proteins to GST-SMN with substitutions at positions 1298-135 (Mu1) was reduced at least 30-fold (Fig. 2A, lane 2; see Fig. 2C for quantitation). Thus, amino acids in proximity to position 130 are crucial for binding of SMN to Sm proteins.
Figure 2. Amino acid substitutions within the tudor domain of SMN strongly interfere with Sm protein binding. (A) Interactions of SMN wild-type and mutant proteins with SmB and -D1. In vitro translated 35S-labeled SmB (upper panel) or SmD1 (lower panel) proteins were tested for binding with immobilized GST-SMN containing the wild-type sequence (lane 1), amino acid substitutions at positions 129-135 (Mu1; lane 2) or 134 [E(134)K; lane 3] or, as a control, with GST alone (lane 4). Following GST pull-down, bound proteins were detected by autoradiography. The input lane represents 50% of translated protein added to assays. (B) Direct binding of native HeLa Sm proteins to SMN1/160 is severely reduced by the SMA-linked point mutation E134K. TPs were tested for binding to immobilized GST-SMN1-160 (lane 1) or GST-SMN1-160/E(134)K (lane 2), and bound proteins were visualized by silver-staining. Input lanes represent 100% of protein added to assays. U1 and U2 snRNP-specific proteins are indicated by asterisks. (C) Quantitation of the Sm protein binding experiment shown in (A).
SMN harbouring an SMA-causing point mutation within the tudor domain fails to interact with Sm proteins
An SMA patient has recently been identified who expresses a mutant SMN protein harbouring a glutamic acid to lysine substitution at position 134 (E134K) (21). This mutation does not lead to an aberrant subcellular localization of SMN (22, our unpublished data). Interestingly, however, this amino acid substitution is located in the SMN tudor domain, which we demonstrate to be crucial for association with Sm proteins. We therefore tested whether the E134K missense mutation affects Sm protein binding. Indeed, GST-SMN containing the E134K mutation did associate only marginally with the in vitro translated Sm proteins B and D1 (Fig. 2A, lane 3, and C). Mutations Mu1 and E134K may affect Sm protein binding by changing the overall structure of SMN rather than specifically altering the Sm binding site. However, this is unlikely since neither SMN association with SIP1 (Fig. 2A, lanes 2 and 3), nor SMN oligomerization (data not shown), was affected by either tudor domain mutation. Importantly, the same effect could be observed when direct binding of HeLa Sm protein binding was tested: the affinity of GST-SMN1-160 for the native HeLa Sm proteins was reduced at least 5-fold when the SMN fragment contained the E134K point mutation (Fig. 2B, lanes 1 and 2). Thus, an SMA-causing mutation within the tudor domain strongly interferes with the direct interaction of SMN with Sm proteins.
Antibodies directed against the tudor domain interfere with U snRNP assembly in vivo
It was next investigated whether interaction of Sm proteins with the tudor domain of SMN is essential for assembly of U snRNPs in vivo. To address this question anti-tudor domain (anti-Tu) antibodies were affinity-purified from a rabbit polyclonal antiserum raised against full-length SMN (see Materials and Methods). Anti-Tu readily detected in western blots GST-fusions containing full-length SMN (Fig. 3A, lane 1) as well as SMN deletion fragments containing all or part of amino acids 90-160 (i.e. GST-SMN1/120, GST-SMN1/160 and GST-SMN36/160; lanes 5, 6 and 7, respectively) but failed to detect SMN fragments comprising the first 90 amino acids or the C-terminus of SMN (i.e. GST-SMN1/60, GST-SMN1/90 and GST-SMN159/294; lanes 3, 4 and 2, respectively). We further tested whether anti-Tu antibody specifically recognizes SMN in X.laevis oocytes as this is a prerequisite for the injection studies described below. As shown in Figure 3B, anti-Tu antibody detected SMN in whole cell extracts (lane 1) and cytoplasmic extracts (lane 2) of X.laevis oocytes but failed to detect SMN in the nuclear fraction (lane 3). Similar results were previously reported using a monoclonal anti-SMN antibody (8). Anti-Tu antibody also detected SMN in affinity-purified U snRNPs but did not cross-react with other U snRNPs (Fig. 3B, lane 4). Thus, anti-Tu antibody exclusively recognizes the tudor domain of SMN but no other proteins of oocyte extracts or U snRNPs. We then tested whether anti-Tu antibody affects the interaction between SMN and Sm proteins (Fig. 3C). Indeed, a 5-fold molar excess of anti-Tu antibody over recombinant GST-SMN protein blocked the interaction of in vitro translated SmB protein to GST-SMN (Fig. 3C, lane 2). This effect was highly specific since the same antibody did not interfere with other SMN-specific interactions, such as oligomerization (Fig. 3C, lane 2) or binding to SIP1 (lane 4). SmB, SMN and SIP1 interacted efficiently with SMN in control experiments (Fig. 3C, lanes 1 and 3).
Figure 3. (A) Affinity-purified anti-SMN antibody (anti-Tu) specifically recognizes the tudor domain. Immunoblot of GST-SMN deletion constructs probed with affinity-purified anti-Tu antibody. GST-fusion protein (0.1 µg) was loaded per lane. (B) Anti-Tu antibody specifically recognizes SMN protein in the cytoplasm of X.laevis oocytes and in purified HeLa U snRNPs. Protein from total oocytes (lane 1) or from oocytes dissected into cytoplasm (lane 2) and nuclei (lane 3) and from purified U snRNPs (lane 4) were fractionated by SDS-PAGE and analyzed by western blotting with anti-Tu antibody. (C) Anti-Tu antibody blocks the interaction between SMN and SmB. Immobilized GST-SMN was incubated with buffer (lanes 1 and 3) or the anti-Tu1 antibody (lanes 2 and 4). Subsequently, 35S-labeled SmB and SMN (lanes 1 and 2) and SIP1 (lanes 3 and 4) were tested for binding to GST-SMN.
The anti-Tu antibody was then tested to determine whether it could specifically interfere with the assembly of U snRNPs in vivo (Fig. 4A). For this we used the X.laevis oocyte system which allows the analysis of U snRNP assembly following microinjection of in vitro transcribed, 32P-labeled U snRNAs (8,23,24). Oocytes received an initial cytoplasmic injection of anti-Tu antibody, a non-immune serum (NIS), or, as a mock control, water. The same oocytes were injected a second time in the cytoplasm with a mixture of three in vitro transcribed and 32P-labeled RNAs, U1 and U5 snRNAs, which are known to assemble on cytoplasmic injection with Sm proteins, and dihydrofolate reductase (DHFR) mRNA, which does not bind to Sm proteins and serves as a control for the specificity of assembly. Incubation was then continued for an additional hour. Thereafter, the oocytes were homogenized and assembly of Sm proteins onto the RNA was analyzed by immunoprecipitation with the anti-Sm monoclonal antibody Y12 (6,23-25). As expected, U1 and U5 snRNAs were immunoprecipitated in the mock control and in oocytes injected with NIS antibody, indicating that the Sm core domain had formed on these RNAs (Fig. 4A, lanes 5 and 6). Sm core assembly was specific for U snRNAs since co-injected DHFR mRNA was not co-immunoprecipitated by the Y12-antibody (Fig. 4A, lanes 5 and 6). Strikingly, injection of either of two different preparations of anti-Tu antibodies almost completely blocked the Sm core assembly onto U1 and U5 snRNA (Fig. 4A, lanes 7 and 8, and B). Thus, an antibody that recognizes the tudor domain of SMN and interferes with Sm protein binding efficiently inhibits the formation of Sm core U snRNP particles in vivo.
Figure 4. Antibodies directed against the tudor domain of SMN affect U snRNP assembly in vivo. (A) Antibodies directed against the tudor domain (anti-Tu1 and anti-Tu2) interfere with assembly of U1 and U5 snRNP in vivo. Oocytes were injected with 32P-labeled U1 and U5 snRNA and DHFR mRNA subsequent to a pre-injection with water (lane 5), non-immune serum (NIS; lane 6), or one of two anti-Tu antibodies (lanes 7 and 8). RNA was co-precipitated from homogenized oocytes with the anti-Sm monoclonal antibody Y12. Lanes 5-8, precipitates; lanes 1-4, corresponding supernatants; lane 9, marker RNAs. (B) Quantitation of U snRNA precipitation by monoclonal antibody Y12 shown in (A).
DISCUSSION
Whereas previous studies have established a role for SIP1 in the assembly of the Sm core domain of U snRNPs, the role of SMN in this process remained elusive (7,8). Here we show that SMN likewise functions as an essential assembly factor in vivo, and provide evidence that the interactions between SMN and Sm proteins are critical for this function.
To gain insight into the mode of action of SMN in U snRNP assembly we identified the sequence in SMN that mediates direct binding to Sm proteins. Several lines of evidence presented in this study support the conclusion that the tudor domain of SMN is necessary and sufficient for Sm protein binding. First, when truncated SMN constructs with extensive deletions at either the N- or C-terminus were tested for binding to SmB protein, only those constructs harbouring the tudor domain maintained binding activity (Fig. 1A). Second, Sm proteins which had been isolated from highly purified U snRNPs bound directly to SMN fragments containing the tudor domain (Fig. 1B). Third, a point mutation in the tudor domain renders the full-length SMN protein incapable to interact with SmB and SmD1, whereas SMN dimerization and interaction with SIP1 were not affected (Fig. 2A). Fourth, antibodies that specifically recognize the tudor domain of SMN strongly reduced SmB binding to SMN but did not interfere with other biochemical activities of SMN, such as oligomerization or SIP1 binding (Fig. 3C). Together, these data strongly suggest that the interaction between SMN and Sm proteins is mediated by the tudor domain of SMN. These results are in contrast to a recent report, which implicated the C-terminus of SMN in the interaction with Sm proteins (7). In this study, peptides corresponding to exon 6 of SMN were shown to compete for SmB binding to SMN in vitro (7). With respect to our study, which delineates the function of the tudor domain from four independent experimental approaches (see above), we feel that the previously reported result may be due to indirect effects caused by the peptide used for competition.
Tudor domains have been found by homology searches in several proteins of higher eukaryotes (18,19). Although the overall primary sequence of tudor domains found in different proteins may be low, they are predicted to form a common fold consisting of a single [alpha]-helix and a small [beta]-sheet of three strands. No function, however, has yet been assigned to any of the known tudor domains. Here we show that the tudor domain of SMN serves as a binding site for Sm proteins of spliceosomal Sm proteins. It may therefore be anticipated that tudor domains found in other proteins are also engaged in specific protein-protein interactions. Interestingly, a recently discovered SMN-related protein implicated in pre-mRNA splicing contains a sequence with striking homology to the tudor domain of SMN. It will be of interest to test whether this protein is engaged in interactions similar to those of SMN (19,26).
Our observation that an antiserum against the tudor domain of SMN efficiently inhibits the formation of the Sm core domain of snRNAs U1 and U5 provides strong evidence for an in vivo function of SMN in U snRNP assembly. Interestingly, a monoclonal antibody against the N-terminus of SMN has been shown to stimulate assembly in vivo (8). This may indicate that distinct domains in SMN mediate different steps in U snRNP assembly. At what step in U snRNP assembly the SMN-Sm protein interactions are required is currently unclear. It is tempting to speculate that SMN is necessary to prepare the Sm proteins for U snRNA binding. This could be accomplished, for example, by releasing Sm proteins from intracellular storage pools, preventing Sm proteins from aberrant aggregation and/or promoting the formation of the hetero-oligomeric Sm protein complexes. It is also conceivable that SMN acts as an assembly platform, since it interacts with Sm proteins and is transiently associated with U snRNA in the cytoplasm (8). Recent studies have demonstrated that the presence of only Sm proteins and U snRNA is sufficient for efficient Sm core assembly in vitro (27), suggesting that SMN plays a regulatory role in Sm core assembly. U snRNP assembly studies carried out in vitro with purified components should help to understand the exact mode of action of SMN and its interacting proteins.
A novel factor, termed pICln, has been put forward as a potential regulator of U snRNP formation (28). This could be inferred from the observations that pICln binds to several Sm proteins and inhibits U snRNP assembly in vivo. Moreover, binding of Sm proteins to SMN and pICln appears to be mutually exclusive. It has therefore been suggested that pICln acts as a negative regulator of U snRNP assembly, whereas SMN appears to counteract this function by actively promoting the assembly (28).
The analysis of the SMA-linked E134K missense mutation in SMN identifies a reduced interaction between Sm proteins and SMN as a biochemical defect in SMA. The E134K substitution is currently the only SMA causing mutation in the tudor domain of SMN. Most other missense mutations or deletions found in SMA patients affect the C-terminus of SMN and have been shown to be defective in SMN oligomerization (3,4,16). It will therefore be interesting to see whether reduced oligomerization of SMN caused by these C-terminal mutations likewise affects the assembly of U snRNPs in the cytoplasm. Alternatively, alterations in the C-terminus of SMN may well affect the nuclear function of SMN in pre-mRNA splicing (6) and thereby contribute to the defects occurring in SMA.
MATERIALS AND METHODS
Plasmid constructs
cDNAs for full-length SMN and SmB were amplified by polymerase chain reaction (PCR) using primers that introduced EcoRI and XhoI sites to the 5[prime] and 3[prime] ends, respectively, and cloned into pET21a (Novagen, Madison, WI). Fragments corresponding to the different regions of SMN were obtained by PCR and subcloned into pGEX 5X-1 (Pharmacia, Uppsala, Sweden) for GST-fusion peptides or into a pET21a-zz-vector. The pET21a-zz-vector was constructed by insertion of the PCR-amplified zz-domain (two copies of the IgG-binding domain of Staphylococcus aureus protein A) into the NheI/BamHI restriction sites. Clones harbouring the tudor domain mutations were generated by PCR methods.
Generation of antiserum and affinity-purification of specific antibodies
A rabbit antiserum against SMN was generated by sequential immunization with 1 mg of zz-tagged SMN. An antibody specific for the tudor domain was then obtained from the final bleeding by successive purification over an affinity resin containing either GST-SMN1-90 or GST-SMN36-159 immobilized on a CNBr-activated Sepharose column (Pharmacia). Bound antibody was eluted in 100 mM glycine (pH 2.3) and dialyzed against phosphate-buffered saline (PBS), pH 8.0. Prior to oocyte injection, the antibody was concentrated to 1 mg/ml in a Centricon microconcentrator (Amicon, Bedford, MA).
Recombinant proteins
For production of 35S-labeled proteins, plasmids containing SMN, SmB or SmD1 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 plasmids containing various SMN fragments were transformed into the Escherichia coli strain BL21(DE3). After induction with 1 mM isopropyl-[beta]-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 either frozen (for zz-tagged proteins) or further purified over GST-Sepharose 4B beads (for GST-tagged proteins; Pharmacia). Bound proteins were eluted with lysate buffer containing 10 mM GST and dialyzed against binding buffer (300 mM NaCl, 50 mM Tris-HCl pH 7.4, 5 mM MgCl2).
Western blot analysis
GST-SMN or fragments of GST-SMN (0.1 µg) were separated on a 12% SDS-polyacrylamide gel and transferred to Hybond C-nitrocellulose (Amersham, Uppsala, Sweden) using a standard blot apparatus. Blots were incubated in blocking solution [Tris-buffered saline (TBS), 5% non-fat milk] for 1 h at room temperature and then incubated with anti-Tu-antibody at a 1:500 dilution for 1 h at room temperature. After extensive washing with TBS containing 0.1% Tween-20, blots were incubated with horseradish peroxidase-coupled 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 enhanced chemiluminescence detection kit (Amersham). Oocyte extracts and affinity purified U snRNPs were obtained by methods described previously (8,20). For western blotting, extracts and U snRNPs containing 10 µg of protein were separated and processed as described above.
Preparation of RNA-free, native snRNP TPs
Native snRNP TPs were prepared as described previously (20) from a preparation of anti-cap immunoaffinity-purified HeLa U snRNPs that contained predominantly U1 and small amounts of U2 snRNPs. Briefly, proteins were dissociated from the U snRNPs in the presence of DEAE-cellulose (DE 53; Whatman, Kent, UK) and EDTA. The supernatant containing the RNA-free proteins was concentrated by dialysis against reconstitution buffer (20 mM HEPES-KOH pH 7.9, 50 mM KCl, 5 mM MgCl2, 5% (v/v) glycerol, 0.2 mM EDTA) containing 30% (w/v) PEG 6000 (Merck, Darmstadt, Germany) to ~0.3 mg/ml, with a final dialysis step in reconstitution buffer.
Binding assays
Approximately 50 µl of GST-Sepharose was incubated for 1 h at 4°C with 1 µg of purified GST-fusion protein. Alternatively, IgG-Sepharose was incubated with cell lysate containing 0.5-1 µg of overexpressed zz-tagged protein. Resin was washed three times in binding buffer. For protein binding, either 3 µl of 35S-labeled protein or 40 µl of HeLa TPs were incubated with the appropriate resin for 1 h at 4°C, and resin was subsequently washed extensively with binding buffer. Bound protein was eluted with SDS-sample buffer and analyzed by SDS-PAGE either by staining with Coomassie or silver, or by autoradiography using Amplify (Amersham). For antibody inhibition experiments, GST-SMN was bound to GST-Sepharose as described above and incubated for 1 h with a 5-fold molar excess of anti-Tu antibody. Resin was washed three times with binding buffer and subsequently incubated with 3 µl of in vitro translated 35S-labeled protein. Subsequent steps were performed as above. Quantitation of binding efficiencies was carried out using the Optimas software package or the NIH Image 1.6 program.
Microinjection in X.laevis oocytes
In vitro transcription of 32P-labeled RNA and injections into X.laevis oocytes were carried out as described (8). In brief, oocytes were incubated at 20°C for 3 h in OR2 buffer containing 0.2% collagenase type II (Sigma). Defolliculated stage V and VI oocytes were collected and kept in small batches for up to 5 days at 20°C. In a typical injection experiment, 30-50 nl of 32P-labeled RNA was injected into the cytoplasm of oocytes. For antibody inhibition experiments, oocytes were pre-injected with 50 nl of the indicated antibody (1 µg/µl) and incubated for 1 h before they received a second injection of radiolabeled RNA. For immunoprecipitation of RNA-protein complexes, the injected oocytes were incubated for 2 h and subsequently homogenized in 300 µl of ice-cold PBS (pH 7.4). The insoluble fraction was pelleted by centrifugation, and the clear supernatant was transferred into a new microtube containing antibodies bound to protein G-Sepharose (Pharmacia). The mixture was incubated with constant shaking for 1 h at 4°C and subsequently washed five times with 1 ml of ice-cold PBS. Bound RNAs were isolated by phenol extraction for 1 h, precipitated with ethanol and analyzed by denaturing gel electrophoresis. Quantitation of RNA was carried using the Optimas software package.
ACKNOWLEDGEMENTS
We are grateful to Drs Michael Sendtner and Iain Mattaj for providing us with plasmids and antibodies, to Gaby Sowa and Igor Weber for technical assistance, and to Bernhard Laggerbauer for critical reading of the manuscript. This work is part of a PhD thesis for the FU Berlin (D.B.). D.B. is funded by the Boehringer Ingelheim fonds. U.F. received a grant from the Deutsche Forschungsgemeinschaft (Hess-Stipendium) and support from the Max-Planck-Gesellschaft.
REFERENCES
+To whom correspondence should be addressed. Tel: + 49 89 8578 2253; Fax: +49 89 8578 3885; Email: ufischer{at}biochem.mpg.de
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 S. J. Kolb, D. J. Battle, and G. Dreyfuss Molecular Functions of the SMN Complex J Child Neurol, August 1, 2007; 22(8): 990 - 994. [Abstract] [PDF]
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 L. L. Almstead and P. Sarnow Inhibition of U snRNP assembly by a virus-encoded proteinase Genes & Dev., May 1, 2007; 21(9): 1086 - 1097. [Abstract] [Full Text] [PDF]
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 J. van Bergeijk, K. Rydel-Konecke, C. Grothe, and P. Claus The spinal muscular atrophy gene product regulates neurite outgrowth: importance of the C terminus FASEB J, May 1, 2007; 21(7): 1492 - 1502. [Abstract] [Full Text] [PDF]
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 C. Carissimi, L. Saieva, F. Gabanella, and L. Pellizzoni Gemin8 Is Required for the Architecture and Function of the Survival Motor Neuron Complex J. Biol. Chem., December 1, 2006; 281(48): 37009 - 37016. [Abstract] [Full Text] [PDF]
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 T. L. Carrel, M. L. McWhorter, E. Workman, H. Zhang, E. C. Wolstencroft, C. Lorson, G. J. Bassell, A. H. M. Burghes, and C. E. Beattie Survival Motor Neuron Function in Motor Axons Is Independent of Functions Required for Small Nuclear Ribonucleoprotein Biogenesis J. Neurosci., October 25, 2006; 26(43): 11014 - 11022. [Abstract] [Full Text] [PDF]
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 S. Chuma, M. Hosokawa, K. Kitamura, S. Kasai, M. Fujioka, M. Hiyoshi, K. Takamune, T. Noce, and N. Nakatsuji Tdrd1/Mtr-1, a tudor-related gene, is essential for male germ-cell differentiation and nuage/germinal granule formation in mice PNAS, October 24, 2006; 103(43): 15894 - 15899. [Abstract] [Full Text] [PDF]
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 A. L. Arkov, J.-Y. S. Wang, A. Ramos, and R. Lehmann The role of Tudor domains in germline development and polar granule architecture Development, October 15, 2006; 133(20): 4053 - 4062. [Abstract] [Full Text] [PDF]
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 S. Hamamoto, H. Nishitsuji, T. Amagasa, M. Kannagi, and T. Masuda Identification of a Novel Human Immunodeficiency Virus Type 1 Integrase Interactor, Gemin2, That Facilitates Efficient Viral cDNA Synthesis In Vivo. J. Virol., June 1, 2006; 80(12): 5670 - 5677. [Abstract] [Full Text] [PDF]
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 B. Renvoise, K. Khoobarry, M.-C. Gendron, C. Cibert, L. Viollet, and S. Lefebvre Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells J. Cell Sci., February 15, 2006; 119(4): 680 - 692. [Abstract] [Full Text] [PDF]
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 T. J. Golembe, J. Yong, and G. Dreyfuss Specific Sequence Features, Recognized by the SMN Complex, Identify snRNAs and Determine Their Fate as snRNPs Mol. Cell. Biol., December 15, 2005; 25(24): 10989 - 11004. [Abstract] [Full Text] [PDF]
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F. Gabanella, C. Carissimi, A. Usiello, and L. Pellizzoni The activity of the spinal muscular atrophy protein is regulated during development and cellular differentiation Hum. Mol. Genet., December 1, 2005; 14(23): 3629 - 3642. [Abstract] [Full Text] [PDF]
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K. B. Shpargel and A. G. Matera Gemin proteins are required for efficient assembly of Sm-class ribonucleoproteins PNAS, November 29, 2005; 102(48): 17372 - 17377. [Abstract] [Full Text] [PDF]
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T. N. Azzouz, R. S. Pillai, C. Dapp, A. Chari, G. Meister, C. Kambach, U. Fischer, and D. Schumperli Toward an Assembly Line for U7 snRNPs: INTERACTIONS OF U7-SPECIFIC Lsm PROTEINS WITH PRMT5 AND SMN COMPLEXES J. Biol. Chem., October 14, 2005; 280(41): 34435 - 34440. [Abstract] [Full Text] [PDF]
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J. Cote and S. Richard Tudor Domains Bind Symmetrical Dimethylated Arginines J. Biol. Chem., August 5, 2005; 280(31): 28476 - 28483. [Abstract] [Full Text] [PDF]
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L. Wan, D. J. Battle, J. Yong, A. K. Gubitz, S. J. Kolb, J. Wang, and G. Dreyfuss The Survival of Motor Neurons Protein Determines the Capacity for snRNP Assembly: Biochemical Deficiency in Spinal Muscular Atrophy Mol. Cell. Biol., July 1, 2005; 25(13): 5543 - 5551. [Abstract] [Full Text] [PDF]
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W. Feng, A. K. Gubitz, L. Wan, D. J. Battle, J. Dostie, T. J. Golembe, and G. Dreyfuss Gemins modulate the expression and activity of the SMN complex Hum. Mol. Genet., June 15, 2005; 14(12): 1605 - 1611. [Abstract] [Full Text] [PDF]
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J. Anne and B. M. Mechler Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuleen Development, May 1, 2005; 132(9): 2167 - 2177. [Abstract] [Full Text] [PDF]
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 F.-M. Boisvert, C. A. Chenard, and S. Richard Protein Interfaces in Signaling Regulated by Arginine Methylation Sci. Signal., February 15, 2005; 2005(271): re2 - re2. [Abstract] [Full Text] [PDF]
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T. J. Golembe, J. Yong, D. J. Battle, W. Feng, L. Wan, and G. Dreyfuss Lymphotropic Herpesvirus saimiri Uses the SMN Complex To Assemble Sm Cores on Its Small RNAs Mol. Cell. Biol., January 15, 2005; 25(2): 602 - 611. [Abstract] [Full Text] [PDF]
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 I. Cusco, M. J. Barcelo, E. del Rio, M. Baiget, and E. F. Tizzano Detection of novel mutations in the SMN Tudor domain in type I SMA patients Neurology, July 13, 2004; 63(1): 146 - 149. [Abstract] [Full Text] [PDF]
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S. Majumder, S. Varadharaj, K. Ghoshal, U. Monani, A. H. M. Burghes, and S. T. Jacob Identification of a Novel Cyclic AMP-response Element (CRE-II) and the Role of CREB-1 in the cAMP-induced Expression of the Survival Motor Neuron (SMN) Gene J. Biol. Chem., April 9, 2004; 279(15): 14803 - 14811. [Abstract] [Full Text] [PDF]
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J. Yong, T. J. Golembe, D. J. Battle, L. Pellizzoni, and G. Dreyfuss snRNAs Contain Specific SMN-Binding Domains That Are Essential for snRNP Assembly Mol. Cell. Biol., April 1, 2004; 24(7): 2747 - 2756. [Abstract] [Full Text] [PDF]
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T. C. Fleischer, U. J. Yun, and D. E. Ayer Identification and Characterization of Three New Components of the mSin3A Corepressor Complex Mol. Cell. Biol., May 15, 2003; 23(10): 3456 - 3467. [Abstract] [Full Text] [PDF]
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G. K. Carnegie, J. E. Sleeman, N. Morrice, C. J. Hastie, M. W. Peggie, A. Philp, A. I. Lamond, and P. T. W. Cohen Protein phosphatase 4 interacts with the Survival of Motor Neurons complex and enhances the temporal localisation of snRNPs J. Cell Sci., May 15, 2003; 116(10): 1905 - 1913. [Abstract] [Full Text] [PDF]
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 U. R. Monani, M. T. Pastore, T. O. Gavrilina, S. Jablonka, T. T. Le, C. Andreassi, J. M. DiCocco, C. Lorson, E. J. Androphy, M. Sendtner, et al. A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy J. Cell Biol., January 2, 2003; 160(1): 41 - 52. [Abstract] [Full Text] [PDF]
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S. E. Whitehead, K. W. Jones, X. Zhang, X. Cheng, R. M. Terns, and M. P. Terns Determinants of the Interaction of the Spinal Muscular Atrophy Disease Protein SMN with the Dimethylarginine-modified Box H/ACA Small Nucleolar Ribonucleoprotein GAR1 J. Biol. Chem., December 6, 2002; 277(50): 48087 - 48093. [Abstract] [Full Text] [PDF]
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 L. Pellizzoni, J. Yong, and G. Dreyfuss Essential Role for the SMN Complex in the Specificity of snRNP Assembly Science, November 29, 2002; 298(5599): 1775 - 1779. [Abstract] [Full Text] [PDF]
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S. Massenet, L. Pellizzoni, S. Paushkin, I. W. Mattaj, and G. Dreyfuss The SMN Complex Is Associated with snRNPs throughout Their Cytoplasmic Assembly Pathway Mol. Cell. Biol., September 15, 2002; 22(18): 6533 - 6541. [Abstract] [Full Text] [PDF]
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J. Baccon, L. Pellizzoni, J. Rappsilber, M. Mann, and G. Dreyfuss Identification and Characterization of Gemin7, a Novel Component of the Survival of Motor Neuron Complex J. Biol. Chem., August 23, 2002; 277(35): 31957 - 31962. [Abstract] [Full Text] [PDF]
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S. Jablonka, B. Holtmann, G. Meister, M. Bandilla, W. Rossoll, U. Fischer, and M. Sendtner Gene targeting of Gemin2 in mice reveals a correlation between defects in the biogenesis of U snRNPs and motoneuron cell death PNAS, July 23, 2002; 99(15): 10126 - 10131. [Abstract] [Full Text] [PDF]
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U. Narayanan, J. K. Ospina, M. R. Frey, M. D. Hebert, and A. G. Matera SMN, the spinal muscular atrophy protein, forms a pre-import snRNP complex with snurportin1 and importin {beta} Hum. Mol. Genet., July 15, 2002; 11(15): 1785 - 1795. [Abstract] [Full Text] [PDF]
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 H. Schenkel, S. Hanke, C. De Lorenzo, R. Schmitt, and B. M. Mechler P Elements Inserted in the Vicinity of or Within the Drosophila snRNP SmD3 Gene Nested in the First Intron of the Ornithine Decarboxylase Antizyme Gene Affect Only the Expression of SmD3 Genetics, June 1, 2002; 161(2): 763 - 772. [Abstract] [Full Text] [PDF]
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S. Lefebvre, P. Burlet, L. Viollet, S. Bertrandy, C. Huber, C. Belser, and A. Munnich A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy Hum. Mol. Genet., May 1, 2002; 11(9): 1017 - 1027. [Abstract] [Full Text] [PDF]
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W. J. Friesen, A. Wyce, S. Paushkin, L. Abel, J. Rappsilber, M. Mann, and G. Dreyfuss A Novel WD Repeat Protein Component of the Methylosome Binds Sm Proteins J. Biol. Chem., March 1, 2002; 277(10): 8243 - 8247. [Abstract] [Full Text] [PDF]
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W. Rossoll, A.-K. Kroning, U.-M. Ohndorf, C. Steegborn, S. Jablonka, and M. Sendtner Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons? Hum. Mol. Genet., January 1, 2002; 11(1): 93 - 105. [Abstract] [Full Text] [PDF]
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W. J. Friesen, S. Paushkin, A. Wyce, S. Massenet, G. S. Pesiridis, G. Van Duyne, J. Rappsilber, M. Mann, and G. Dreyfuss The Methylosome, a 20S Complex Containing JBP1 and pICln, Produces Dimethylarginine-Modified Sm Proteins Mol. Cell. Biol., December 15, 2001; 21(24): 8289 - 8300. [Abstract] [Full Text] [PDF]
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M. D. Hebert, P. W. Szymczyk, K. B. Shpargel, and A. G. Matera Coilin forms the bridge between Cajal bodies and SMN, the Spinal Muscular Atrophy protein Genes & Dev., October 15, 2001; 15(20): 2720 - 2729. [Abstract] [Full Text] [PDF]
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K. W. Jones, K. Gorzynski, C. M. Hales, U. Fischer, F. Badbanchi, R. M. Terns, and M. P. Terns Direct Interaction of the Spinal Muscular Atrophy Disease Protein SMN with the Small Nucleolar RNA-associated Protein Fibrillarin J. Biol. Chem., October 12, 2001; 276(42): 38645 - 38651. [Abstract] [Full Text] [PDF]
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S. Jablonka, M. Bandilla, S. Wiese, D. Buhler, B. Wirth, M. Sendtner, and U. Fischer Co-regulation of survival of motor neuron (SMN) protein and its interactor SIP1 during development and in spinal muscular atrophy Hum. Mol. Genet., March 1, 2001; 10(5): 497 - 505. [Abstract] [Full Text] [PDF]
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B. Laggerbauer, D. Ostareck, E.-M. Keidel, A. Ostareck-Lederer, and U. Fischer Evidence that fragile X mental retardation protein is a negative regulator of translation Hum. Mol. Genet., February 1, 2001; 10(4): 329 - 338. [Abstract] [Full Text] [PDF]
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L. Pellizzoni, B. Charroux, J. Rappsilber, M. Mann, and G. Dreyfuss A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II J. Cell Biol., January 8, 2001; 152(1): 75 - 86. [Abstract] [Full Text] [PDF]
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P. J. Young, N. t. Man, C. L. Lorson, T. T. Le, E. J. Androphy, A. H.M. Burghes, and G. E. Morris The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in self-association and SIP1 binding Hum. Mol. Genet., November 1, 2000; 9(19): 2869 - 2877. [Abstract] [Full Text] [PDF]
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G. Meister, D. Buhler, B. Laggerbauer, M. Zobawa, F. Lottspeich, and U. Fischer Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins Hum. Mol. Genet., August 12, 2000; 9(13): 1977 - 1986. [Abstract] [Full Text] [PDF]
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L. Campbell, K. M. D. Hunter, P. Mohaghegh, J. M. Tinsley, M. A. Brasch, and K. E. Davies Direct interaction of Smn with dp103, a putative RNA helicase: a role for Smn in transcription regulation? Hum. Mol. Genet., April 12, 2000; 9(7): 1093 - 1100. [Abstract] [Full Text] [PDF]
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S. Hannus, D. Buhler, M. Romano, B. Seraphin, and U. Fischer The Schizosaccharomyces pombe protein Yab8p and a novel factor, Yip1p, share structural and functional similarity with the spinal muscular atrophy-associated proteins SMN and SIP1 Hum. Mol. Genet., March 22, 2000; 9(5): 663 - 674. [Abstract] [Full Text] [PDF]
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N. Owen, C. L. Doe, J. Mellor, and K. E. Davies Characterization of the Schizosaccharomyces pombe orthologue of the human survival motor neuron (SMN) protein Hum. Mol. Genet., March 22, 2000; 9(5): 675 - 684. [Abstract] [Full Text] [PDF]
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S. Paushkin, B. Charroux, L. Abel, R. A. Perkinson, L. Pellizzoni, and G. Dreyfuss The Survival Motor Neuron Protein of Schizosacharomyces pombe. CONSERVATION OF SURVIVAL MOTOR NEURON INTERACTION DOMAINS IN DIVERGENT ORGANISMS J. Biol. Chem., July 28, 2000; 275(31): 23841 - 23846. [Abstract] [Full Text] [PDF]
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W. J. Friesen and G. Dreyfuss 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., August 18, 2000; 275(34): 26370 - 26375. [Abstract] [Full Text] [PDF]
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J. Wang and G. Dreyfuss A Cell System with Targeted Disruption of the SMN Gene. FUNCTIONAL CONSERVATION OF THE SMN PROTEIN AND DEPENDENCE OF Gemin2 ON SMN J. Biol. Chem., March 23, 2001; 276(13): 9599 - 9605. [Abstract] [Full Text] [PDF]
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L. R. Saunders, D. J. Perkins, S. Balachandran, R. Michaels, R. Ford, A. Mayeda, and G. N. Barber Characterization of Two Evolutionarily Conserved, Alternatively Spliced Nuclear Phosphoproteins, NFAR-1 and -2, That Function in mRNA Processing and Interact with the Double-stranded RNA-dependent Protein Kinase, PKR J. Biol. Chem., August 17, 2001; 276(34): 32300 - 32312. [Abstract] [Full Text] [PDF]
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J. Wang and G. Dreyfuss Characterization of Functional Domains of the SMN Protein in Vivo J. Biol. Chem., November 21, 2001; 276(48): 45387 - 45393. [Abstract] [Full Text] [PDF]
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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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