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Human Molecular Genetics Advance Access originally published online on September 13, 2005
Human Molecular Genetics 2005 14(20):3099-3111; doi:10.1093/hmg/ddi343
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Unrip, a factor implicated in cap-independent translation, associates with the cytosolic SMN complex and influences its intracellular localization

Matthias Grimmler, Simon Otter, Christoph Peter, Felicitas Müller, Ashwin Chari and Utz Fischer*

Department of Biochemistry, Theodor Boveri Institute, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany

* To whom correspondence should be addressed. Tel: +49 9318884029; Fax: +49 9318884028; Email: utz.fischer{at}biozentrum.uni-wuerzburg.de

Received June 21, 2005; Revised August 3, 2005; Accepted September 7, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Spliceosomal Uridine-rich small ribonucleo protein (U snRNP) assembly is an active process mediated by the macromolecular survival motor neuron (SMN) complex. This complex contains the SMN protein and six additional proteins, named Gemin2–7, according to their localization to nuclear structures termed gems. Here, we provide biochemical evidence for the existence of another, yet atypical, SMN complex component, termed unr-interacting protein (unrip). This abundant factor has been previously shown to form a complex with unr, a protein implicated in cap-independent translation of cellular and viral mRNA. We show that unrip is integrated into a complex with unr or with the SMN complex in vivo in a mutually exclusive manner. In the latter case, unrip is recruited to the active SMN complex via a stable interaction with Gemin7. However, unlike SMN and Gemins, unrip localizes predominantly to the cytoplasm and is absent from gems/Cajal bodies. Interestingly, RNAi-induced reduction of unrip protein levels leads to enhanced accumulation of SMN in the nucleus as evident by the increased formation of nuclear gems/Cajal bodies. Our data identify unrip as the first component of the U snRNP assembly machinery that associates with the SMN complex in a compartment-specific way. We speculate that unrip plays a crucial role in the intracellular distribution of the SMN complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The spliceosomal Uridine-rich small nuclear ribonucleoprotein particles (U snRNPs) are essential components of the spliceosome, the macromolecular machinery that assembles on introns of pre-mRNAs and catalyses the splicing reaction. Each U snRNP consists of one (U1, U2, U5) or two (U4 and U6) small nuclear RNAs (snRNAs) associated with a set of the seven Sm proteins B/B', D1, D2, D3, E, F and G, and a large number of particle-specific factors (recently reviewed in 1Go). U snRNPs form in vivo in a highly ordered and regulated pathway that is initiated by the post-transcriptional export of the U snRNA from the nucleus to the cytoplasm. Subsequently, the seven Sm proteins bind to the Sm site, a single-stranded sequence motif found in the snRNAs U1, U2, U4 and U5, leading to the formation of the ring-shaped Sm core domain (2Go,3Go). The Sm core domain provides a binding site for a methyltransferase (termed Tgs1p/PIMT) that catalyses the formation of the m2,2,7-trimethylguanosine cap (m3G-cap) of the U snRNA (4Go,5Go). Sm core domain and the m3G-cap both act as nuclear localization signals that target the assembled particles to the nucleus (6Go–8Go).

Although binding of Sm proteins onto U snRNAs can occur spontaneously in vitro when isolated Sm proteins are incubated with U snRNA, recent studies have indicated that this process requires ATP and the assistance of a large number of trans-acting factors (9Go–12Go). These factors are organized in two functional units, termed survival motor neuron (SMN) and PRMT5 complexes that mediate distinct functions in the assembly pathway. In the cytoplasm, the Sm proteins first bind to the PRMT5 complex, which contains the arginine-methyltransferase PRMT5. This enzyme subsequently modifies arginines in the RG-rich tails of the Sm proteins B/B', D1 and D3 to symmetric dimethylarginines (10Go,13Go,14Go). Arginine methylation increases the affinity of Sm proteins for SMN, thus allowing their transfer onto the SMN complex (15Go–17Go). In a final step, Sm proteins are loaded onto the Sm site of the U snRNA, which leads to the formation of the Sm core domain (10Go–12Go). The finding that the cap-hypermethylase Tgs1p and U snRNP-transport factors associate with SMN further suggests that the SMN complex is also involved in these latter steps of U snRNP biogenesis (18Go–21Go). In fact, biochemical studies have shown that the SMN complex accompanies the mature U snRNPs to the nucleus (20Go). Post-translational modifications of SMN (and possibly also of other assembly factors) may regulate the different functions of the SMN complex in the biogenesis pathway of U snRNPs (22Go).

SMN interacts with a large number of proteins in vivo (23Go). However, when the SMN complex was isolated from cultivated HeLa cells under stringent conditions, only six stably associated components were found to be part of the assembly machinery. As these proteins localize not only to the cytoplasm but also to the nuclear gemini of coiled bodies (gems), they have been collectively named ‘Gemins’ (24Go). More recent studies showed that in many, but not all cells these nuclear structures are equivalent to the well-known nuclear Cajal bodies (25Go). Upon purification, we and others have identified an additional factor that is associated with SMN in vivo, termed unr-interacting protein (unrip) (10Go,20Go). Unrip is a WD-repeat protein that forms a complex with unr (upstream of N-ras), a factor implicated in cap-independent translational regulation of mRNAs (26Go,27Go).

Here, we show that unrip is the eighth major component of the assembly-active SMN complex but differs from other Gemins in that it is absent from the nucleus. Interestingly, RNA-interference (RNAi)-induced reduction of this factor leads to an increase in the number of gems/Cajal bodies, raising the hypothesis that it may modulate the subcellular distribution of the active SMN complex. Finally, we provide evidence that the interaction with the SMN complex occurs via binding to Gemin7. This interaction is mutually exclusive with binding of unrip to unr, thus allowing unrip to be part of two independent complexes in vivo. Our data raise the hypothesis that unrip interacts with the SMN complex in a reversible and compartment-specific manner, thereby regulating its localization and/or activity in U snRNP assembly.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
A Sub-fraction of cellular unrip is specifically associated with the SMN complex
Although previous studies have established the interaction of unrip with unr in vivo (26Go,27Go), its proposed function within the SMN complex remained unclear. To resolve this issue, monospecific polyclonal antibodies directed against human unrip were generated (Fig. 1A). Using this antibody and the previously reported monoclonal antibody 7B10 directed against the N-terminus of human SMN (28Go), immunoprecipitation studies were performed with HeLa cytosolic extract. SDS–PAGE analysis of the anti-unrip precipitate revealed a large number of proteins, including two prominent ones with apparent molecular weights of ~ 40 and 100 kDa. Mass spectrometry (data not shown) and immunoblotting identified these proteins as unrip and unr, respectively (Fig. 1B, lanes 3, 4 and Fig. 1C lane 2). As expected, affinity purification with 7B10 recovered the SMN complex, consisting of SMN, Gemin2–7 and the Sm proteins (Fig. 1B, lane 4 and Fig. 1C, lane 3). Furthermore, unrip was readily detected in the SMN complex confirming earlier data (Fig. 1B and 1C) (10Go,20Go). More importantly, a comparison of both purifications revealed the entire SMN complex to be present in the unrip-immunoprecipitation (Fig. 1B, SMN complex components are indicated by dots and, in part, are also detected by western blot shown in Fig. 1C). None of these proteins were enriched in the pre-immune serum control purification, illustrating that these interactions are specific (Fig. 1C, lane 1). Thus, highly enriched SMN complex contains unrip and affinity-purified unrip contains the SMN complex.



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  		Figure 1. Unrip is part of the SMN complex in vivo. (A) Detection of unrip and SMN in HeLa cytosolic extract by western blotting using affinity-purified monoclonal anti-SMN antibody 7B10 (lane 1) and rabbit anti-unrip antibodies (lane 2). (B) Cytosolic extract from HeLa cells was immunoprecipitated with anti-unrip and anti-SMN antibody. Proteins were eluted from the beads, resolved by SDS–PAGE and analysed by silver staining (lanes 3 and 4). The unrip pre-immune serum was used for a control immunoprecipitation (lane 2). Lane 1 shows a molecular weight marker. Dots indicate known proteins of the SMN complex. An asterisk marks the unr protein, which can be detected only in the unrip immunoprecipitation. The right panel shows enlarged parts of the 40 and 100 kDa range of the gel. (C) The immunoprecipitated complexes shown in (A) were resolved by SDS–PAGE and analysed by western blotting with the indicated antibodies. (D) Unrip partially co-fractionates with the SMN complex. HeLa cytosolic extract (lane 1) was fractionated on a Superdex 200 pg gel filtration column. Fractions were separated by SDS–PAGE (lanes 2–20) and analysed by western blotting using antibodies directed against SMN, unrip and Gemin3. The indicated molecular masses were determined by an independent chromatography with a gel filtration size standard.

 
Next, we asked whether unr, the known major interactor of unrip in vivo (26Go), was likewise a component of the SMN complex. Interestingly, in the 7B10 immunoprecipitate unr could be detected neither by silver staining (see Fig. 1B, for enlargement of this portion of gel) nor by immunoblotting with an antibody raised against human unr (Fig. 1C, lane 3). Thus, unrip, but not unr, interacts with the SMN complex in cytosolic extract.

To further strengthen the view that unrip is at least part of two complexes, HeLa cytosolic extract was fractionated by gel filtration chromatography and fractions were probed on western blots with different antibodies. As shown in Figure 1D, SMN and Gemin3 elute in a high molecular weight range (~660 kDa–1 MDa), which corresponds to the size of the SMN complex. Unrip, in contrast, elutes in a much more diffuse pattern ranging from ~150 kDa (the calculated mass of the unr/unrip heterodimer) to ~1 MDa (the mass of the SMN complex). Together, these data suggest that unrip not only binds to unr but also interacts specifically with the SMN complex.

Association of unrip with cytoplasmic SMN complex
A hallmark of SMN and its associated proteins is their subcellular localization to both the cytoplasm and the nuclear gems/Cajal bodies (24Go,29Go). Our finding that unrip is a major component of the cytoplasmic SMN complex led us to investigate whether it likewise localizes in gems/Cajal bodies as observed for the other complex components. This question was addressed by indirect immunofluorescence in cultivated HeLa cells. Endogenous unrip and SMN were detected using monospecific antibodies. In line with earlier findings, the antibody 7B10 detected SMN both in the cytoplasm and in the nuclear foci (Fig. 2A, c) (24Go,30Go). A partial co-localization of SMN and unrip in the cytoplasm was observed, consistent with the finding that a fraction of cellular unrip is part of the SMN complex (Fig. 2A, d, red and yellow merge). In striking contrast, unrip in the same cells could be detected neither in gems/Cajal bodies nor in other regions of the nucleus (Fig. 2A, b). Consequently, an overlay of both images revealed no co-localization in this compartment (Fig. 2A, d). To confirm these results by biochemical means, we prepared nuclear and cytosolic extract from HeLa cells and determined the amount of SMN and unrip in these fractions by western blotting. In extracts, which were normalized for the amount of SMN, unrip was detected almost exclusively (to >90%) in the cytosolic fraction (Fig. 2B, lane 1). The small amount of unrip in the nuclear fraction most likely originates from cytoplasmic contaminations. SMN, in contrast, was detected in both compartments in similar quantities (compare lanes 1 and 2). Likewise, immunoprecipitation of SMN co-precipitated significant amounts of unrip only from the cytosolic but not from the nuclear extract (data not shown). Thus, unrip associates with the SMN complex only in the cytoplasm, whereas the nuclear complex contains little, if any, unrip.



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  		Figure 2. Unrip localizes predominantly to the cytoplasm but is absent from nuclear foci. (A) Localization of SMN and unrip in HeLa cells by immunofluorescence. Affinity-purified polyclonal rabbit anti-unrip (b) and monoclonal anti-SMN antibody 7B10 (c) were used as primary antibodies. Rhodamine-labeled anti-rabbit and fluorescein-labeled anti-mouse antibodies were used as secondary antibodies. Co-localization of proteins is shown by overlay of both images (yellow merge, d). (a) shows the analysed HeLa cells in differential interference contrast (DIC) mode. (B) HeLa cytosolic (lane 1) and nuclear extract (lane 2) were resolved by SDS–PAGE and analysed by immunoblotting using affinity-purified anti-unrip antibodies (upper panel) and anti-SMN antibody 7B10. The protein amount loaded in each lane was normalized for SMN.

 
Unrip-containing SMN complex is active in U snRNP assembly
The association of unrip with cytoplasmic but not with nuclear SMN complex was interesting in the light of earlier findings that only the former is active in U snRNP assembly (22Go). Therefore, we tested whether unrip played a crucial role during the formation of U snRNPs using a previously reported in vitro assembly assay. In this assay, in vitro transcribed and 32P-labeled U1 snRNA is incubated with cytosolic extract and assembly is subsequently monitored by native gel electrophoresis (10Go). We first asked whether cytosolic extracts lacking unrip could still promote formation of functional U1 snRNP. Indeed, assembly of the Sm core domain was strongly reduced in extract that had been immunodepleted of ~95% of endogenous unrip (Fig. 3A, lanes 2 and 3). This became evident by the impaired formation of a previously reported ‘M-complex’, which is super-shifted by the anti-Sm antibody Y12 (Fig. 3A, compare lanes 5 and 6). Formation of a complex marked by an asterisk was not affected. This complex forms SMN independently and consists of U1 snRNA and the U1-specific protein A only (10Go). Note that a non-specific complex is also retarded to the same position. In contrast, untreated or extract treated with control antibodies showed efficient formation of U1 snRNP (Fig. 3A, lanes 4 and 7). Thus, depletion of unrip from the extract severely impairs U snRNP assembly. We next tested by western blotting whether removal of unrip also had an impact on the abundance of other components of the assembly machinery. Indeed, along with the diminution of unrip also SMN, Gemin2 and 3 were co-depleted from the extract, whereas the level of Sm proteins remained largely unchanged (Fig. 3B). All attempts to dissociate unrip from the rest of the SMN complex, which would allow for the specific depletion of merely unrip, failed even at very stringent conditions (1.5 M NaCl or 0.3 M KCl/ 0.5 M NaSCN were tested) (Fig. 3B compare lanes 1–3 with lanes 4–6 and 7–9). These data strongly suggested that unrip is part of the assembly machinery of U snRNPs. To verify this directly, SMN complex isolated either via immunoprecipitation with 7B10 antibody or via anti-unrip antibody was assessed for formation of the U1 snRNP in vitro. As shown in Figure 3C, both complexes efficiently generated U1 snRNP cores, as evident by the appearance of complex M (Fig. 3C, lanes 2–5), whereas a control purification with a non-related antibody failed to promote U1 snRNP formation (lanes 6 and 7). In conclusion, these data show that unrip is part of the SMN complex active in U snRNP assembly.



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  		Figure 3. Unrip is associated with the SMN complex, active in U snRNP assembly. (A) Cytosolic extracts in PBS [lanes 1–3 in (B)] were incubated with 32P-labeled U1 snRNA and analysed by native gel electrophoresis. Complex ‘M’ represents the assembled Sm core domain, the band indicated with an asterisk resembles a complex that is formed independent of the SMN complex (lanes 2–4). To verify Sm core assembly, Y12 antibody was added to assembly reactions prior to analysis by native gel electrophoresis. The M-complex is super-shifted to the origin of the gel, illustrating assembly of the Sm core domain (lanes 5–7). Lane 1 shows U1 snRNA in the absence of extract. (B) Immunodepletion of unrip under different salt conditions. HeLa cytosolic extracts are shown either before depletion (lanes 1, 4 and 7), after immunodepletion with unrip-antibody (lanes 2, 5 and 8), or after a control-depletion with unrip pre-immuneserum (lanes 3, 6 and 9). Depletions were performed in cytosolic extract containing either 130 mM NaCl (PBS, lanes 1–3), 1.5 M NaCl (1.5 M, lanes 4–6) or 300 mM KCl/500 mM NaSCN (NaSCN, lanes 7–9). Extracts were separated by SDS–PAGE and analysed by western blotting using antibodies directed against unrip, SMN, Gemin2, Gemin3 and the anti-Sm antibody Y12. (C) Cytosolic PBS extract from HeLa cells was immunoprecipitated with anti-unrip antiserum, anti-SMN antibody 7B10 or a control antibody. The immunoprecipitates were then incubated with 32P-labeled U1 snRNA and subsequently analysed by native gel electrophoresis (lanes 2, 4 and 6). The specificity of Sm core formation was assessed by addition of the Sm antibody Y12 to the assembly reaction (lanes 3, 5, 7). Lane 1 shows the U1 snRNA in the absence of extract.

 
Because depletion of unrip by biochemical means simultaneously led to reduction of other essential assembly factors, the question whether unrip was necessary for assembly activity remained open. To circumvent SMN complex co-depletion, we used RNAi to selectively reduce unrip proteins levels. As shown in Figure 4A, silencing of unrip was nearly complete 72 h after transfection (lane 2), whereas minor amounts of the protein were expressed at later time points (compare lane 2 with lanes 3 and 4). Importantly, silencing of unrip did not affect the expression levels of other components of the SMN complex, such as SMN, Gemin3 or Sm proteins, thus allowing the analysis of unrip reduction directly (Fig. 4B). Cytosolic extracts derived from untransfected (lanes 3 and 4) and unrip-siRNA transfected HeLa cells (lanes 1 and 2) were analysed for U snRNP assembly by native gel electrophoresis. Even though unrip was reduced in RNAi-treated cells by >90%, no significant reduction of the assembly activity was observed when compared to control extract (lanes 3 and 4). This finding suggests that unrip, although being a component of the active SMN complex, is not essential for the assembly reaction per se.



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  		Figure 4. Silencing of unrip leads to an accumulation of the SMN complex in nuclear gems/Cajal bodies. (A) Reduction of unrip protein levels in HeLa cells by RNAi. Cells were transfected with siRNA directed against unrip for 72, 96 and 120 h. Whole cell extracts obtained from these cells (lanes 2, 3, and 4, respectively) and control cells (lane 1) were resolved by SDS–PAGE and analysed by immunoblotting with monospecific antibodies directed against SMN, Gemin3, unrip, Sm B/B' and Sm D1, respectively. (B) U snRNP assembly activity of extracts derived from unrip-silenced cells. Cytosolic extracts derived from cells treated with unrip siRNA for 72 h (lane 1) or control extract (lane 3) were assessed for their competence in U snRNP assembly by native gel electrophoresis, using 32P-labeled U1 snRNA. Addition of monoclonal antibodies Y12 leads to a super-shift of complex M, verifying Sm core assembly (lanes 2 and 4). Lane 5 shows U1 snRNA in the absence of extract. (C) Silencing of unrip leads to an accumulation of SMN complex in the nucleus. HeLa cells were treated with siRNA directed against unrip as described earlier and separated into a cytosolic (C) and a nuclear fraction (N). Successful compartment separation was assessed by immunodetection of {alpha}Tubulin and Histone H4 as marker proteins for cytoplasm and the nucleus, respectively. SMN, Gemin2 and 3 were analysed by monospecific antibodies. (D) Localization of the SMN complex. HeLa cells grown on cover slides were treated with unrip siRNA for 72 h. Overexpression of HA-SMN was achieved by transient transfection. Localization of SMN was analysed by indirect immunofluorescence in control-treated (d) and unrip-silenced cells (e) using monoclonal mouse anti-SMN 7B10 as a primary antibody. Overexpressed HA-SMN was detected using anti-HA (clone 16B12, Babco) as a primary antibody (f). The secondary antibody was a rhodamine-labeled anti-mouse antibody. Nucleus and cytoplasm are indicated by DAPI stain and DIC, respectively.

 
RNAi-induced reduction of unrip causes nuclear accumulation of the SMN complex
On the basis of the data described earlier, we next tested whether unrip contributed to the U snRNP biogenesis by influencing the intracellular localization of the SMN complex. Interestingly, immunofluorescence of cells treated with unrip-RNAi revealed a significant increase in SMN and Gemin2-positive nuclear structures (i.e. gems and/or Cajal bodies, Fig. 4D e and Supplementary Material, Section A). Whereas non-transfected control cells contained 1.8±0.12 nuclear foci (Fig. 4D d), their number was increased in unrip-deprived cells more than 3-fold (5.9±0.22 Cajal bodies/cell, see also Statistical Analysis in Supplementary Material Section B). Two lines of evidence suggest that the higher gem/Cajal body number reflected an increased level of SMN complex in the nucleus: Firstly cells that were transfected with a plasmid that allows for overexpression of haemagglutinin (HA)-tagged SMN exhibited a strong increase in nuclear gems/Cajal bodies (13.4±2.53), reminiscent of the effect observed in unrip-deprived cells (Fig. 4D f). Localization of endogenous unrip was not altered in these cells, excluding non-specific effects caused by the overexpression of HA-SMN (Supplementary Material, Section C); furthermore, overexpression of HA-unrip leads to a decrease in SMN-positive nuclear foci (Supplementary Material, Section C). Secondly, when unrip-deprived cells were separated into a nuclear and cytosolic fraction, a significant increase of SMN, Gemin2 and 3 was observed in the nuclear fraction when compared with control cells (Fig. 4C, lanes 3 and 4). Interestingly, this coincided with a decrease of these factors in the cytosol (lanes 1 and 2). Together, our data suggest that unrip influences the intracellular distribution of the SMN complex.

Interaction with Gemin7 incorporates unrip into the SMN complex
As the interaction of unrip with the SMN complex is very stable in vivo and even withstands 1.5 M salt (discussed earlier), we anticipated efficient binding to one or several Gemins and/or Sm proteins. This was addressed by in vitro protein-binding assays using recombinant glutathione S-transferase (GST)–unrip immobilized to a glutathione–Sepharose matrix and 35S-methionine-labeled SMN complex proteins, i.e. SMN, Gemin2–7 and Sm proteins. After extensive washing, bound proteins were eluted from the resin, resolved by SDS–PAGE and detected by autoradiography. As shown in Figure 5A, only Gemin7 (lane 21) but none of the other core-factors of the SMN complex (i.e. SMN and Gemin2–6) bound to GST–unrip. Binding was specific, as this interaction was not observed with a GST-control protein (Fig. 5A, lane 20). In addition, we detected a weak but reproducible binding of SmB, D1, D2, D3 and E to GST–unrip (Fig. 5A, lane 25–39).



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  		Figure 5. Recruitment of unrip to the SMN complex is mediated by Gemin7. (A) Recombinant GST or GST–unrip fusion protein was immobilized on glutathione–Sepharose resin and incubated with the indicated in vitro translated 35S-labeled proteins. After extensive washing, the bound proteins were eluted and resolved by SDS–PAGE. The upper panel shows a autoradiography of the binding assay, the middle and lower panels show the Coomassie-stained gel of the corresponding experiment. The input lanes show 10% of the indicated, radiolabeled protein used in the experiment. (B) Recombinant GST (lane 2), GST–Gemin7 (lane 3) or a complex composed of GST–Gemin7 and His–Gemin6 (lane 4) was immobilized on glutathione-Sepharose resin and incubated with in vitro translated 35S-labeled unrip (10% of the labeled unrip used in this experiment is shown in lane 1). After washing, bound proteins were separated by SDS–PAGE and visualized either by Coomassie staining (upper panel) or by autoradiography (lower panel). (C) Direct binding of Gemin7 to unrip. GST–Gemin7 was expressed in E. coli either alone (lane 2) or in combination with His-tagged unrip (lane 4) and purified on a glutathione–Sepharose resin. As a control, His–unrip was co-expressed and purified with GST (lane 3). Lane 1 shows His–unrip purified on a nickel resin. Proteins were resolved by SDS–PAGE and visualized by Coomassie staining.

 
To verify the interaction between unrip and Gemin7, GST–Gemin7 was expressed in Escherichia coli either alone or in a complex with His–Gemin6 and incubated with in vitro translated and 35S-labeled unrip. An efficient association was observed to both, GST–Gemin7 and the GST–Gemin7/His–Gemin6 complex, but not to GST–protein alone (Fig. 5B). This interaction is direct, rather than mediated by another factor of the reticulocyte lysate, as a complex composed of GST–Gemin7 and His–unrip can be co-purified from bacteria using glutathione–Sepharose (Fig. 5C). To further analyse binding of unrip to Sm proteins, recombinant Sm hetero-oligomers B/D3, D1/D2 and F/E/G were immobilized to CNBr-activated Sepharose and incubated with in vitro translated, 35S-labeled unrip. In this assay, we failed to detect significant binding of unrip, leaving open whether these interactions contribute to the incorporation of unrip into the SMN complex (data not shown).

To further characterize the mode of interaction between unrip and Gemin7, in vitro binding assays with truncation mutants of both proteins were performed (Fig. 6A). Initially, we wished to determine the sequence within Gemin7 that mediates binding to unrip. As shown in Figure 6B (lane 3 and 4), recombinant GST–Gemin7 and a truncation consisting of the first 56 amino acids (Gemin71–56) bound specifically to full-length unrip, whereas a C-terminal fragment (Gemin757–131) (lane 5) failed to bind. Therefore, the N-terminus of Gemin7 is necessary and sufficient for binding to unrip. These results were confirmed by in vitro binding assays using recombinant unrip as bait and in vitro translated Gemin7 as prey. As shown in Figure 6B, a GST–fusion of full-length unrip and a C-terminal fragment encompassing amino acids 195–350 (unrip195–350) bound efficiently to Gemin7 and Gemin71–56 (lanes 8, 10, 13 and 15). In contrast, a fragment containing the putative WD-repeats failed to bind in the same assay (lanes 9 and 14). The C-terminal fragment of Gemin7 (Gemin757–131) did not bind any of the unrip truncations (lanes 18-20). Next, we asked whether the very C-terminal tail (amino acids 295–350) of unrip is sufficient for binding to Gemin7. In vitro binding assays using unrip295–350 showed that this truncation mutant failed to bind to any Gemin7-construct (Fig. 6B, lanes 25, 30 and 35). Thus, neither the putative WD-repeat (unrip1–295) nor the C-terminal tail of unrip (unrip295–350) alone is sufficient for binding to Gemin7.



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  		Figure 6. Identification of domains involved in the Gemin7–unrip interaction. (A) Schematic presentation of the truncations of Gemin7 and unrip used for the in vitro binding assays in (B). Hatched areas mark the putative interacting regions between the two proteins. (B) Recombinant GST (lanes 2, 7, 12, 17, 22, 27 and 32), GST–fusions of Gemin7 (lanes 3–5) and GST–fusions of unrip (lanes 8–10, 13–15, 18–20, 23–25, 28–30 and 33–35) were immobilized on glutathione–Sepharose resin and incubated with the indicated in vitro translated 35S-labeled proteins. After extensive washing, the bound proteins were eluted, resolved by SDS–PAGE and visualized by autoradiography. The input lanes (1, 6, 11, 16, 21, 26 and 31) show 10% of the indicated radiolabeled protein used in each experiment.

 
We therefore conclude that unrip is recruited to the SMN complex, predominantly if not exclusively by an association with Gemin7. The domains involved in this interaction are the N-terminal 56 amino acids of Gemin7 and the C-terminal part of unrip encompassing amino acids 195–350.

Gemin7 and unr compete for binding to unrip
Our biochemical work clearly indicates that unrip interacts with the SMN complex via Gemin7 and is also associated with unr (26Go). Therefore, the absence of unr in purified SMN complexes raised the question whether the interaction of unrip with unr prevents its association with the SMN complex and vice versa. This was assessed by in vitro binding assays using immunopurified unrip. For this, unrip immunoprecipitated from HeLa cytosolic extract was incubated with either 35S-labeled Gemin7 (Fig. 7A, upper panel) or unr (lower panel). As a control, unrip pre-immune serum was used. After extensive washing, bound proteins were eluted, resolved by SDS–PAGE and detected by autoradiography. As shown in Figure 7A, both Gemin7 and unr, i.e. the in vivo binding partners of unrip, bound efficiently to the unrip-immunoprecipitate but not to the control (compare lanes 2 and 3). We then asked whether the interaction of unr with unrip can be competed by an excess of a purified recombinant Gemin6/7 complex (the heterodimer was used rather than Gemin7 alone as the latter tends to aggregate and may hence associate non-specifically with unrip in this assay). Indeed, the interaction of unr to unrip could be efficiently prevented upon addition of rising amounts of the Gemin6/7 heterodimer to the binding assay (Fig. 7B, lanes 2–6). Bovine serum albumin (BSA) as a control in similar amounts had no effect (lane 7).



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  		Figure 7. Interaction of unrip with Gemin7 impairs binding of unrip to unr. (A) Unrip was affinity-purified from cytosolic HeLa extract. Subsequently, in vitro translated 35S-labeled Gemin7 (upper panel) or unr (lower panel) was added, and binding of labeled proteins was analysed by autoradiography of the dried SDS–PAGE gel. As a control, pre-immune serum was used (lane 2). Lane 1 shows 10% of radiolabeled input Gemin7 and unr. (B) Immunoprecipitated unrip (~1 µg) from HeLa cytosolic extract was incubated with radiolabeled unr (0.3 fmol) and increasing concentrations (0, 13, 25, 38 and 50 ng/µl) of recombinant, purified Gemin7/Gemin6 complex (upper panel, lanes 2–6). The resin subsequently was washed, proteins were eluted from the matrix, resolved by SDS–PAGE and bound unr was detected by autoradiography. As a control, 50 ng/µl BSA was used as competitor protein (lane 7). In the lower panel, immunoprecipitated unrip (~1 µg) was incubated with radiolabeled Gemin7 (2.4 fmol) and increasing concentrations (0, 13, 25, 50 and 125 ng/µl) of recombinant GST-unr (lanes 9–13). After washing of the resin and SDS–PAGE of the bound proteins, the 35S-labeled Gemin7 was detected by autoradiography. As a control, 125 ng/µl BSA was used as competitor protein (lane 14). Ten percent of 35S-labeled input used in the experiment is shown in lane 1 and 8, respectively.

 
Finally, we analysed whether recombinant unr could compete for the interaction between unrip and 35S-labeled Gemin7. Increasing amounts of recombinant unr gradually displaced Gemin7 from unrip (Fig. 7B, lower panel, lanes 9–13), although the observed competition was less efficient compared with the inverse experiment (Fig. 7B, upper panel, lanes 2–6). Thus, these in vitro binding studies indicate that binding of unrip to either unr or Gemin7 is mutually exclusive.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Biochemical studies have revealed a large number of factors that are associated with SMN in vivo (reviewed in 23Go). However, among these interactors, only a subset appears to be stably and stoichiometrically bound. These factors, collectively named ‘Gemins’, are functionally associated with the SMN complex, the entity that promotes the assembly of spliceosomal U snRNPs. Although the principal role of the SMN complex as a whole in the biogenesis of U snRNPs is well established, only limited information is available regarding the contribution of individual components to the assembly reaction. We have provided several lines of evidence here that unrip, a WD-repeat protein and interacting partner of the translation factor unr, is a major component of the SMN complex. Firstly by immunoprecipitation with monospecific anti-unrip antibodies, we recover not only unr, but also the SMN complex. Consistent with our previous data (10Go), isolated SMN complex contains significant levels of unrip. Secondly, depletion of unrip from cytosolic extract abolishes its competence to promote formation of U snRNP particles. Furthermore, this procedure simultaneously co-depletes other factors of the assembly machinery, such as SMN, Gemin2 and 3. Thirdly, we identify Gemin7, a known component of the SMN complex (31Go), as the primary and direct binding partner for unrip. In fact, association of unrip with the SMN complex is stable up to 1.5 M NaCl or 0.3 M KCl/ 0.5 M NaSCN, suggesting high affinity binding in the cytosol. These data strongly argue that unrip is recruited to the SMN complex via an interaction with Gemin7. Our findings are in good agreement with a recent report published by Pellizzoni- and co-workers (32Go) providing evidence for a interaction of unrip with the SMN complex. This study also reports binding of unrip to Sm proteins. Although we are also able to detect weak interactions between recombinant unrip and in vitro translated Sm proteins, immobilized recombinant Sm proteins heterodimers failed to interact with in vitro translated unrip in the same assay. It is therefore questionable whether the Sm proteins are in fact interaction partners of unrip in vivo.

We show that the interaction between Gemin7 and unrip is mediated via an N-terminal sequence element in Gemin7 that overlaps with a motif previously implicated in SMN-binding (33Go). Given that unrip is a stoichiometric component of the cytosolic SMN complex, one could imagine that unrip and SMN can bind simultaneously to Gemin7. We have attempted to test this possibility, but failed to see a significant binding of SMN to Gemin7 and the Gemin6/7 dimer. Therefore, further studies will be required to determine the mode of interaction between these factors.

The stable association of unrip with the SMN complex prevented selective depletion of this protein from extracts without simultaneously reducing the levels of other assembly factors. To circumvent this problem, we employed RNAi to specifically reduce unrip expression without affecting other components of the SMN complex. Extracts derived from these cells assembled U1 snRNPs as efficiently as the control extract (Fig. 4B). Although these in vitro studies suggest that unrip is not essentially required for the assembly reaction per se, the consequence of unrip-depletion in vivo is currently less clear. To address this question, an experimental setup to study the biogenesis of U snRNPs in vivo is highly demanded. Such a test system has been established in Xenopus laevis oocytes, but an equivalent one in somatic cells is currently not available. The development of this tool will be a major technical challenge for future studies on the in vivo function of the SMN complex.

A hallmark of the SMN complex is its localization not only in the cytoplasm but also in the nuclear gems/Cajal bodies. Although the localization of the SMN complex in the cytoplasm correlates well with its function in U snRNP assembly, it is currently unclear why this complex can also be detected in the nucleus. It is hypothesized that the SMN complex accompanies the assembled U snRNP to the nucleus where both entities dissociate. Gems, structures which often overlap with Cajal bodies (also termed coiled bodies), may be sites where U snRNPs and SMN complex are separated and the latter is re-directed to the cytoplasm (21Go,34Go). The predominant localization of unrip to the cytoplasm could indicate that it either dissociates from the SMN complex prior to nuclear import or rapidly returns to the cytoplasm after import along with the SMN complex. Thus, unrip may participate in the activation of the SMN complex after its return from the nucleus. Alternatively and not mutually exclusive with the first scenario, unrip may help to retard the SMN complex in the cytoplasm while the assembly reaction is in progress. Consistent with the latter model is our observation that RNAi-induced reduction of unrip appears not to interfere with the assembly process per se. Rather, it leads to an increase in the nuclear pool of the SMN complex, indicating that the presence of unrip shifts the steady-state distribution of the SMN complex to the cytoplasm. Further studies are required to address how unrip influences the subcellular trafficking of the SMN complex.

Our biochemical work, presented here, shows that only a fraction of the endogenous unrip is associated with the SMN complex, whereas the rest forms the previously described unr/unrip dimer. In functional assays, this dimer has been linked to the cap-independent translation of cellular and viral mRNAs containing internal ribosome entry sites (IRES) (26Go,27Go). The role of unrip in this scenario currently remains unclear. Our finding that the binding of unrip to unr and Gemin7 is mutually exclusive argues that overlapping regions of unrip are recognized by both proteins. Understanding the role of unrip in the context of U snRNP assembly may thus provide useful insights into its function in cap-independent translation and vice versa. In conclusion, our identification of unrip as a major, yet atypical, component of the SMN complex has set the stage for detailed examinations of both SMN-mediated U snRNP assembly and IRES-driven translation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
DNA constructs
Full-length cDNAs corresponding to the open-reading frames of unrip, unr (mRNA variant 1), Gemin6 and Gemin7 were amplified from a human brain Marathon cDNA library (Clontech). For in vitro translation and expression of recombinant proteins, unrip, unr, Gemin6 and Gemin7 were subcloned into pET28a vector (Novagen). For expression of GST–fusion proteins unrip and Gemin7 were subcloned into pGEX6P-1 (Amersham). Truncations of unrip and Gemin7 were generated with specific primers and subcloned into pGEX6P-1 or pET28a vector, respectively. Plasmids encoding the Sm proteins B, D1, D2, D3, E, F and G, SMN and Gemin2–5 have been described previously (10Go,28Go,35Go,36Go).

Recombinant proteins and in vitro protein binding assays
Full-length human Gemin6, Gemin7, unrip and truncated versions thereof were expressed as GST–fusion proteins in E. coli Rosetta(DE3) pLysS (Novagen). E. coli harboring the respective plasmids were cultured in superbroth medium. Protein expression was induced at mid-logarithmic phase by the addition of 1 mM IPTG. Expression was performed for 5 h at 16°C. Bacteria were harvested by centrifugation, resuspended in lysis buffer [25 mM NaCl, 20 mM Tris/HCl pH 8.0, 1 mM DTT, 0.01% IgePal and protease inhibitors Leupeptin, PepstatinA, Aprotonin (10 µg/ml each) and AEBSF (0.1 mM)] and lysed by sonication. GST–fusion proteins were purified on glutathione–Sepharose resin (Amersham) following the manufacturer's instructions. Recombinant GST–Gemin7/His-tagged-Gemin6 complex was obtained by co-expression of proteins in E. coli strain Rosetta(DE3) pLysS following procedures described earlier. The protein complex was purified by two consecutive affinity purification steps on glutathione–Sepharose as the first step and Pro-bond nickel chelating resin (Invitrogen) as the second step. 35S-methionine-labeled proteins were produced using a TNT-T7 Quick Coupled Transcription/Translation System (Promega). In vitro translated proteins were incubated with ~ 2µg purified GST–fusion proteins, immobilized on a glutathione–Sepharose resin (Amersham) and allowed to bind in lysis buffer at 4°C for 1 h. After washing the resin three times with lysis buffer, bound proteins were eluted by boiling in 2x SDS sample buffer, resolved by SDS–PAGE and analysed by Coomassie staining. Labeled proteins were detected by autoradiography of the dried gel.

Immunofluorescence microscopy
HeLa cells were grown on cover slides in DMEM/10% FCS and were washed once in phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde for 7 min and permeabilized in 0.2% Triton X-100/PBS for 5 min on ice. After blocking with 2% BSA in PBS, cells were incubated for 1 h with monoclonal mouse anti-SMN antibody 7B10 or affinity-purified polyclonal rabbit unrip-antibody. As secondary antibody, fluorescein- or rhodamine-conjugated secondary anti-mouse or anti-rabbit antibodies were used and analysed with a 63x oil immersion lens on a Zeiss Axiovert 200M microscope. Localization of overexpressed, HA-tagged SMN was analysed with a mouse monoclonal HA-antibody (clone 16B12, Babco) and a secondary rhodamine anti-mouse antibody. SMN and Gemin2 double-labeling experiments were performed using affinity-purified rabbit anti-SMN and mouse monoclonal anti-Gemin2 antibody (clone4, BD Transduction Laboratories) as primary antibodies. For SMN and unrip co-immunofluorescence, monoclonal SMN 7B10 and affinity-purified rabbit unrip antibody were used. Secondary antibodies were rhodamine-labeled anti-rabbit and fluorescein-labeled anti-mouse antibodies, respectively.

Preparation of HeLa cell extracts and antibody production
To obtain cytosolic extract, active in U snRNP assembly, 20l cultured suspension HeLa cells were harvested by centrifugation for 5 min at 1.500 g and washed once in PBS. The cell pellet (~15 g) was resuspended in 2.5 volumes of PBS containing 0.01% IgePal. Cells were lysed in a Dounce homogenizer, S (B. Braun) by 20 strokes. Cytosolic extract was obtained by centrifugation in a swing out rotor at 17.000 g for 10 min. Complete clarification of the extract was achieved by filtration through a 0.45 µm low protein binding filter. Cytosolic and nuclear extracts were prepared following a protocol of Dignam et al. (37Go). The separation of nuclear or cytosolic extract of siRNA-treated cells was achieved with a Qproteome Cell Compartment Kit (Qiagen) according to the manufacturer's protocol. Compartment separation was analysed by immunodetection of cytoplasmic marker protein {alpha}Tubulin (anti-{alpha}Tubulin antibody, clone B5-1-1, Sigma) and Histone H4 as a nuclear marker protein (anti-Histone H4 antibody, Cell Signaling Technology), respectively.

Antibodies against unrip and unr were raised by injection of recombinant full-length human proteins into rabbits. Antibodies were affinity-purified on columns with the respective covalently linked antigen. Immunoprecipitation and detection of SMN in western blots were performed with the mouse monoclonal antibody 7B10 (28Go).

Immunoprecipitations and immunodepletions
Immunoprecipitations and immunodepletions were carried out in HeLa cytosolic extract, using antibodies covalently linked to protein A–Sepharose. To attempt dissociation of unrip from the SMN complex, immunodepletion was carried out in extracts either brought to 1.5 M NaCl or to 0.3 M KCl/0.5 M NaSCN. For comparison, untreated extract was diluted with PBS to the final resulting volume.

RNA interference
Unrip levels were reduced by transfection of a mixture of two double-stranded 21 nt long siRNA (sequences: 5'-AAACUGUUACGCAUAUAUGACTT-3' and 5'-AACUUAUGGACGAUCUAUUGCTT-3', purchased from IBA Nucleic Acids Synthesis, Göttingen) with OligofectamineTM (Invitrogen) following the protocol of the manufacturer. Silencing of unrip was assayed by western blotting of cell extracts, 48, 72, 96 and 120 h after transfection, using a monospecific unrip antiserum. Transient transfection of adherent HeLa cells with a N-terminally HA-tagged SMN (subcloned in pcDNA3.1 vector, Invitrogen) was carried out with EffecteneTM (Qiagen) according to the protocol of the manufacturer and analysed 48 h after transfection.

In vitro assembly of U snRNPs
To analyse U snRNP assembly in vitro, 3 µl HeLa cytosolic extract (containing ~10 mg/ml protein) or immunoprecipitated SMN complex were incubated at 37°C for 45 min with 25 fmol 32P-U1 RNA, 2 µg tRNA, 1 mM ATP and 1 µl RNasin in a final volume of 20 µl. The reactions were analysed by native gel electrophoresis as described previously (10Go). Analysis of the assembly activity of unrip-silenced cells was carried out with cytosolic extract prepared with a Qproteome Cell Compartment Kit (Qiagen) according to the manufacturer's protocol. Assembly reaction was performed as described earlier.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We are indebted to R. Lührmann and J. Steitz for providing reagents and E. Dinkl for technical help. This work was supported by grants of the German Research Foundation (DFG, FOR426 and SFB 581) and families of SMA (fsma).

Conflict of Interest statement: Authors have declared no conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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