FEBS Letters 579 (2005) 2348–2354 FEBS 29461

Unrip is a component of SMN complexes active in snRNP assembly

Claudia Carissimia, Jennifer Baccona,b, Marco Stracciaa, Pieranna Chiarellac, Alessio Maiolicad

Alan Sawyerc, Juri Rappsilberd, Livio Pellizzonia,*

a Dulbecco Telethon Institute, Institute of Cell Biology (CNR), 00016 Monterotondo Scalo, Rome, Italyb University of Pennsylvania School of Medicine, 19104 Philadelphia, USA

c EMBL Monoclonal Antibody Core Facility, 00016 Monterotondo Scalo, Rome, Italyd The FIRC Institute for Molecular Oncology Foundation, 20139 Milan, Italy

Received 8 March 2005; accepted 13 March 2005

Available online 25 March 2005

Edited by Gianni Cesareni

Abstract A macromolecular complex containing survival ofmotor neurons (SMN), the spinal muscular atrophy protein,and Gemin2–7 interacts with Sm proteins and snRNAs to carryout the assembly of these components into spliceosomal small nu-clear ribonucleoproteins (snRNPs). Here we report the charac-terization of unr-interacting protein (unrip), a GH-WD proteinof unknown function, as a component of the SMN complex thatinteracts directly with Gemin6 and Gemin7. Unrip also binds asubset of Sm proteins, and unrip-containing SMN complexesare necessary and sufficient to mediate the assembly of spliceos-omal snRNPs. These results demonstrate that unrip functions inthe pathway of snRNP biogenesis and is a marker of cellularSMN complexes active in snRNP assembly.� 2005 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.

Keywords: Survival of motor neurons; Spinal muscularatrophy; Small nuclear ribonucleoprotein; unr-interactingprotein

1. Introduction

Spliceosomal small nuclear ribonucleoproteins (snRNPs)

are the essential ribonucleoprotein particles that carry out

the removal of introns from precursor mRNAs in eukaryotes.

The major spliceosomal snRNPs consist of one snRNA mol-

ecule (U1, U2, U4/U6 or U5), a common set of seven Sm

proteins as well as additional snRNP-specific proteins [1].

The snRNP biogenesis pathway in higher eukaryotes has a

cytoplasmic as well as a nuclear phase and requires the func-

tion of two multiprotein complexes, the PRMT5 complex and

the survival of motor neurons (SMN) complex [2,3]. In the

cytoplasm, Sm proteins associate first with the PRMT5 com-

plex, which symmetrically dimethylates specific arginine resi-

dues in a subset of Sm proteins to increase their affinity for

Abbreviations: SMN, survival of motor neurons; SMA, spinal muscu-lar atrophy; unr, upstream of N-ras; unrip, unr-interacting protein;snRNP, small nuclear ribonucleoproteins

*Corresponding author. Fax: +39 06 90091259.E-mail address: livio.pellizzoni@ibc.cnr.it (L. Pellizzoni).

0014-5793/$30.00 � 2005 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2005.03.034

SMN, and afterward with the SMN complex [4–7]. The

SMN complex bound to the Sm proteins interacts with newly

exported snRNAs and mediates the ATP-dependent forma-

tion of the Sm core, an heptameric ring of Sm proteins,

around a single stranded region of the snRNA called the

Sm site [8–13]. Following hypermethylation of the 5 0 cap

and shortening of the 3 0 end, newly assembled snRNPs are

imported in the nucleus where they function in pre-mRNA

splicing [1].

The SMN complex is a large macromolecular machine con-

taining SMN and six additional integral components, called

Gemin proteins (Gemin2–7), which are held together by a

network of protein–protein interactions [3]. The SMN com-

plex interacts directly and independently with specific

domains of both Sm proteins and snRNAs and ensures their

efficient association into snRNPs [2,3]. The SMN complex

functions as a chaperone of ribonucleoprotein particles by

imposing a stringent control on the specificity of the assembly

process [11].

In addition to Sm proteins and snRNAs, an increasing

number of proteins have been shown to interact with the

SMN complex either in vivo, in vitro or both [2,14]. Many

of these proteins are believed to associate transiently with

subsets of the SMN complex, yet in most cases their inter-

action with SMN has not been adequately characterized

either biochemically or functionally. It is therefore unclear

whether they represent integral components of the SMN

complex or targets of its activity. The need for a detailed

characterization of the full spectrum of SMN interactions

goes beyond its relevance for understanding the pathway

of snRNP biogenesis and extends into the ongoing search

for the pathogenic mechanism behind the devastating neuro-

muscular disease spinal muscular atrophy (SMA), which

results from homozygous deletions or mutations in the

SMN1 gene [15].

Here we have generated a monoclonal antibody against

native SMN complexes and demonstrate, using both

co-immunoprecipitation experiments and analysis by mass

spectrometry, that unr-interacting protein (unrip) is a com-

ponent of the SMN complex. In vitro binding experiments

indicate that Gemin6 and Gemin7 bind directly to unrip

and bring it into the SMN complex. Furthermore, we show

that unrip also interacts with a subset of Sm proteins, and

unrip-containing SMN complexes mediate snRNP assembly.

blished by Elsevier B.V. All rights reserved.

C. Carissimi et al. / FEBS Letters 579 (2005) 2348–2354 2349

Our findings reveal that unrip functions in the pathway of

snRNP biogenesis and is a marker of the active pool of

SMN complexes.

2. Materials and methods

2.1. DNA constructs and antibodiesThe pcDNA3 plasmids containing myc-tagged SMN, Gemin2–7 and

Sm proteins as well as HA-tagged unrip were described previously[9,16–21]. To generate GST- and His-tagged unrip fusion proteins,the unrip open reading frame was excised from the EcoRI and XhoIsites of pcDNA3 and cloned into the same sites of pGEX 5X andPET 28 vectors, respectively. The antibodies used were anti-unrip (rab-bit polyclonal), anti-SMN clone 8 (BD Transduction Laboratories),anti-Gemin2 2E17 [19], anti-Gemin3 12H12 [17], anti-Gemin4 17D10[18], anti-Gemin5 10G11 [21], anti-Gemin6 [13], anti-pICln clone 32(BD Transduction Laboratories), anti-coilin clone 56 (BD Transduc-tion Laboratories), anti-tubulin (Sigma), and purified mouse and rab-bit immunoglobulins (Sigma).

Fig. 1. 7F3 is a novel anti-SMN monoclonal antibody. (A) Silver stainingtagged SMN (SMN) and of non-specific proteins from parental HeLa cells (vitro translated, [35S]methionine-labeled, SMN and Gemin2–7 (GMN2–7) pr1% empigen. 10% of the input (in vitro translation) and immunoprecipitat[35S]methionine-labeled HeLa extracts were immunoprecipitated with 7F3 aempigen and analyzed by SDS–PAGE and autoradiography. (D) Indirect imcells double labeled using 7F3 and anti-coilin antibodies.

2.2. Antibody productionOne Balb/c female was primed with Immuneasy adjuvant (Qiagen)

and native SMN complexes (40 lg) purified from the HeLa flag-SMN cell line as described previously [20], and was boosted twiceat 2 week intervals. Following fusion of SP2 myeloma cells withmouse splenocytes, hybridoma supernatants were spotted onto ami-nosilane modified slides coated with SMN complexes, or non-specificproteins as a control (see Fig. 1A), and analyzed using a LS400Scanner (Tecan) and the GenePix Pro 4.1 software as described pre-viously [22].

2.3. Protein sequencing by mass spectrometryBands were excised from silver-stained polyacrylamide gels and in-

gel digested with trypsin. Tryptic peptides were analyzed by nano-elec-trospray tandem mass spectrometry as described previously [23].

2.4. Cell culture and immunofluorescence analysisHeLa cells were cultured as described previously [20]. For in vivo

labeling of proteins, HeLa cells were incubated overnight with100 lCi of [35S]methionine in DMEM without methionine supple-mented with 10% FBS. Double-label indirect immunofluorescence

analysis of SMN complexes purified from HeLa cells expressing flag-control) [20]. Known SMN complex components are indicated. (B) Inoteins were immunoprecipitated with 7F3 antibodies in the presence ofes (7F3 IP) were analyzed by SDS–PAGE and autoradiography. (C)ntibody or mouse immunoglobulins (control) in the presence of 1%munofluorescence analysis by confocal laser microscopy of HeLa PV

2350 C. Carissimi et al. / FEBS Letters 579 (2005) 2348–2354

experiments were carried out on HeLa PV cells plated on glass cover-slips and images were collected with a Leica TCS 4D confocal micro-scope as described previously [17].

2.5. Protein production and in vitro binding experimentsIn vitro translated proteins were produced in the presence of

[35S]methionine (Amersham) using a coupled transcription–translationsystem according to the manufacturer�s protocol (Promega). His-tagged unrip and GST-tagged fusion proteins were expressed in Esch-erichia coli BL21(DE)pLys cells (Invitrogen) and purified, respectively,using nickel chelation chromatography (Novagen) or glutathione–Se-pharose (Amersham) according to the manufacturer�s protocols. Forin vitro binding experiments, 3 lg of GST or GST-tagged proteinsbound to glutathione–Sepharose beads were incubated either with invitro translated [35S]methionine-labeled proteins or with 1 lg of puri-fied His-tagged unrip in RSB-100 buffer (100 mM NaCl, 10 mMTris–HCl, pH 7.4, 2.5 mM MgCl2) containing 0.1% NP40, EDTA-freeprotease inhibitor cocktail (Roche) and phosphatase inhibitors(50 mM NaF, 0.2 mM Na3VO4) for 2 h at 4 �C. Following extensivewashing with the same buffer, bound proteins were eluted by boilingin SDS–PAGE sample buffer.

2.6. Sucrose gradient centrifugation and immunoprecipitation

experimentsHeLa extracts were prepared in RSB-100 buffer containing 0.1%

NP40 and protease and phosphatase inhibitors as previously described[20]. For sucrose gradient centrifugation experiments, HeLa extractswere centrifuged on 10 ml of 10–30% sucrose gradients in RSB-100for 4 h at 38000 rpm in a SW 41 rotor at 4 �C. Immunoprecipitationexperiments were carried out for two hours at 4 �C using antibodiesbound to protein G–Sepharose (Sigma) in RSB-100 buffer containing0.1% NP40 (or 1% empigen when indicated) and protease and phos-phatase inhibitors. Following five washes with the same buffer, boundproteins were eluted by boiling in SDS–PAGE sample buffer. For allexperiments, proteins were analyzed by SDS–PAGE on 12% polyacryl-amide gels.

2.7. In vitro assembly of snRNPsHeLa extract preparation, immunodepletions and snRNP assembly

reactions were performed as described previously [11]. Protein com-plexes were immunopurified from these extracts with anti-unrip oranti-SMN antibodies bound to protein G–Sepharose for one hour at4 �C in reconstitution buffer (20 mM HEPES–KOH, pH 7.9, 50 mMKCl, 5 mM MgCl2, 0.2 mM EDTA, and 5% glycerol) containing0.01% NP40 and washed five times with the same buffer. Assemblyreactions with HeLa extracts or beads-bound protein complexes werecarried out for one hour at 30�C and contained 10000 cpm of in vitrotranscribed [32P]UTP-labeled snRNA, 2.5 mM ATP and 10 lM E. colitRNA in a volume of 20 ll. After addition of heparin and urea to afinal concentration of 5 mg/ml and 2 M, respectively, samples wereanalyzed by electrophoresis on native gels and autoradiography.

3. Results

3.1. 7F3 is a novel anti-SMN monoclonal antibody

We generated a novel monoclonal antibody against SMN

(7F3) by immunizing mice with native SMN complexes puri-

fied from HeLa cells that express flag-tagged SMN (Fig. 1A)

[20]. 7F3 poorly recognizes denatured SMN on Western blot

(data not shown). To further characterize the specificity of

7F3, in vitro translated [35S]methionine-labeled SMN and Ge-

min2–7 proteins were analyzed by immunoprecipitation with

7F3 antibody under conditions that disrupt most protein–pro-

tein interactions. Fig. 1B shows that SMN is the only compo-

nent of the SMN complex specifically recognized by 7F3.

Under similar conditions, 7F3 specifically immunoprecipitates

a single protein corresponding to SMN from [35S]methionine-

labeled HeLa extracts (Fig. 1C). Mapping experiments indicate

that 7F3 recognizes a large epitope encoded by exon 2b and

exon 3 of SMN (data not shown). Furthermore, immunofluo-

rescence analysis of HeLa cells with 7F3 antibodies shows the

expected subcellular localization for SMN both in the cyto-

plasm and in nuclear Gems (Fig. 1D), which are found both

associated with and separate from Cajal bodies [24]. We con-

clude that 7F3 is a novel monoclonal antibody that recognizes

SMN in its native conformation.

3.2. Unrip and SMN are associated in vivo

Next, we analyzed by SDS–PAGE and silver staining the

protein composition of SMN complexes immunoprecipitated

with 7F3 from HeLa extracts (Fig. 2A). In addition to the

known integral components of the SMN complex, we found a

prominent 38 kDa protein that we identified by mass spectrom-

etry as unrip, a member of the GH-WD repeat protein family

[25]. Consistent with these results, unrip was found in SMN

immunoprecipitates from nuclear extracts of HeLa cells [9].

Unrip was originally identified in HeLa cells as an interacting

partner of unr, a cytoplasmic RNA binding protein involved

in the internal initiation of translation of viral RNAs, although

a direct role for unrip in translation has not been demonstrated

[25]. LC-MS/MS analysis indicated that the unr protein is not

present in purified SMN complexes (data not shown). We

sought to carry out the biochemical and functional character-

ization of unrip because its function is unknown and its rela-

tionship with the SMN complex remains elusive. Western

blot analysis shows that anti-unrip antibodies (a kind gift of

Utz Fischer) specifically recognize purified His-tagged unrip

and a single band of the expected size in HeLa extracts (Fig.

2B). To confirm that SMN and unrip associate in vivo, we per-

formed immunoprecipitation experiments from HeLa extracts.

Fig. 2C demonstrates that SMN and unrip efficiently and spe-

cifically coimmunoprecipitate each other. Gemin proteins also

are found in these immunoprecipitates (data not shown).

Importantly, unrip does not associate with pICln or other com-

ponents of the PRMT5 complex. These experiments demon-

strate that unrip and SMN are associated in vivo.

3.3. Unrip does not localize in Gems

SMN localizes in the cytoplasm and in the nucleus, where is

highly concentrated in Gems [24], and all the Gemin proteins

share a similar subcellular localization [16–21]. In light of its

association with SMN, we investigated the subcellular localiza-

tion of unrip in HeLa cells by immunofluorescence experi-

ments. Fig. 2D shows that unrip is predominantly localized

in the cytoplasm but also in the nucleus. Interestingly, how-

ever, unrip does not localize with SMN in Gems. Although

we cannot formally exclude that the epitope recognized by un-

rip antibodies is inaccessible in these nuclear structures, these

findings indicate that unrip interacts with a subset but not all

of cellular SMN.

3.4. Unrip is a component of the SMN complex

SMN and Gemin proteins are tightly associated in large

macromolecular complexes [14]. We further investigated

whether unrip is a component of these SMN complexes by

immunoprecipitation experiments. Western blot analysis of

HeLa extracts fractionated by sucrose gradient centrifugation

shows that unrip, SMN and Gemin proteins are broadly dis-

tributed and partially overlap between 20S and 80S (Fig.

3A). To identify which complexes contained SMN and unrip,

Fig. 2. Unrip is associated with SMN. (A) SDS–PAGE and silver staining analysis of immunoprecipitates with 7F3 antibodies or mouseimmunoglobulins (control) from HeLa extracts. Known protein components are indicated. Asterisks mark immunoglobulin chains. (B) HeLa extractand purified His-tagged unrip were analyzed by SDS–PAGE and Western blot with anti-unrip antibodies. (C) HeLa extracts wereimmunoprecipitated using mouse immunoglobulins (control) or antibodies against unrip, SMN and pICln proteins. 5% of the input (HeLa) andthe immunoprecipitates (IP) were analyzed by SDS–PAGE and Western blot with antibodies against the proteins indicated on the right. (D) Indirectimmunofluorescence analysis by confocal laser microscopy of HeLa PV cells double labeled using 7F3 and anti-unrip antibodies.

C. Carissimi et al. / FEBS Letters 579 (2005) 2348–2354 2351

each fraction was immunoprecipitated using 7F3 antibodies.

Western blot analysis of immunoprecipitates demonstrates

that unrip is associated with the macromolecular complexes

containing SMN and Gemin proteins (Fig. 3B). Noticeably,

a large proportion of unrip and Gemin3–6 proteins that frac-

tionate in complexes smaller then 30S are not associated with

SMN and represent distinct pools of these proteins. These re-

sults identify unrip as a component of SMN complexes.

3.5. Unrip interacts directly with Gemin6 and Gemin7

To identify the interactions responsible for unrip association

with the SMN complex, we performed in vitro binding exper-

iments. We incubated in vitro translated [35S]methionine-la-

beled HA-unrip with several SMN complex components

fused to GST. Fig. 4A shows that unrip binds to Gemin7 very

efficiently and to Gemin6 weakly. No interaction was observed

with Gemin3, Gemin4 and Gemin5 (data not shown). To ex-

clude the possibility that these interactions were mediated by

components of the reticulocyte lysate, we performed direct in

vitro binding experiments with purified recombinant proteins.

Fig. 4B demonstrates that unrip interacts directly and specifi-

cally with both Gemin6 and Gemin7. These results indicate

that Gemin6 and Gemin7 mediate unrip association with the

SMN complex.

3.6. Unrip-containing SMN complexes mediate snRNP

assembly

Next, we investigated if unrip interacts with Sm proteins, the

best-characterized targets of SMN complex function [2,3]. To

do so, in vitro translated [35S]methionine-labeled Sm proteins

were incubated with GST-unrip or GST as a control. Fig.

4C shows that unrip interacts efficiently with SmB, SmD2

and SmD3, and weakly with SmE. The interaction with Sm

proteins suggests that unrip may function in snRNP assembly.

To test this possibility, we carried out immunodepletetion

experiments from HeLa extracts competent for snRNP assem-

bly with antibodies against unrip, SMN or rabbit immuno-

globulins as a control. Western blot analysis shows the

Fig. 3. Unrip is a component of the SMN complex. HeLa extract wasanalyzed by centrifugation on a 10–30% sucrose gradient. (A)Fractions were analyzed by SDS–PAGE and Western blot withantibodies against the proteins indicated on the right. (B) Fractionswere analyzed by immunoprecipitation with 7F3 antibodies and boundproteins analyzed as in (A). Sedimentation (S) values are indicated.

2352 C. Carissimi et al. / FEBS Letters 579 (2005) 2348–2354

specific and efficient depletion of unrip and SMN with their

respective antibodies (Fig. 5A). Consistent with their associa-

tion as well as the presence in distinct complexes, unrip deple-

tion reduces SMN levels by approximately half in extracts

whereas most unrip is still present in SMN-depleted extracts.

We then analyzed these extracts by snRNP assembly and

found that unrip depletion impairs Sm core formation (Fig.

5C), indicating that unrip-containing SMN complexes are nec-

essary for efficient assembly of snRNPs.

Previous studies have demonstrated that purified SMN

complexes containing Sm proteins mediate Sm core assembly

in the absence of extracts [10,11]. To find out if unrip-asso-

ciated SMN complexes are sufficient for snRNP assembly,

we isolated unrip- and SMN-containing protein complexes

by immunoprecipitation from HeLa extracts. Mock immu-

noprecipitations with rabbit immunoglobulins served as a

control. Western blot analysis demonstrates the presence of

unrip and SMN in both immunoprecipitates (Fig. 5B).

Importantly, these purified protein complexes are competent

to carry out the Sm core formation on U1 but not on

U1DSm (Fig. 5D), an snRNA that bears a mutated Sm site

and is used as a control of specificity. Collectively, these re-

sults indicate that unrip-containing SMN complexes are

both necessary and sufficient for the efficient assembly of

spliceosomal snRNPs.

4. Discussion

Dissecting out the process of snRNP assembly has been an

ongoing interest in the fields of RNA processing and, more re-

cently, of SMA since the identification of SMN as the SMA

disease gene product. Characterization of all the components

of the SMN complex is necessary for understanding its func-

tion at the molecular level and, possibly, to uncover the molec-

ular defect of SMA. Fig. 6 shows a schematic representation of

the SMN complex that takes into account previous and pres-

ent findings. It represents an arbitrary simplification, as the ex-

act stoichiometry and spatial organization of the individual

components are unknown. SMN forms high order oligomers,

a feature impaired in SMA [26,27], and interacts directly with

Gemin2, Gemin3, Gemin5 and Gemin7 [16,17,19,21]. Gemin3

and Gemin7 mediate the interactions of Gemin4 and Gemin6,

respectively [16,18]. Here we have shown that unrip is brought

to the SMN complex by the direct interaction with Gemin6

and Gemin7.

We have demonstrated that unrip is a component of the

SMN complex. Unrip association with SMN is very stable

and, similar to that of Gemin proteins, withstands high salt

concentrations known to dissociate most SMN interactions

with its targets (data not shown) [16,20]. However, immun-

odepletion experiments and lack of unrip staining in Gems

indicated that unrip and SMN are also found separately,

thereby revealing that unrip association with SMN is

restricted to a subset of SMN complexes. Importantly,

unrip-containing SMN complexes are those necessary and

sufficient for Sm core formation in vitro. Therefore, unrip

may be considered as a marker of SMN complexes active in

snRNP assembly.

Consistent with its role in snRNP assembly, and similar to

most SMN complex components [2,3,14], unrip interacts with

a specific subset of Sm proteins and may provide additional

contacts for the recruitment of Sm proteins to the SMN com-

plex. Interestingly, unrip bears similarity to the 36 kDa com-

ponent of the eIF3 multisubunit complex that is critical for

stabilizing the eIF3 structure [25]. Unrip may function simi-

larly as a cofactor of the SMN complex by supporting the

structural organization and/or conformational changes likely

associated with the formation of the Sm core on the Sm site.

Although unrip does not localize in Gems, immunoprecipita-

tion experiments from cytoplasmic and nuclear fractions indi-

cated that unrip and SMN are associated in both subcellular

compartments of HeLa cells (data not shown). Since the

SMN complex accompanies snRNPs throughout their biogen-

esis and plays a direct role in their nuclear import [28], one

possibility is that unrip detachment occurs after nuclear import

and facilitates the dissociation of the SMN complex and

snRNPs.

Fig. 4. Unrip interacts with Gemin6, Gemin7 and a subset of Sm proteins. (A) In vitro translated [35S]methionine-labeled unrip was incubated eitherwith GST or the indicated GST-fusion proteins immobilized on beads. The input (10%) and bound unrip were analyzed by SDS–PAGE andautoradiography. (B) Purified His-tagged unrip was incubated with GST, GST-Gemin6 or GST-Gemin7 immobilized on beads. The input (5%) andbound unrip were analyzed by SDS–PAGE and Western blot. (C) In vitro translated [35S]methionine-labeled Sm proteins were incubated with GSTor GST-unrip immobilized on beads. The input (10%) and bound proteins were analyzed by SDS–PAGE and autoradiography.

Fig. 5. Unrip-containing SMN complexes mediate snRNP assembly.(A) Western blot analysis of HeLa extracts depleted with anti-unrip(Dunrip) or anti-SMN (DSMN) antibodies and rabbit immunoglobu-lins (mock). (B) Western blot analysis of protein complexes immuno-purified from HeLa extracts with anti-unrip, anti-SMN and rabbitIgGs (mock). (C) The extracts in A were used for snRNP assemblyreactions with radioactive U1, electrophoresis on native gels andautoradiography. (D) Beads-bound protein complexes as in B wereused for snRNP assembly reactions with radioactive U1 or U1DSm,electrophoresis on native gels and autoradiography. RNP complexescontaining Sm proteins (Sm, snRNP) or the U1A protein areindicated.

Fig. 6. Unrip is a component of the SMN complex. A schematicrepresentation of the SMN complex depicts its known components andtheir reciprocal interactions (see text for details).

C. Carissimi et al. / FEBS Letters 579 (2005) 2348–2354 2353

Acknowledgments:We are indebted to Drs. G. Dreyfuss and U. Fischerfor providing several reagents and antibodies. We thank Drs. F. Gaba-nella and P. Amatucci for their help. This work was supported by Tele-thon-Italy (TCP 02011). J.B. is an MSTP scholar. L.P. is an EMBOYoung Investigator and an Assistant Telethon Scientist.

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