UNCL, the mammalian homologue of UNC-50, is an inner nuclear membrane RNA-binding protein

Brain Research 877 (2000) 110–123www.elsevier.com/ locate /bres

Interactive report

UNCL, the mammalian homologue of UNC-50, is an inner nuclear1membrane RNA-binding protein

a,d , b c c*Jamie Fitzgerald , Derek Kennedy , Nareerat Viseshakul , Bruce N. Cohen ,b a dJohn Mattick , John F. Bateman , John R. Forsayeth

aMurdoch Childrens Research Institute, and Department of Paediatrics, University of Melbourne, Royal Children’s Hospital, Parkville 3052,Australia

bThe Centre for Molecular and Cellular Biology, and Department of Biochemistry, University of Queensland, Queensland 4072, AustraliacDivision of Biomedical Sciences, University of California Riverside, Riverside, CA 92521, USA

dDepartment of Anesthesia, University of California San Francisco, San Francisco, CA 94143-0542, USA

Accepted 19 July 2000

Abstract

We isolated a mammalian homologue of the C. elegans gene unc-50 that we have named UNCL. The 777 kb rat UNCL cDNA encodesa 259 amino acid protein that is expressed in a wide variety of tissues with highest mRNA levels in brain, kidney and testis. Hydropathyplot analysis and in vitro translation experiments with microsomal membranes indicate that UNCL is a transmembrane protein.Hemagglutinin tagged UNCL was stably transfected into SaOS-2 osteosarcoma cells and exhibited a nuclear rim staining pattern whichwas retained following extraction with 1% Triton X-100, suggesting that UNCL localizes to the inner nuclear membrane. UNCL-HA wasextractable in 350 mM NaCl, suggesting that UNCL is not associated with the nuclear matrix. Homopolymer RNA-binding assaysperformed on in vitro translated UNCL protein and ‘structural modeling by homology’ suggest that UNCL binds RNA via anamino-terminal RNA Recognition-like Motif. Since unc-50 is required for expression of assembled muscle-type nicotinic receptors in thenematode we investigated whether UNCL had a similar function for mammalian nicotinic receptors. When UNCL was co-expressed withneural nicotinic receptors in Xenopus oocytes or COS cells it increased expression of functional cell surface receptors up to 1.6-fold. Weconclude that UNCL is a novel inner nuclear membrane protein that associates with RNA and is involved in the cell-surface expression ofneuronal nicotinic receptors. UNCL plays a broader role because UNCL homologues are present in two yeast and a plant species, none ofwhich express nicotinic receptors and it is also found in tissues that lack nicotinic receptors. 2000 Elsevier Science B.V. All rightsreserved.

Theme: Neurotransmitters, modulators, transporters, and receptors

Topic: Acetylcholine receptors: nicotinic

Keywords: Neuronal nAChR; RNA-binding; Inner nuclear membrane; unc-50

1. Introduction transfected cells have suggested that the efficiency ofAChR assembly in non-muscle cells is much lower than in

The expression and assembly of nicotinic acetylcholine muscle cells. Moreover, certain genetic variants of thereceptors (nAChR) in muscle has been intensively studied C2C12 myoblast line differentiate into myotubes butfor a number of years [29,31,45,56]. Reconstitution experi- synthesize AChR at a rate 3–5% of that observed inments in both permanently [8,22] and transiently [30,31] wild-type myotubes [6,7]. Many of the AChR subunit

cDNAs cloned from the central nervous system can beexpressed in Xenopus oocytes, although few can be

1Published on the World Wide Web on 10 August 2000. expressed with any appreciable efficiency in a variety of*Corresponding author. Murdoch Childrens Research Institute, Royal

mammalian cells [24]. It appears that cell lines commonlyChildren’s Hospital, Rm S342, Flemington Rd, Parkville 3052, Victoria,used for transfection lack some property necessary forAustralia. Tel.: 161-3-9345-6263; fax: 161-3-9345-7997.

E-mail address: [email protected] (J. Fitzgerald). efficient assembly of some subtypes of nicotinic receptor.

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02692-5

J. Fitzgerald et al. / Brain Research 877 (2000) 110 –123 111

Our goal, therefore, was to identify proteins or factors that ency of 0.13SSC/0.1% SDS at 508C, and positive plaquesfunction to increase cell surface expression levels of rescreened until a pure clone obtained.nAChRs.

Some years ago, genetic variants of the nematode, C. 2.3. In vitro transcription and translationelegans, were isolated [10] that were resistant to theacetylcholinesterase inhibitor, levamisole [39]. A sub-class Cell-free translations were carried out in the presence orof these variants had mutations in loci that did not map to absence of canine microsomal membranes (Promega) usingacetylcholine receptor subunit genes. In animals homo- the TNT coupled transcription and translation systemzygous for recessive mutations in the unc-50 gene, muscle (Promega) according to the manufacturer’s instructions.acetylcholine receptors fail to assemble as demonstrated by Five hundred ng of UNCL and 100 ng of luciferase werethe lack of levamisole-binding activity detectable in ex- transcribed using T7 polymerase and 500 ng of G3BPtracts of the organism [38]. Unc-50 does not encode a cDNA was transcribed using T3 polymerase. In onenAChR subunit but it appears to be required for nAChR experiment the translated material was treated with al-assembly. kaline carbonate (pH 11.5) to disrupt microsomal mem-

We screened the Expressed Sequence Tag database branes [25]. Samples were centrifuged in a benchtop(dbEST) at the National Center for Biotechnology In- centrifuge at 14,000 rpm for 30 min at 48C and loaded onformation (NCBI) for mammalian homologues of unc-50. 13% SDS-PAGs separately as pellet and supernatant andSeveral human EST clones were identified that were subjected to fluorography.homologous to the unc-50 cDNA. A full-length rat cDNAwas isolated by library screening that we named UNCL.

2.4. Northern analysisOur results indicate that UNCL is an inner nuclearmembrane protein that binds RNA and is involved in the

Approximately 5 mg poly (A)-selected mRNA wassurface expression of some combinations of neuronal

electrophoresed on a formaldehyde gel, transferred to anAChR subunits.

nylon membrane [52] and probed with UNCL cDNA8labeled to a specific activity of 5310 cpm/mg by random-

priming. The membrane was allowed to hybridize for 24 h2. Materials and methods at 42–458C and washed to a stringency of 0.13SSC/0.1%

SDS at 508C.2.1. Expression vectors and cell lines

2.5. Cell extractionThe partial human UNCL clone, TEST1CO9, was

subcloned into the expression vector pMT23, a derivative Transfected SaOS-2 cells were grown to 80% con-of pMT2 [16]. GAP120 SH3-domain-binding protein fluency in six-well multi-chamber slides in DMEM con-

2(G3BP) is in pBluescript SK (Stratagene). A hemag- taining 10% fetal calf serum. Control cells were washedglutinin (HA) tag with the amino acid sequence with PBS and fixed directly in 4% paraformaldehyde inCYPYDVPDYASL was added to the C-terminus of UNCL PBS for 10 min on ice and permeabilized with 0.5% Triton(UNCL-HA) immediately prior to the stop codon by PCR. X-100 (TX-100) in PBS for 4 min on ice. The other cellsThe resulting PCR product was cloned into the expression were extracted in 1% TX-100 in PBS or 1% TX-100 plusvector pRC/CMV and stably transfected into SaOS-2 350 mM NaCl in PBS for 10 min. Following extraction,osteosarcoma cells using the Lipofectamine (Boehringer cells were washed twice with PBS and fixed in 4%Mannheim) reagent according to manufacturer’s instruc- paraformaldehyde.tions. Rat a4 and b2 nAChR subunit cDNAs were clonedinto pCDL-SRa296 [9,15,59] and human M1 muscarinic

2.6. Immunofluorescent stainingreceptor cDNA was in the expression vector, pSG5(Stratagene).

Fixed cells were blocked in 0.2% BSA in PBS for 1 h atroom temperature. Cell were then incubated with either

2.2. cDNA library screening anti-hemagglutinin monoclonal antibody, HA.11 (BabCO),or anti-KDEL monoclonal antibody (StressGen) diluted 1

5A lgt10 rat brain cDNA library (6310 clones) was in 150 in 0.2% BSA in PBS for 1 h at room temperature.screened according to Sambrook et al. [52], except that After three 10 min PBS washes, anti-mouse IgG conju-filters were prehybridized in Church buffer [12] at 658C for gated to FITC diluted 1 in 150 in PBS was added for 1 h at2 h. Two hundred ng of TEST1CO9, a 1283 bp fragment room temperature. Next cells were washed three times inof the partial human UNCL cDNA, was random-primer PBS, rinsed in distilled water, mounted in Vectashield

32 7labeled with [ P] dCTP to a specific activity of 1.3310 mounting medium containing DAPI, and examined using acpm/mg DNA [52]. The filters were washed to a string- Zeiss Axioplan 2 fluorescent microscope. Images were

112 J. Fitzgerald et al. / Brain Research 877 (2000) 110 –123

3processed with IPLab software v3.2.4 (Scientific Image UNCL cDNA. Determination of specific [ H]acetylcholineProcessing) on a G3 Macintosh computer. binding was performed by a filtration assay as described

[24]. The level of non-specific binding was determined by2.7. RNA-binding assay the addition to some assay tubes of 1 mM unlabeled

3nicotine. Specific [ H]acetylcholine binding was calculatedRNA homopolymers (ICN Pharmaceuticals) were cou- by subtracting the non-specific from the total binding.

pled to agarose-adipic acid hydrazide (Amersham Phar-macia Biotech.) according to the manufacturer’s instruc-tions. Translated UNCL protein was added to 100 ml of 3. Resultshomopolymer-agarose and 500 ml of BB binding buffer50

(50 mM NaCl; 50 mM Tris–HCl, pH 8.0; 0.02% (v/v) 3.1. Isolation of UNCL cDNANP-40) and rotated for 1 h at 48C. The binding reactionwas washed three times with binding buffer or buffer The C. elegans unc-50 cDNA sequence (Genbankcontaining 250 mM NaCl for 15 min each at 48C and accession number Z48055) was used to screen the dbESTeluted into loading buffer containing 2 M urea and 10 mM using the GENINFO (R) Experimental BLAST NetworkDTT at 658C for 10 min. Eluted proteins were electrophor- service [2] at the NCBI (http: / /www.ncbi.nlm.nih.gov/esed on a 13% SDS-PAG and subjected to fluorography. BLAST/ ). Two human expressed sequence tag cDNAFor quantitation of protein bound to RNA homopolymers, sequences (ESTs) with significant homology to unc-50dried gels were scanned using a Molecular Dynamics were identified (Z21289 and T19459). We obtained a 1283Storm phosphoimager and band intensity determined using bp partial human cDNA corresponding to EST Z21289ImageQuaNT v4.2 (Molecular Dynamics). which was designated TEST1CO9 and found by confirmat-

ory sequencing to be homologous to unc-50. In order to2.8. Structural modeling recover a full-length mammalian clone the TEST1CO9

cDNA was used as a probe to screen a rat brain cDNACo-ordinates were assigned from the crystal structure of library. Following three rounds of duplicate replica screen-

the U1A N-terminal RRM (U1A) [49] to the secondary ing, two candidate rat UNCL clones were identified. Thestructural regions of each protein according to the align- first clone, UNCL-1, was approximately 0.8 kb in size andment shown in Fig. 1A. All suitable loop structures were hence too small to contain the entire reading frame basedobtained by searching the Brookhaven Protein Databank on alignment of the unc-50 and the human UNCL se-for proteins containing loops of the same length joining quences. However, the second clone, UNCL-2, was 1.1 kbsimilar secondary structural units. The resultant crude in size, and large enough to contain the UNCL cDNA.model was then refined by energy minimization using UNCL-2 was amplified by high fidelity PCR with lgt10Biosym’s CVFF forcefield to a convergence criterion of primers and subcloned into pMT23. Sequencing of severalless than 1.0 kcal /molA (maximum derivative). The ac- clones revealed that UNCL-2 contained the entire UNCLcuracy and validity of this model was tested by the method coding region of 777 bp, 162 bp of 39 untranslated regionof Eisenberg [18,42]. and 133 bp of 59 untranslated region. Primers flanking the

UNCL coding region were used to amplify a cDNA2.9. Xenopus oocyte expression fragment that deleted all the 39 and 59 untranslated

sequence and improved the Kozak consensus for transla-Xenopus oocytes were surgically isolated by a previous- tion. The 777 bp product was cloned into pMT23 [16] and

ly published protocol [50] in accordance with the NIH and pCRII (Vitrogen) for subsequent experiments.University of California guidelines. Two groups of oocytes The Genbank/EMBL accession number U96638 hasfrom a single Xenopus were injected with RNA on the been assigned to the UNCL nucleotide /protein sequence.same day. One group was injected with RNA for the Human ESTs that represent UNCL sequence map toneuronal nAChR subunits, 50 ng of a3b4 per oocyte. A chromosome 2q11 according to human UniGene mapsecond group was injected with 50 ng of a3b4 RNA and 5 (http: / /www.ncbi.nlm.nih.gov/UniGene/Hs.Home.html)ng of UNCL RNA. The functional response of the a3b4 at NCBI and have been assigned UniGene number Hs.receptors was measured 2 days later by voltage-clamping 13370.whole oocytes at 250 mV and bath application of 1 mMacetylcholine. 3.2. UNCL is highly conserved

2.10. Transfection of COS cells The UNCL cDNA contains an open reading frame of777 nucleotides that codes for a 259 amino acid protein

COS-7 cells were transiently transfected by a modified with a predicted molecular weight of 30 kDa (Fig. 1A).DEAE-dextran /adenovirus method as described previously Alignment of predicted protein sequences from rat, human,[23]. 1 mg/ml of neuronal nicotinic or muscarinic subunit D. melanogaster, C. elegans, S. cerevisae and S. pombe,cDNAs was transfected together with 0, 0.1, or 1 mg/ml of and partial sequence from a plant species, A thaliana,


Fig. 1. Amino acid sequence of UNCL. (A) Predicted amino acid sequence of rat UNCL. Transmembrane domains predicted by hydropathy plot analysisare underlined. N-terminal region with homology to LEM domain [40] is double underlined. (B) UNCL sequences are highly conserved. Alignment of rat(Genbank accession number U96638), human (AF077038), D. melanogaster (G7299067), C. elegans (Q10045), S. cerevisae (P36125), S. pombe(G2104463), and partial A. thaliana (AC006298) UNC-50/UNCL protein sequences. The A. thaliana protein sequence is a putative translation of an ESTclone and represents the amino-terminal 121 residues of UNCL. An asterisk marks amino acids that are identical in at least four species. Alignmentgenerated using CLUSTALV software package [32] with minor changes performed visually. (C) Hydropathy plot of the rat UNCL protein sequence usingthe Kyte-Doolittle algorithm [37] at http: / /www.bmb.psu.edu/nixon/webtools /hydro /default.htm with a window size of 19 amino acids. Predictedtransmembrane domains are numbered 1–5.


heart, kidney and testis. The level of UNCL mRNA washighest in brain, kidney and testis and lowest in heart andall tissues expressed a single sized species, suggesting thatthe transcript is not alternatively spliced. The widespreadexpression pattern for UNCL mRNA indicated by theNorthern blot is also supported by the origin of UNCLclones in the EST database which demonstrate that UNCLis expressed in many tissues not represented on ourNorthern blot such as placenta, aorta, and mammary gland.

3.4. UNCL is a transmembrane protein

Although UNCL lacks a well defined signal sequenceFig. 1. (continued)

that would identify it as a secreted protein, hydrophobicityplot analysis (Fig. 1C) suggests that UNCL protein con-

revealed a striking degree of conservation between the tains several transmembrane domains. To determineseven proteins (Fig. 1B). The conservation between whether UNCL is an integral membrane protein, UNCLspecies is represented by sequence homologies of 46% cDNA was transcribed and translated in vitro with andbetween rat and nematode, 34% between rat and S. pombe, without canine pancreatic microsomes (Fig. 3). In the40% between rat and A. thaliana with human and rat absence of microsomes, the majority of translated proteinsequences sharing 92% homology and 87% identity. These remained in the supernatant following microcentrifugation.sequences clearly share homology over a large evolution- However, when the translation reaction was carried out inary distance, suggesting that UNCL function is conserved. the presence of dog pancreas microsomal membranes theThis is further supported by the observation that all majority of UNCL pelleted with microsomes, indicatingsequences identified so far contain several regions of that UNCL is either secreted into microsomes or insertedvirtually identical motifs, of which in the microsomal membrane. Following treatment withYRKQTKDQWARDDPAFLVLLS is the longest. We con- carbonate at pH 11.5 [25], UNCL protein remained withclude, therefore, that the rat, human, drosophila, yeast and the pelleted microsomes, strongly suggesting that UNCL ispartial A. thaliana UNCL sequences deposited in Genbank an integral membrane protein.represent homologues of the C. elegans gene, unc-50. In the absence of microsomal membranes, UNCL mi-

Although the putative protein sequence does not contain grated as a doublet of approximately 26 and 27 kDa ina recognizable signal sequence, hydropathy plot analysisindicates that up to five hydrophobic regions .18 residuesin length are present in the central region and C-terminusof the protein sequence [37], suggesting that UNCL mayfunction as a transmembrane protein (Fig. 1C). It isunclear, without detailed topological studies, as to whichdomains are transmembrane.

3.3. UNCL is widely expressed in rat tissues

The UNCL cDNA was used as a probe in a Northern1blot of poly A RNA from rat tissues. As shown in Fig. 2,

the UNCL cDNA probe detected a 1.2 kb mRNA in brain,

Fig. 3. UNCL is a transmembrane protein. UNCL cDNA was transcribed35and translated in vitro in the presence of [ S]-methionine with and

without microsomal membranes. Lane 1, control reaction without inputFig. 2. UNCL mRNA expression in multiple tissues. Northern blot of UNCL cDNA. Pellet (lane 2) and supernatant (lane 3) fractions of inpoly (A) RNA isolated from various rat tissues probed with full-length rat vitro translated UNCL cDNA. Pellet (lane 4) and supernatant (lane 5)UNCL cDNA. 1, brain; 2, heart; 3, kidney; 4, liver; 5, skeletal muscle; 6, fractions of translations performed in the presence of microsomes. Pellettestis. The migration position of a 1.35 kb mRNA marker is indicated on fraction (lane 6) of translation performed in the presence of microsomesthe left. followed by alkaline carbonate treatment.


Fig. 4. UNCL localizes to the inner nuclear membrane. (A) Extraction of cells expressing UNCL-HA. SaOS-2 osteosarcoma cells transfected withUNCL-HA cDNA were fixed (no extraction) or extracted with 1% Triton X-100 (1%TX-100) in PBS or 1% Triton X-100 with 350 mM NaCl(1%TX-1001salt) and mounted in DAPI-mounting solution. Cells shown in upper panels were stained for UNCL-HA with HA.11 and in lower panelswith KDEL antibody. Below each image is the corresponding DAPI stained images. Images stained for UNCL-HA were exposed for 3 s and KDEL 5 s. AllDAPI images were exposed for 200 ms. (B) Alignment of UNCL N-terminus to LEM domain present in three INM proteins. The N-terminal rat UNCLsequence from 1 to 44 was aligned visually to the LEM domain alignment (adapted from Ref. [40]) containing segments of human LAP-2b (accessionnumber P42167), human emerin (P50402), human MAN1 (AF112299) and rat UNCL (U96638). Asterisks show amino acids in UNCL that are identical orconserved in at least two INM proteins. Below alignment is a segment of HCMV gB (accession number P06473) that shows similarity to the LEM domain.Alignment was performed visually. Amino acids conserved in at least two INM proteins are underlined. Numbers flanking amino acid sequences indicatepositions of putative LEM domains within each protein.

size, although following translation with microsomes only predicted UNCL protein. Our conclusion is that the firstthe 27 kDa is apparent in the microsome pellet. The lower transmembrane domain of UNCL acts as an internal signalband of the doublet could be due either to translation sequence.initiation from a second methionine located 10 amino acidsfrom the N-terminal methionine (see Fig. 1A) in the in 3.5. UNCL localises to the inner nuclear membranevitro translation reaction or to some other unspecifiedprocessing step. In the in vitro translation system, the To determine the intracellular sub-localization ofsignal peptide does not appear to be cleaved since the UNCL, SaOS-2 cells transfected with UNCL-HA cDNAproduct translated in the presence of microsomal mem- were immunostained with the HA antibody, HA.11 (Fig.branes is the same size as in the absence of membranes. 4A). In untreated cells stained for UNCL-HA the nuclearThis is consistent with the lack of a well defined signal rim and an ER-like reticular structure surrounding thesequence (Fig. 1A) present at the amino terminus of the nucleus was stained. In cells stained for KDEL-containing


ER proteins a reticular staining pattern with very little were washed in buffer containing either low salt (50 mMnuclear staining was observed. Treatment with 1% Triton NaCl) or high salt (250 mM NaCl); the bound materialextracts proteins from the ER network and the outer was eluted and electrophoresed on a 13% SDS-poly-nuclear membrane that is contiguous with the ER, but does acrylamide gel. When the UNCL/homopolymer complexesnot extract inner nuclear membrane (INM) and nuclear were washed in a low salt buffer UNCL bound non-pore proteins [3,20,21,28,51]. Triton extraction resulted in specifically to all homopolymer–sepharose complexes andthe loss of UNCL-HA staining from the reticular ER also to sepharose alone (lanes 5–9). However, when thenetwork but staining was retained in the nucleus, sug- binding complex was washed in high salt buffer, UNCLgesting that UNCL is a component of the INM but not the associated only with polyG RNA sepharose (lanes 10–14).outer nuclear membrane. The ER staining observed in As controls, a known RNA-binding protein, GAP 120untreated cells probably represents ‘back-up’ of UNCL SH3-domain-binding protein (G3BP) [36], and a proteinprotein, suggesting the INM may be saturable which has that does not bind RNA, luciferase, were translated, addedpreviously been reported for INM proteins in transfected to homopolyG sepharose and washed in high salt. Ascells [40,57,58,19]. However, the possibility that UNCL expected, a proportion (26%) of the translated G3BPresides in the ER in addition to the nucleus cannot be bound to polyG RNA (lane 4) as determined by den-discounted. We have also observed occasional intranuclear sitometry. In contrast, none of the luciferase bound polyGstaining which may represent non-membrane degradation (lane 2). In comparison, 5.3% of total translated UNCLfragments and has been noted previously for an INM bound to polyG. These data suggest that UNCL bindsprotein [40]. In contrast, KDEL staining was absent RNA and may have specificity for RNA rich in guaninefollowing 1% Triton X-100 extraction, demonstrating this ribonucleotides.procedure was effective at solubilizing ER membranes. A potential RNP-1 [11] motif was identified visually inExtraction of cells with 1% Triton X-100 and 350 mM the primary amino acid sequence of rat UNCL at aminoNaCl solubilizes INM proteins but not proteins that acids 98–102. Structural modeling by homology was usedassociate with nuclear matrix components, such as lamins to determine whether a complete RRM could be identified[51]. Following salt extraction, no UNCL-HA staining was [17,34,47], a methodology recently used to identify apresent in nuclei, suggesting that UNCL does not interact cryptic RRM in the Wilm’s tumor suppressor protein WT1directly with the nuclear matrix. [35]. The rat UNCL sequence was aligned visually to

In support of the finding that UNCL is an INM protein known RRM containing proteins [5] to identify the overallis the observation that a region resembling the LEM secondary structure, b–a–b–b–a–b, required for a func-domain, which is found in several other INM proteins [40], tional RRM-type motif [33]. Once the secondary structuralis present at the N-terminus of UNCL. Although the regions within the rat UNCL sequence were identified,precise function of LEM domains has not been defined it is they were aligned with those identified in the crystalpresent in three INM proteins, lamina-associated structure of U1A [49] as shown in Fig. 5B. The proteinpolypeptide-2, and at the N-termini of emerin, and MAN1. was subsequently modeled to the U1A N-terminal RRMThe region of UNCL with the highest homology spans and the resulting folded protein of rat UNCL is shown as aamino acids 19–42 and encompasses the residues ribbon structure in Fig. 5C. The accuracy and validity ofAGAKRYKYLRRLFRF where the overall pattern of this model was tested by the method of Eisenberg [18,42].positively charged residues is conserved with the LEM Briefly, the probability of finding each amino acid in thedomain sequence (Fig. 4B). This region is conserved in all assigned environment is calculated from a 3D–1D scoringUNCL homologues, although there is less amino acid table derived from a statistical analysis of known proteinsimilarity with the C-terminal end of the LEM motif. We structures [43]. The amino acid sequence of the protein isconclude from these experiments and observations that then compared with the 3D profile generated from theUNCL is a transmembrane protein that localizes to the environment classifications. The expected score for aINM. protein is given by: Scalc5exp(20.83311.0083length),

where length is the number of residues. Based on studies3.6. UNCL is an RNA-binding protein of all co-ordinate entries in the Brookhaven Databank

derived by X-ray, NMR and computational methods, alongInitial inspection of the primary protein sequence sug- with a number of deliberately misfolded proteins, Eisen-

gested that UNCL may contain an RNA Recognition-type berg et al. [18] suggest that a cutoff of ,0.453Scalc isMotif (RRM) with redundant RNP-1 and RNP-2 consensus useful for identifying grossly misfolded structures. Modelssequences in the amino terminus [47]. Therefore, we asked with scores between 0.453Scalc and Scalc are consideredwhether UNCL could bind to RNA in vitro. UNCL mRNA correct, although it is still possible that locally misfolded

35was translated in vitro and the [ S]-labeled protein was regions occur. Examination of profile score in a movingadded to each of four RNA homopolymers: polyA, polyC, window scan of 10 residues is suggested to identify locallypolyG and polyU, each coupled to sepharose (Fig. 5A). misfolded regions. An Eisenberg analysis of rat UNCLNext, the UNCL–homopolymer–sepharose complexes gave a profile score of 17.81 (0.453Scalc515.39). This


value strongly suggests that the modeled region of UNCL (reviewed in Ref. [55]). However, there is no sequencecan attain a structure similar to that of the U1A N-terminal similarity between these b–a–b–b–a–b type structuresRRM. within a species let alone between species (sequence

Data derived from the crystal structure of U1A indicates alignments reviewed in Ref. [5]). The lack of conservedthat residues Y13, N16, E19, K22, K23, Q54, F56, K80, residues may be reflected in the use of redundant residuesR83, Q85 and D92 are potentially involved in the inter- between the unc-50-related proteins shown in Fig. 1B.action with the RNA substrate [47–49]. The 3D modeledstructure of rat UNCL shows that the spatial position andfunction of U1A Y13 may be replaced in UNCL by F57 3.7. Effect of UNCL on expression of neuronal nAChRsand U1A F56 may be replaced by UNCL F99. Thephenylalanine residue at position 99 in UNCL is conserved The mutant unc-50 was originally identified on the basisas either tyrosine or tryptophan in the other unc-50 /UNCL of its failure to assemble nicotinic acetylcholine receptorshomologues. All of these amino acids can potentially in the nematode, C. elegans. We investigated whetherpresent their respective phenol side chains for ring stacking UNCL played a role in the expression of mammalianinteractions with an RNA substrate (see Fig. 5C). It should nicotinic acetylcholine receptors (AChRs). In our ex-be noted that the general b–a–b–b–a–b structure may be perience, most neuronal AChRs assemble poorly in mam-typical of RRMs but it is not limited to this family of malian cells. In contrast, the Xenopus oocyte can directproteins. It is generally considered that this structure assembly of a broader array of subtypes. We began byprovides an anti-parallel binding platform for RNA–pro- studying the effect of UNCL on the expression of thetein interactions, as is the case for the ribosomal S6 protein ganglionic subtype of neuronal nicotinic receptors, a3b4,

Fig. 5. RNA-binding domain of UNCL. (A) UNCL binds to homopolyG RNA. UNCL, luciferase or G3BP cDNA was transcribed and translated in vitro in35the presence of [ S]-methionine, incubated with one of four RNA homopolymers coupled to agarose and washed with either low or high salt buffers. Lane

1, total luciferase translation. Lane 2, luciferase bound to homopolyG RNA. Lane 3, total G3BP translation. Lane 4, G3BP bound to homopolyG RNA.Lanes 5 and 10, UNCL bound to agarose. Lanes 6 and 11, UNCL bound to homopolyA RNA. Lanes 7 and 12, UNCL bound to homopolyC RNA. Lanes 8and 13, UNCL bound to homopolyG RNA. Lanes 9 and 14, UNCL bound to homopolyU RNA. Complexes in lanes 5–9 were washed in low salt buffer.Complexes in lanes 2, 4, and 10–14 were washed in high salt buffer. Molecular weights are indicated on left. (B) Alignment of rat UNCL to human U1Aused for 3D structural modelling. RNP-1 and RNP-2 motifs are indicated above the alignment, whereas the conserved secondary structures have beenboxed and their structural type indicated below the alignment. Numbers indicate the amino acid position within the protein. (C, see next page) Comparativeribbon structures of UNCL (green) and U1A (yellow). The UNCL ribbon represents the 3D model of the proposed RRM lying between amino acids 54 and129, side chains, potentially involved in ring stacking interactions within the RNA docking surface, have been indicated (F99 not shown). The U1A ribbonstructure represents the solved crystal structure from amino acids 10 to 86, side chains involved in RNA interactions within the RNA docking surface areshown. Below each ribbon structure is its respective Eisenberg analysis profile. The red horizontal line (only seen for U1A) indicates the cutoff for aminoacids occupying a potentially unfavourable position as determined by the Eisenberg analysis. No amino acids are determined as being unfavourable forUNCL. The amino acids below the cutoff in U1A represent residues lying outside the minimal RRM consensus (amino acids 87 to 116).


Fig.

5.(c

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in Xenopus oocytes. One group of oocytes was injected tional to the number of active surface receptors, UNCLwith a3b4 nAChR RNA plus UNCL RNA in a w/w ratio increased the amount of functional receptors on the surface1 UNCL:10 a3b4 and a matched control group was when present in small amounts. These data show, however,injected with an equal amount of a3b4 RNA without that low levels of UNCL can enhance the rate of assemblyUNCL. After 2 days, the functional response of the and/or insertion of a3b4 nAChRs into the plasma mem-oocytes was tested by voltage-clamping at 250 mV and brane of Xenopus oocytes.applying 1 mM acetylcholine. Initially, the acetylcholine To confirm this result and to test the effect of UNCL onresponse of the UNCL-injected group was |160% greater the a4b2 receptor subtype which can be expressed inthan the controls (P , 0.05) (Fig. 6A). However, after an mammalian cells, we co-expressed UNCL with thisadditional 2 days, the difference in current was not nAChR subunit combination in COS cells (Fig. 6B).

3significant. Since the voltage-clamped response is propor- Specific [ H]-acetylcholine binding was measured in ex-

Fig. 6. Effect of UNCL on nicotinic and muscarinic acetylcholine receptor expression. (A) Current induced by 1 mM ACh in Xenopus oocytes expressinga3b4 receptors. Oocytes were injected with nicotinic a3 and b4 subunit mRNAs alone (white bars), or with UNCL, a3 subunit, and b4 subunit mRNAs(black bars). Height of bars indicates mean response current (nA).Voltage5250 mV. Numbers in parentheses indicate the number of oocytes used for eachcondition. (B) Effect of UNCL on a4b2 nicotinic receptor expression in transfected COS cells. (C) Effect of UNCL on M1 human muscarinic receptor

3expression in transfected COS cells. Specific binding is dpms310 . Each data point is the mean of triplicate measurements and error bars are s.e.m. andeach experiment repeated at least twice. *P , 0.05 compared to control mean.


tracts of COS cells transiently expressing the a4b2 with our finding that UNCL is an RNA-binding protein. Inreceptor in the presence and absence of various amounts of transfected cells UNCL localizes to the ER in addition toinput UNCL cDNA. The expression level of receptor the nucleus, although it has been noted that the INM isincreased when UNCL was added at a w/w ratio of 1 saturable [51] and INM proteins destined for the nuclearUNCL DNA:10 receptor subunit DNAs (P , 0.05). When envelope can ‘back-up’ in the ER of transfected cellsUNCL was added in a 1:1 w/w ratio, surface receptor [51,52,58,19,40]. Our data strongly suggest that UNCL isexpression fell below the control levels. Therefore, a slight an INM protein but we cannot exclude the possibility thatoverexpression of UNCL increases a4b2 nAChR expres- UNCL plays a role in both the ER and nuclear membranession, but even modest overexpression of UNCL has an like Ire1p, a transmembrane protein that interacts withinhibitory effect on receptor expression. Inhibition of RNA and localizes to both ER and nuclear membranesnicotinic receptor expression could be due to either an [54].inhibitory effect on receptor expression or a generalized In support of an INM subcellular localization is thetoxic effect. We investigated this possibility by expressing presence of a sequence at the amino-terminus of UNCLthe human M1 muscarinic receptor (mAChR) in the that resembles the LEM domain found in the nucleo-presence of various amounts of UNCL. When UNCL plasmic portion of LAP-2, emerin and MAN1 [40]. ThecDNA was co-transfected into COS cells at a ratio of 1 nucleoplasmic location suggests that these modules act asUNCL:10 mAChR (Fig. 6C), acetylcholine binding was protein or nucleic acid interaction domains and the findingnot significantly affected. However, as for the nAChRs, that in LAP-2 the LEM domain is distinct from theequal amounts of UNCL and M1 mAChR cDNA sig- lamin-binding and chromosome-binding domains [26,27]nificantly reduced surface receptor (P , 0.05). We con- suggests that LEM may interact with a previously un-cluded from these experiments that UNCL is involved in identified target. It is interesting to note that an INM-expression of neuronal nicotinic receptors when expressed targeting sequence has been described in the humanat low levels. At high relative levels, UNCL probably cytomegalovirus glycoprotein B protein which is translo-exerts a toxic non-specific effect on protein expression. cated to the INM following viral infection [46]. The

critical sequence is a DRLRHR motif which when fused tothe membrane protein CD8 is sufficient for nuclear mem-brane targeting. This motif resembles part of the LEM

4. Discussion domain corresponding to the arginine and lysine-richsegment at the carboxyl-terminal end of the domain (see

In order to improve the transfection efficiency of Fig. 4B). Because of this sequence similarity and the factneuronal nAChRs in non-neuronal cells we sought to that it is clearly present in three of six INM proteins ofidentify factors important for nAChR expression and different function it seems reasonable to speculate thatbecame aware of a previously uncharacterized C. elegans LEM domains function as INM localization signals.gene, unc-50. Defects in this gene result in an uncoordi- The finding that UNCL binds RNA was unexpected butnated phenotype and, significantly, the unc-50 mutant was derives from two lines of evidence. First, UNCL proteinfound to be devoid of surface nicotinic receptors. We specifically binds polyG RNA in vitro in a manner thatsearched the EST database at NCBI and identified a resists stringent washing. Second, by means of ‘structuralmammalian homologue of unc-50 which we have termed modeling by homology’, the N-terminal is predicted toUNCL (UNC1L, Roman numeral for 50) and sought to fold into a structure consistent with an RRM-type folddetermine its role in nicotinic receptor expression. found in many RNA-binding proteins. It should be noted

In vitro translation experiments show that UNCL is a that, although the crystal structure of U1A was used as atransmembrane protein and Triton X-100 extraction of template for the structural modeling, it does not suggesttransfected cells indicate that UNCL is present in the inner that the two proteins function in a similar manner. Thenuclear membrane (INM). To date, six proteins have been U1A crystal structure is a convenient template for predict-localised to the INM in mammals: lamin B receptor (LBR) ing the tertiary b–a–b–b–a–b structure of putative[60,61,14], lamina-associated polypeptides (LAP) -1 [53] RRM-like molecules. However, this fold is not strictlyand -2 [21], emerin [4], nurim [51] and MAN1. LBR limited to this family of proteins. It has been speculated[61,62,57,58,19] and LAP-2 [21] interact directly with that the RRM fold shares distant evolutionary conservationspecific lamins within the nuclear matrix and with chro- with ribosomal RNA-binding proteins such as L12, L30matin, while LAP-1 [53] and emerin [20,44] bind to [33] and S6 [41,47]. Given that the N-terminal region oflamins. Since nuclear matrix-associated proteins are gener- the putative RNA-binding motif of UNCL is not strictlyally resistant to extraction in high salt [51] our data show conserved between rat and C. elegans, it is most likely thatthat UNCL does not interact directly with the nuclear the RNA-binding region is topographically more like thoselamina or chromatin because UNCL is readily extractable of ribosomal proteins. The RNA-binding domain, betweenin high salt buffer. This suggests that UNCL does not have amino acids 54 and 129, overlaps the first two of fivea structural function like LBR and LAP-2 and is consistent putative transmembrane domains predicted by hydropathy


plot analysis. Although we provide evidence that UNCL is Acknowledgementsa transmembrane protein it is unknown whether these twohydrophobic regions function as transmembrane domains We gratefully acknowledge the generosity of Dr. R.or as hydrophobic domains within an RNA-binding mod- Horvitz (MIT, MA) and Dr. M. Hengartner (Cold Springule. This issue requires further investigation. Harbor Laboratories, NY) in providing us with the se-

The phenotype of the unc-50 mutant suggests that the quence of unc-50 without which this study could not haverole of the unc-50 protein is restricted to nicotinic receptor been undertaken. We thank Dr. N. Affara (Cambridge, UK)assembly since no other molecular defects were reported for the partial human clone TEST1CO9 and Dr. W. Sadee[38]. Although our data certainly indicate a role for UNCL (UCSF, CA) for M1 muscarinic receptor cDNA. We wouldin neuronal nicotinic receptor expression as we have also like to thank the Drug Design and Developmentdemonstrated in transfected COS cells and oocytes, a Centre of the University of Queensland for the kind use ofbroader function for UNCL is likely. First, homologues of their facilities to perform the protein modeling. Thanks areunc-50 are present in two yeast and a plant species that also due to Dr. R. Edwards (UCSF, CA) for providing usentirely lack nicotinic receptors. Second, Northern analysis with the lGT10 rat brain cDNA library and to theshows that UNCL is expressed in mammalian tissues that members of the Anesthesia Research Laboratory at UCSFdo not express neuronal or muscle-type nicotinic receptors for their comments and suggestions. This work waswith most abundant expression in the brain, kidney and supported by grants to J.R.F. from the NIH (R01-testis, although there is a low but detectable level in DA08373), to B.N.C. from the American Heart Associa-muscle. The brain has a high concentration of neuronal tion ([0405895) and to J.F.B. from the National Healthnicotinic receptors but it is unknown why UNCL is and Medical Research Council of Australia. J.F. is sup-expressed at higher levels in kidney and testis. If UNCL ported by the Royal Children’s Hospital Research Institute,regulates nicotinic receptor expression one would expect D.K. is supported by a Dora Lush Fellowship from theUNCL expression in muscle since muscle-type nicotinic National Health and Medical Research Council of Aus-receptors are concentrated at the neuromuscular junction. tralia, and J.M. is supported by a Special Research CentreThis is clearly not the case and with the realisation that grant from the Australian Research Council.UNCL is probably present in all eukaryotes suggests thatUNCL performs a highly conserved and fundamentalbiochemical role involved in, but not restricted to, the

Referencessurface expression of nAChRs.It is not clear how UNCL acts to regulate nicotinic

[1] J. Abelson, C.R. Trotta, H. Li, tRNA splicing, J. Biol. Chem. 273receptor expression, although we suggest that UNCL is not(1998) 12685–12688.specific to nicotinic receptors and participates in a basic

[2] S.F. Altschul, W. Gish, W. Miller, E.W. Myers, D.J. Lipman, Basiccellular process that takes place at the nuclear envelopelocal alignment tool, J. Mol. Biol. 215 (1990) 403–410.

and involves an interaction with an RNA species. There is [3] S.M. Bailer, H.M. Eppenberger, G. Grifiths, E.A. Nigg, Characteri-no evidence that the known INM proteins bind RNA, zation of a 54-kD protein of the inner nuclear membrane: evidence

for a cell cycle-dependent interaction with the nuclear lamina, J.although at least two integral membrane endonucleasesCell Biol. 114 (1991) 389–400.involved in RNA splicing localize to the nuclear envelope.

[4] S. Bione, E. Maestrini, S. Rivella, M. Mancini, S. Regis, G. Romeo,In yeast, transfer RNA (tRNA) splicing takes place at theD. Toniolo, Identification of a novel X-linked gene responsible for

nuclear membrane by a mechanism that involves tRNA Emery–Dreifuss muscular dystrophy, Nat. Genet. 8 (1994) 323–endonuclease, which cut tRNAs at intron–exon 327.

[5] E. Birney, S. Kumar, E.R. Krainer, Analysis of the RNA-recognitionboundaries, and tRNA ligase which ligates together themotif and RS and RGG domains: conservation in metazoan pre-resultant two RNA fragments (for review, see Ref. [1]).mRNA splicing factors, Nucleic Acids Res. 21 (1993) 5803–5816.tRNA endonuclease is an integral membrane protein that is

[6] R. Black, D. Goldman, S. Hochschwender, J. Lindstrom, Z.W. Hall,present at the inner nuclear envelope and tRNA ligase is Genetic variants of C2 muscle cells that are defective in synthesis ofpresent both within the nucleus and at the nuclear mem- the a-subunit of the acetylcholine receptor, J. Cell Biol. 105 (1987)

1329–1336.brane [13]. A second endonuclease, Irep1, which is also a[7] R.A. Black, Z.W. Hall, Use of a replica technique to isolate muscleprotein kinase, participates in splicing of the HAC1

cell lines defective in expressing the acetylcholine receptor, Proc.transcription factor at the nuclear membrane [54]. Al-Natl. Acad. Sci. USA 82 (1985) 124–128.

though UNCL is clearly not an endonuclease one possi- [8] P. Blount, M.M. Smith, J.P. Merlie, Assembly intermediates of thebility is that it is involved in nuclear splicing reactions mouse muscle nicotinic acetylcholine receptor in stably transfected

fibroblasts, J. Cell Biol. 111 (1990) 2601–2611.perhaps by functioning as an RNA docking protein at the[9] J. Boulter, J. Connolly, E. Deneris, D. Goldman, S. Heinemann, J.nuclear envelope or transporting RNAs laterally along the

Patrick, Functional expression of two neuronal nicotinic acetyl-INM to nuclear pores for splicing or export. The RNAcholine receptors from cDNA clones identifies a gene family, Proc.

targets of UNCL are not known but they may be mRNAs Natl. Acad. Sci. USA 84 (1987) 7763–7767.that code for proteins such as molecular chaperones [10] S. Brenner, The genetics of Caenorhabditis elegans, Genetics 77required for assembly of nicotinic receptor complexes. (1974) 71–94.


[11] C.G. Burd, G. Dreyfuss, Conserved structures and diversity of software for multiple sequence alignment, CABIOS 8 (1992) 189–functions of RNA-binding proteins, Science 265 (1994) 615–621. 191.

[12] G.M. Church, W. Gilbert, Genomic sequencing, Proc. Natl. Acad. [33] D.W. Hoffman, C.C. Query, B.L. Golden, S.W. White, J.D. Kenne,Sci. USA 81 (1984) 1991–1995. RNA-binding domain of the A protein component of the U1 small

nuclear riboprotein analyzed by NMR spectroscopy is structurally[13] M.W. Clark, J. Abelson, The subnuclear localization of tRNA ligasesimilar to ribosomal proteins, Proc. Natl. Acad. Sci. USA 88 (1991)in yeast, J. Cell Biol. 105 (1987) 1515–1526.2495–2499.[14] J.-C. Courvalin, K. Lassoued, H.J. Worman, G. Bloebel, Identifica-

[34] D.J. Kenan, C.C. Query, J.D. Keene, RNA recognition: towardstion and characterization of autoantibodies against the nuclearidentifying determinants of specificity, Trends Biochem. Sci. 16envelope lamin B receptor from patients with primary biliary(1991) 214–220.cirrhosis, J. Exp. Med. 172 (1990) 961–967.

[35] D. Kennedy, T. Ramsdale, J. Mattick, M. Little, An RNA recogni-[15] E.S. Deneris, J. Connolly, J. Boulter, E. Wada, K. Wada, L.W.tion motif in Wilm’s tumour protein WT1 revealed by structuralSwanson, J. Patrick, S. Heinemann, Primary structure and expres-modelling, Nat. Genet. 12 (1996) 329–331.sion of beta 2: a novel subunit of neuronal nicotinic acetylcholine

receptors, Neuron 1 (1988) 45–54. [36] D. Kennedy, S.A. Wood, T. Ramsdale, P.P. Tam, K. Steiner, J.S.Mattick, Identification of a mouse orthologue of the human ras-[16] A.J. Dorner, M.J. Krane, R.J. Kaufman, Reduction of endogenousGAP-SH3-domain binding protein and structural confirmation thatGRP78 levels improves secretion of a heterologous protein in CHOthese proteins contain an RNA recognition motif, Biomed. Pept.,cells, Mol. Cell. Biol. 8 (1988) 4063–4070.Prot. Nucleic Acids 2 (1997) 93–99.[17] G. Dreyfuss, M.J. Matunis, S. Pinol-Roma, C.G. Burd, hnRNP

[37] J. Kyte, R.F. Doolittle, A simple method for displaying theproteins and the biogenesis of mRNA, Annu. Rev. Biochem. 62hydropathic character of a protein, J. Mol. Biol. 157 (1982) 105–(1993) 289–321.131.[18] D. Eisenberg, M. Wesson, M. Yamashita, Interpretation of protein

[38] J.A. Lewis, J.S. Elmer, J. Skimmimg, S. McLafferty, J. Fleming, T.folding and binding with atomic parameters, Chem. Struct. 29AMcGee, Cholinergic receptor mutants of the nematode Caenor-(1989) 217–221.habditis elegans, J. Neurosci. 7 (1987) 3059–3071.[19] J. Ellenberg, E.D. Siggia, C.L. Smith, J.F. Presley, H.J. Worman, J.

[39] J.A. Lewis, C.H. Wu, H. Berg, J.H. Levin, The genetics ofLippincott-Schwartz, Nuclear membrane dynamics and reassemblylevamisole resistance in the nematode C. elegans, Genetics 95in living cells: targeting of an inner nuclear membrane protein in(1980) 905–928.interphase and meiosis, J. Cell Biol. 138 (1997) 1193–1206.

[40] F. Lin, D.L. Blake, I. Callebaut, I.S. Skerjanc, L. Holmer, M.W.[20] J.A. Ellis, M. Craxton, J.A. Yates, J. Kendrick-Jones, AberrantMcBurney, M. Paulin-Levasseur, H.J. Worman, MAN1, an innerintracellular targeting and cell cycle-dependent phosphorylation ofnuclear membrane protein that shares the LEM domain with lamina-emerin contribute to the Emery–Dreifuss muscular dystrophy phe-associated polypeptide 2 and emerin, J. Biol. Chem. 257 (2000)notype, J. Cell Sci. 111 (1998) 781–792.4840–4847.[21] R. Foisner, L. Gerace, Integral membrane proteins of the nuclear

envelope interact with lamins and chromosomes, and binding is [41] M. Lindahl, L.A. Svensson, A. Liljas, S.E. Sedelnikova, I.A.modulated by mitotic phosphorylation, Cell 73 (1993) 1267–1279. Eliseikina, N.P. Fomenkova, N. Nevskaya, S.V. Nikonov, M.B.

Garber, T.A. Muranova, Crystal structure of the ribosomal protein[22] J.R. Forsayeth, A.J. Franco, A.B. Rossi, J.B. Lansman, Z.W. Hall,S6 from Thermus thermophilus, EMBO J. 13 (1994) 1249–1254.Expression of functional mouse muscle acetylcholine receptors in

Chinese hamster ovary cells, J. Neurosci. 10 (1990) 2771–2779. [42] R. Luthy, J.U. Bowie, D. Eisenberg, Assessment of protein modelswith three-dimensional profiles, Nature 356 (1992) 83–85.[23] J.R. Forsayeth, P.D. Garcia, Adenovirus-mediated transfection of

cultured cells, Biotechniques 17 (1994) 354–359. [43] R. Luthy, A.D. McLachlan, D. Eisenberg, Secondary structure-basedprofiles: use of structure-conserving scoring tables in searching[24] J.R. Forsayeth, E. Kobrin, Formation of oligomers containing theprotein sequence databases for structural similarity, Proteins Struct.beta-3 and beta-4 subunits of the rat nicotinic receptor, J. Neurosci.Funct. Genet. 10 (1991) 229–239.17 (1997) 1531–1538.

[44] S. Manilal, N. Thi Man, C.A. Sewry, G.A. Morris, The Emery–[25] Y. Fujiki, A.L. Hubbard, S. Fowler, P.B. Lazarow, Isolation ofDreifuss muscular dystrophy protein, emerin, is a nuclear membraneintracellular membranes by means of sodium carbonate treatment:protein, Hum. Mol. Genet. 5 (1996) 801–808.application to endoplasmic reticulum, J. Cell Biol. 93 (1982) 97–

102. [45] J.P. Merlie, J. Lindstrom, Assembly in vivo of mouse muscleacetylcholine receptor: identification of an a-subunit species that[26] K. Furukawa, C.E. Fritze, L. Gerace, The major nuclear envelopemay be an assembly intermediate, Cell 34 (1983) 747–757.targeting domain of LAP2 coincides with its lamin binding region

but is distinct from its chromatin interaction domain, J. Biol. Chem. [46] G.A. Meyer, K.D. Radsak, Identification of a novel signal sequence273 (1998) 4213–4219. that targets transmembrane proteins to the nuclear envelope inner

membrane, J. Biol. Chem. 275 (2000) 3857–3866.[27] K. Furukawa, C. Glass, T. Kondo, Characterization of the chromatinbinding activity of lamina-associated polypeptide (LAP) 2, Bio- [47] K. Nagai, C. Oubridge, N. Ito, J. Avis, P. Evans, The RNP domain:chem. Biophys. Res. Commun. 238 (1997) 240–246. a sequence-specific RNA-binding domain involved in processing

and transport of RNA, Trends Biochem. Sci. 20 (1995) 235–240.[28] Y. Gerace, Y. Ottaviano, C. Kondor-Koch, Identification of a majorpolypeptide of the nuclear pore complex, J. Cell Biol. 95 (1982) [48] K. Nagai, C. Oubridge, N. Ito, T.H. Jessen, J. Avis, P. Evans,826–837. Crystal structure of the U1A spliceosomal protein complexed with

its cognate RNA hairpin, Nucleic Acids Symp. Ser. 34 (1995) 1–2.[29] W.N. Green, T. Claudio, Acetylcholine receptor assembly: subunitfolding and oligomerization occur sequentially, Cell 74 (1993) [49] C. Oubridge, I. Nobutoshi, P.R. Evans, C.H. Teo, K. Nagai, Crystal

˚57–69. structure at 1.92 A resolution of the RNA-binding domain of theU1A spliceosomal protein complexed with an RNA hairpin, Nature[30] Y. Gu, J.R. Forsayeth, S.M. Verrall, X.-M. Yu, Z.W. Hall, Assembly372 (1994) 432–438.of the mammalian muscle acetylcholine receptor in transfected COS

cells, J. Cell Biol. 114 (1991) 799–807. [50] M.W. Quick, J. Naeve, N. Davidson, H.A. Lester, Incubation with[31] Y. Gu, A. Franco Jr., P.D. Gardner, J.B. Lansman, J.R. Forsayeth, horse serum increases viability and decreases background neuro-

Z.W. Hall, Properties of embryonic and adult muscle acetylcholine transmitter uptake in Xenopus oocytes, Biotechniques 13 (1992)receptors transiently expressed in COS cells, Neuron 5 (1990) 358–362.147–157. [51] M.M. Rolls, P.A. Stein, S.S. Taylor, E. Ha, F. McKeon, T.A.

[32] D.G. Higgins, A.J. Bleasby, R. Fuchs, CLUSTALV: Improved Rapoport, A visual screen of a GFP-fusion library identifies a new


type of nuclear envelope membrane protein, J. Cell Biol. 146 (1999) [58] B. Soullam, H.J. Worman, Signals and structural features involved in29–43. integral membrane protein targeting to the inner nuclear membrane,

[52] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A J. Cell Biol. 130 (1995) 15–27.Laboratory Manual, Cold Spring Harbor Laboratory Press, New [59] Y. Takebe, M. Seiki, J. Fujisawa, M. Hoy, K. Yokota, K. Arai, M.York, 1989. Yoshida, N. Arai, SR alpha promoter: an efficient and versatile

[53] A. Senior, L. Gerace, Integral membrane proteins specific to the mammalian cDNA expression system composed of the simian virusinner nuclear membrane and associated with the nuclear lamina, J. 40 early promoter and the R-U5 segment of human T-cell leukemiaCell Biol. 107 (1988) 2029–2036. virus type 1 long terminal repeat, Mol. Cell. Biol. 8 (1988) 466–

[54] C. Sidrauski, P. Walter, The transmembrane kinase Ire1p is a 472.site-specific endonuclease that initiates mRNA splicing in the [60] H.J. Worman, J. Yuan, G. Blobel, S.D. Georgatos, A lamin Bunfolded protein response, Cell 90 (1997) 1031–1039. receptor in the nuclear envelope, Proc. Natl. Acad. Sci. USA 85

[55] H. Siomi, G. Dreyfuss, RNA-binding proteins as regulators of gene (1988) 8531–8534.expression, Curr. Opin. Gen. Dev. 7 (1997) 345–353. [61] H.J. Worman, C.D. Evans, G. Bloebel, The lamin B receptor of the

[56] M.M. Smith, J. Lindstrom, J.P. Merlie, Formation of the a-bun- nuclear envelope inner membrane: a polytopic protein with eightgarotoxin binding site and assembly of the nicotinic acetylcholine potential transmembrane domains, J. Cell Biol. 111 (1990) 1534–receptor subunits occur in the endoplasmic reticulum, J. Biol. Chem. 1542.262 (1987) 4367–4376. [62] Q. Ye, H.J. Worman, Primary structure analysis and lamin B and

[57] B. Soullam, H.J. Worman, The amino-terminal domain of the lamin DNA binding of human LBR, an integral protein of the nuclearB receptor is a nuclear envelope targeting signal, J. Cell Biol. 120 envelope inner membrane, J. Biol. Chem. 269 (1994) 11306–11311.(1993) 1093–1100.

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UNCL, the mammalian homologue of UNC-50, is an inner nuclear membrane RNA-binding protein