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Brief Communication 1243

ETR-1, a homologue of a protein linked to myotonic dystrophy, is essential for muscle development in Catherine A. Milne and

Post-transcriptional gene processing by RNA-binding BLAST analysis indicated that ETR-1 is most similar to a proteins (RBPs) has crucial roles during development subfamily of ELAV-like RBPs. ETR-1 shares 74% iden- [1,2]. Here, we report the identification of ETR-1 tity with CUG-bp [7], its closest homologue, within the (ELAV-type RNA-binding protein), a muscle-specific RRMs (excluding the tether region) whereas it has only RBP in the nematode Caenorhabditis elegans. ETR-1 28% identity with Drosophila ELAV over the same regions is related to the family of RBPs defined by the protein (see Supplementary material). ELR-1 [14], the only other ELAV, which is essential for neurogenesis in the fruit known ELAV-like protein in C. elegans is more closely fly Drosophila; members of the family possess two related to Drosophila ELAV, with 59% identity in the consecutive RNA recognition motifs (RRMs) separated RRMs. ELR-1 has only 29% identity with ETR-1. ELR-1 from a third, carboxy-terminal RRM by a tether region and vertebrate family members closely related to ELAV of variable length [3–6]. Its closest homologue, share neuronal expression patterns that suggest a conser- CUG-binding protein (CUG-bp), is a human RBP that vation of function [3,14,15]. More distant family members has been implicated in the disease myotonic like ETR-1 and CUG-bp share a similar structural organi- dystrophy and binds CUG repeats in the 3′ sation but have more diverged RRMs and different untranslated region (UTR) of the mRNA for myotonic expression patterns ([4,7]; this paper). dystrophy protein kinase (DMPK) [7,8]. Inactivation of etr-1 by RNA-mediated interference resulted in The etr-1 gene is located on the left arm of C. elegans chro- embryonic lethality. Embryos failed to elongate and mosome II (predicted gene T01D1.2) [16]. Our cDNA pre- became paralysed, a phenotype characteristic of dicts a protein, which we call ETR-1a, containing three C. elegans Pat mutants, which are defective in muscle RRMs in an arrangement characteristic of ELAV-like pro- formation and function [9]. The data indicate that etr-1 teins, but with an unusually long tether of 280 amino acids is essential for muscle development in C. elegans, rich in glutamine and alanine. Comparison of the sequence perhaps by playing a role in post-transcriptional of our clone (from a yeast two-hybrid cDNA library created processing of some muscle component, and thus by Bob Barstead) with a previously identified cDNA suggesting a possible conservation of gene function (GenBank accession number U53931) revealed that there with human CUG-bp. are at least two alternatively spliced forms of ETR-1, which differ in the glutamine-rich tether region (see Sup- Address: Medical Research Council, Laboratory of Molecular , Hills Road, Cambridge CB2 2QH, UK. plementary material). Evidence from expressed sequence tags (ESTs) suggests there may be additional isoforms [16]. Correspondence: Jonathan Hodgkin E-mail: [email protected] We analysed etr-1 expression by fusing 4 kb of upstream Received: 10 August 1999 sequence and the first exon of etr-1 to sequences encoding a Revised: 16 September 1999 green fluorescent protein (GFP)–β-galactosidase reporter. Accepted: 16 September 1999 Transgenic animals expressed this reporter specifically in Published: 25 October 1999 muscle (Figure 1). Strong expression was observed in muscle cells in embryos. Adult expression was seen in stri- Current Biology 1999, 9:1243–1246 ated body-wall muscle along the length of the animal, espe- cially in the head. Strong expression was also seen in the 0960-9822/99/$ – see front matter © 1999 Elsevier Science Ltd. All rights reserved. vulval muscles. Additional expression was observed in intestinal muscle, anal sphincter muscle and the sex-spe- Results and discussion cific muscles of the male tail (data not shown). No expres- ETR-1 was identified as a protein that interacted in a sion was observed in pharyngeal muscle. This expression yeast two-hybrid screen with FOX-1 (feminising signal on pattern suggests that ETR-1 has a muscle-specific function. X), an RRM-containing protein involved in primary sex determination in C. elegans [10–13], but further evidence Next, we used RNA-mediated interference (RNAi) [17] to of a physical interaction in vitro was not detected. Further- inhibit the function of etr-1. To avoid potential interfer- more, neither overexpression nor reduced expression of ence with other RRM-containing proteins, we targeted etr-1 had any visible sex-specific effects on worms (data the unique tether region between RRM2 and RRM3. not shown). Instead, ETR-1 turned out to be an essential This region was chosen because there were no significant factor for muscle development in C. elegans. matches to other proteins in the sequence databases and 1244 Current Biology Vol 9 No 21

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Current Biology Current Biology

Expression pattern of etr-1. GFP fluorescence pattern of animals RNAi with sequences encoding the tether region in etr-1 results in an carrying a rol-6 marker and a non-integrated extrachromosomal array of embryonic lethal phenotype. (a,b) Representative wild-type animals at a GFP–lacZ reporter (derived from plasmid pD96.04, a gift from A. Fire) (a) the three-fold stage (just before hatching) and (b) the L1 larval fused to promoter elements of etr-1. In early embryos, muscle cells are stage (immediately after hatching). (c,d) Animals produced by parents initially seen as a continuous sheet on the lateral side of the embryo. that had been injected with the etr-1 dsRNA (prepared as described in Between 300 and 350 min, starting from the anterior, the sheet begins [17] and injected into the gonad of wild-type hermaphrodites at a to separate as the cells move to form two dorsal and two ventral muscle concentration of approximately 1 mg/ml). In (a), the arrest at the quadrants [40]. (a,b) Dorsolateral view of GFP expression in an two-fold stage results in the characteristic crescent-shaped wild-type approximately 300 min embryo is seen in a muscle sheet which is just embryo. (c) An RNAi embryo of approximately the same age, which has beginning to separate at the anterior. (c,d) 1.5-fold-stage embryos arrested at the two-fold stage and remained encased in the egg shell. (approximately 430 min) express GFP in the four muscle quadrants The animal in (d) hatched from the egg and was able to feed, but along the length of the embryo (two of which are seen clearly in this remained paralysed and never developed further. The scale bar example). (e) Adult animals show expression in body-wall muscle, seen represents 10 µm and applies to all panels. Anterior is to the top left. here in stripes along the length of the adult hermaphrodite. The four muscle quadrants spiral along the length of the animal because of the presence of the rol-6 marker. Anterior is to the left. (f,g) Ventral view of vulval muscle. (h,i) Intestinal muscle seen in lateral view with the ventral lev-11 and unc-52, respectively [18–26]. As an RBP, ETR- side to the left. (j,k) Lateral view of anal sphincter muscle (arrowhead) 1 is unlikely to have a structural role in muscle but it is with dorsal at the top. (b,d,g,i,k) The corresponding Nomarski images of clearly essential for proper muscle development. This the fluorescence images shown in (a,c,f,h,j). suggests that ETR-1 may post-transcriptionally regulate some essential structural component of muscle. An example of post-transcriptional processing affecting because inactivating it should affect all isoforms of etr-1. muscle development has been observed previously. Injection of double-stranded RNA (dsRNA) complemen- MEC-8, an RBP with two RRMs, is required in C. elegans tary to sequences encoding the tether region of ETR-1 for alternative splicing of unc-52 (perlecan), a component caused embryonic lethality. After morphogenesis began of the extracellular matrix that links the muscle to the and the egg transformed from an ovoid shape into a cylin- hypodermis [9,26,27]. drical worm, the embryo doubled over but ceased to elon- gate further. A number of animals hatched and were able To examine how muscle-specific proteins are perturbed to feed but remained paralysed at the two-fold stage when etr-1 is inactivated, RNAi and wild-type embryos of (Figure 2). This phenotype is characteristic of a class of comparable ages were fixed and stained with antibodies C. elegans mutants called Pat (paralysed elongation arrest at against perlecan (MH2), vinculin (MH24), integrin the two-fold stage) [9]. Muscle contraction is thought to (MH25), M line (MH42) and myosin B (DM5-8) [28–30] play a role in elongation, and a failure to correctly form (Figure 3). Perlecan, vinculin and integrin are all compo- muscle results in pre-elongation (two-fold) arrest [9,18]. nents of the dense body, which anchors muscle thin fila- ments and forms an attachment to the hypodermis. Most Pat mutants cloned to date encode structural or Myosin makes up part of the thick filament and is functional components of muscle. Examples include anchored by the M line (reviewed in [31]). All samples myosin A, β-integrin, troponin C, vinculin, tropomyosin were also stained with MH27 [29], which recognises band and perlecan, encoded by myo-3, pat-3, pat-10, deb-1, desmosomes. For all antibodies that were tested, protein Brief Communication 1245

Figure 3

Localisation of muscle-specific antibodies in wild-type and etr-1 (RNAi) embryos. Embryos (a) (b) (c) (d) (e) of similar ages were stained for (a,f,k,p) M line (MH42) [29], (b,g,l,q) perlecan (MH2) [28], (c,h,m,r) myosin B (DM5-8) [30], (d,i,n,s) integrin (MH25) [29] and (e,j,o,t) vinculin (MH24) [29]. (a–e) Nomarski (f) (g) (h) (i) (j) and (f–j) corresponding fluorescence images of wild-type embryos. (k–o) Nomarski and (p–t) corresponding fluorescence images of embryos from parents injected with dsRNA corresponding to the ETR-1 tether region. Embryos were fixed and stained with antibody (k) (l) (m) (n) (o) as described in [40] except that, directly after treatment with paraformaldehyde, slides were placed on dry ice for 5 min, then embryos were freeze-fractured by popping off the frozen coverslip and placed in methanol for (p) (q) (r) (s) (t) 4 min. All embryos were also stained with MH27. Antibodies were detected with fluorescein isothiocyanate (FITC)-labelled sheep anti-mouse secondary antibody. The grid pattern seen in some embryos is specific for MH27, which recognises band Current Biology desmosomes. The other antibodies gave their own characteristic staining patterns in the absence of MH27. Animals are oriented with dorsal to the top and anterior to the left.

product was observed, although muscle formation was C10H11.9). The characteristic CUG repeat seen in the 3′ somewhat disrupted compared with the wild type. In etr-1 UTR of human DMPK does not appear in either the (RNAi) embryos probed with MH2 and MH24, disconti- C. elegans homologue, nor in mup-2, the troponin T equiv- nuities in staining could be seen and the MH24 staining alent in C. elegans [37]. C10H11.9 is the C. elegans gene appeared weaker than in the wild type. MH2, MH24, let-502, expressed in seam cells [38] so is unlikely to be a MH25 and MH42 staining in RNAi embryos was clumpier target of ETR-1. The DMPK homologue K08B12.5 and than in the wild type. DM5-8 staining appeared less dis- troponin T remain targets worth investigation, however, turbed. Many of the RNAi embryos showed muscle fibres as the sequences of the RRMs in ETR-1 are sufficiently that pulled away from the hypodermis of the animal, different from those of CUG-bp to suggest an altered reflecting a possible defect in muscle attachment. binding preference.

These results strongly suggest that ETR-1 is an RBP The proteins tested in the antibody study are clearly essential for muscle development in C. elegans. Its closest present in etr-1 (RNAi) embryos so ETR-1 is not essential homologue, CUG-bp, has been implicated in myotonic for their translation. ETR-1 may, however, affect the ratio dystrophy. CUG-bp is thought to affect processing or of different isoforms for one or more of the proteins transport of RNA messages containing the CUG repeat because the antibodies used may not be isoform specific. sequence [32]; one of its known targets is the CUG It also remains possible that ETR-1 is not acting on any of repeat in the 3′ UTR of DMPK, which is greatly the muscle components directly but is affecting the trans- expanded in patients with myotonic dystrophy. Binding lation or splicing of some other regulatory component. of CUG-bp to these expanded repeats alters the subcel- ETR-1 may also regulate itself, as shown for Drosophila lular localisation of DMPK [33,34]. Mice lacking DMPK Sex-lethal [39]. develop late onset, progressive skeletal myopathy but do not show all the abnormalities reported in muscles of Our results show that ETR-1 is essential for muscle human patients with myotonic dystrophy [35]. Other development in C. elegans and is closely related to CUG- messages may also be affected; for example, splicing of bp, a protein implicated in a human muscle-wasting cardiac troponin T (cTNT), which contains a CUG- disease. Further investigation into the role of ETR-1 repeat sequence, is misregulated in the tissue of patients should provide significant information about the process with myotonic dystrophy [36]. Two potential DMPK of muscle assembly in C. elegans. Parallels with CUG-bp homologues have been identified in C. elegans (K08B12.5, may also provide insights into the human disease. 1246 Current Biology Vol 9 No 21

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Supplementary results and discussion S3. Lu X, Timchenko NA, Timchenko LT: Cardiac elav-type RNA-binding protein (ETR-3) binds to RNA CUG repeats expanded in myotonic Other proteins closely related to CUG-bp include the dystrophy. Hum Mol Genet 1999, 8:53-60. Drosophila protein Bruno [S1], the Xenopus protein Eden [S2] and a human protein, ETR-3, which is expressed in cardiac muscle and binds the CUG-repeat expansion in myotonic dystrophy patients and a CUG sequence within its own message [S3].

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Figure S1

Alignment of the amino-acid sequence of ETR-1a 1 ------ETR-1a with the alternatively spliced form ETR-1b 1 ------CUG-bp 1 ------ETR-1b (GenBank accession number Bruno 1 MFTSRASFLANRRMIFDFSEKNNDIYLDAFGMSDKSSSATNSLPNSPIHSNNNNPSLLNNNNNNSNGTSSNNSLNVGNNN MDFIMANTGAGGGVDTQAQLMQSAAAAAAVAATNAAAAPVQNAAAVAAAAQLQQQQVQQAILQVQQQQTQQAVAAAAAAV U53931), the human protein CUG-bp, and the ELAV 1 Drosophila proteins Bruno and ELAV. The ETR-1a 1 ------MSGAVLPAVLVVPKMESVVVPIAESTVINAKDTTPSTAADNDVSPSPSEPDTDAIKMFVG-QIP initial Clustal W alignment was refined by eye. ETR-1b 1 ------MSGAVLPAVLVVPKMESVVVPIAESTVINAKDTTPSTAADNDVSPSPSEPDTDAIKMFVG-QIP CUG-bp 1 ------MNGTLDHPDQPDLDAIKMFVG-QVP Blue, identical amino acids; grey, similar amino Bruno 81 SNPSLGGTNSNALVSVGSNGIMSAAGLVNNNNNPCSANRNVVAMVDDDACFRLDTDATVTYGEKEPDPDNIKMFVG-QVP TQQLQQQQQAVVAQQAVVQQQQQQAAAVVQQAAVQQAVVPQPQQAQPNTNGNAGSGSQNGSNGSTETRTNLIVNYLPQTM acids. Where only ETR-1a and ETR-1b were ELAV 81 2 identical, the sequences were left unshaded. ETR-1a 64 RQWNETDCRRLFEKYGSVFSCNILRDKS--TQAS------KGCCFVTFYHRKDAIEAQGALHNIKVIEGMHHPVQ ETR-1b 64 RQWNETDCRRLFEKYGSVFSCNILRDKS--TQAS------KGCCFVTFYHRKDAIEAQGALHNIKVIEGMHHPVQ Black underline, positions of RRMs; boxes CUG-bp 25 RTWSEKDLRELFEQYGAVYEINVLRDRSQNPPQS------KGCCFVTFYTRKAALEAQNALHNMKVLPGMHHPIQ Bruno 160 KSMDESQLREMFEEYGAVHSINVLRDKA--TGIS------KGCCFVTFYTRRAALKAQDALHNVKTLNGMYHPIQ numbered 1 and 2, positions of the RNP1 and ELAV 161 TE-DEIRSL--FSSVGEIESVKLIRDKSQVYIDPLNPQAPSKGQSLGYGFVN-YVR-----PQDAEQAVNVLNGLRL--Q RRM1 RNP2 sequences, which are the mostly highly 1 ETR-1a 131 MKPADTE--NRNE-RK----LFIGQLSKKHNEENLREIFAKFGHI--EDCSVLRDQDGKSRGCAFVTFTNRSCAVVATKE conserved parts of the RRMs. The arrangement ETR-1b 131 MKPADTE--NRNE-RK----LFIGQLSKKHNEENLREIFAKFGHI--EDCSVLRDQDGKSRGCAFVTFTNRSCAVVATKE CUG-bp 94 MKPADSEKNNAVEDRK----LFIGMISKKCTENDIRVMFSSFGQI--EECRILRGPDGLSRGCAFVTFTTRAMAQTAIKA of the three RRMs in all five proteins is Bruno 227 MKPADSE--NRNE-RK----LFVGMLNKKLNENDVRKLFEVHGAI--EECTVLRDQNGQSKGCAFVTFATKHAAISAIKV characteristic of ELAV family members. ELAV 230 NKTIKVSFARPSSDAIKGANLYVSGLPKTMTQQELEAIFAPFGAIITSRILQNAGNDTQTKGVGFIRFDKREEATRAIIA 2 RRM2 1 Significant differences include an extended ETR-1a 202 MH-HSQTMEGCSAPLVVKFADTQKDKDVKTKSMITGSGAGSPKGGAASLLQNLNPALLQQLGGGQNYQAVASLLSLINGQ ETR-1b 202 MH-HSQTMEGCSAPLVVKFADTQKDKDVKTKSMITGSGAGSPKGGAASLLQNLNPALLQQLGGGQNYQAVASLLSLINGQ amino-terminal domain in ELAV and Bruno and CUG-bp 168 MH-QAQTMEGCSSPMVVKFADTQKDK--EQKRMAQQLQQQMQQISAASVWGNL--AGLNTLG------PQYLALL--- Bruno 298 TLSQNKIMEGCTSPLVVKFADTQKEK--EQK-KIQQIQAN-----LWNLASNIN----IPLGQ------TATSVT--- the extended tether region of ETR-1. ETR-1b is ELAV 310 -L-N GTTPSSCTDPIVVKFSNTPGSTS-----KIIQPQ------slightly larger than ETR-1a (592 versus 584 ETR-1a 281 QGQQQQHQQQQNVLGILGTVFA------amino acids, respectively) and contains a ETR-1b 281 QGQQQQHQQQQNVLGILGTVLSALGKLTEGGDDASAKSSSEKPRHQALMTSPAPTATSSTSSSASHHHQQHQQQLSQQQQ CUG-bp 232 ------QQTASSGNLNTLSS------slightly higher glutamine and serine content Bruno 355 ------TPILP------(29 vs 17 glutamines, 14 vs 9 serines) in the ELAV 341 ------118 amino acid alternatively spliced region. ETR-1a 303 ------GNPLLGNPAMAAQNQFDAITMAQIAHQQQMLALQGFAVQQGAPSQQQQGLFLAHQYSTNYSTGSPDSASN ETR-1b 361 QQQHPQQQGLGNPLLGNPAMAAQNQFDAITMAQIAHQQQMLALQGFAVQQGAPSQQQQGL------CUG-bp 246 ------LHPMGGLNAMQLQN-LAALAAAASAAQNTPSGTNALTTSS-SP------LSVLTSSGS-----S Bruno 360 ---NPPQQ--PSPVLGADAITPASIQLLQQLQAVGLQHQLLQALTGLGAQQ------SSSATDT--S ELAV 341 ------

ETR-1a 373 ATDSSSDDNNVMNNRLTPTSKMAQYGYGPLIPRHPAQLYLQQRFPATAAAYVPGLAG-GMAGAKTTSPVAASLANHQQIA ETR-1b 421 ------AG-GMAGAKTTSPVAASLANHQQIA CUG-bp 297 PSSSSS------NSVNP---IASLG------ALQTLAGATAGLNVGSLAG-MAALNGGLG--SSGLSNGTGST Bruno 414 AVAAAG------LLTP------MTVQNLAAIAAMTQPSLSNAAAAAARATSPGSAQLTNTAALL ELAV 341 ------LPAFLNPQLVRRIGGAMH-T-PV------NKGL

ETR-1a 452 LTPFAGGA-AALDHFQAMQQYALLANLQATGG-VGVQATTSAAQMVGNGDVK-GPDGAN-LFIYHLPQDFGDSDLINTFA ETR-1b 445 LTPFAGGA-AALDHFQAMQQYALLANLQATGG-VGVQATTSAAQMVGNGDVK-GPDGAN-LFIYHLPQDFGDSDLINTFA CUG-bp 352 MEALT------QAYSGIQQYAAAA-LPTLYN----QNLLTQQSIGAAGSQKEGPEGAN-LFIYHLPQEFGDQDLLQMFM Bruno 466 WSDPNPMASAYMSTAAGLPQFGSASALSTSP---LASVALSAAAAAAAGKQIEGPEGCN-LFIYHLPQEFTDTDLASTFL ELAV 366 ARF------SPMAGDMLDVMLPNGLGAAAAAATTLAS-GPGGAYPIFIYNLAPETEEAALWQLFG

2 ETR-1a 528 PFGGILSAKVFIDKVTNLSKCFGFVSYENAQSATNAISAMNGFQIGSKRLKVQLKVDRGN--PYNR ETR-1b 521 PFGGILSAKVFIDKVTNLSKCFGFVSYENAQSATNAISAMNGFQIGSKRLKVQLKVDRGN--PYNR CUG-bp 419 PFGNVVSAKVFIDKQTNLSKCFGFVSYDNPVSAQAAIQSMNGFQIGMKRLKVQLKRSKNDSKPY Bruno 542 PFGNVISAKVFIDKQTSLSKCFGFVSFDNPDSAQVAIKAMNGFQVGTKRLKVQLKKPKDS-KPY ELAV 424 PFGAVQSVKIVKDPTTNQCKGYGFVSMTNYDEAAMAIRALNGYTMGNRVLQV-FKTNKAK

RRM3 1 Current Biology