Proc. Natl. Acad. Sci. USAVol. 93, pp. 3232-3237, April 1996Developmental Biology

Human myosin VIIA responsible for the Usher 1B syndrome: Apredicted membrane-associated motor protein expressed indeveloping sensory epithelia

(deafness/retinitis pigmentosa/unconventional myosins/band 4.1 family/alternative mRNA splicing)

DOMINIQUE WEIL*, GALLIA LEVY*, IMAN SAHLYt, FABIENNE LEVI-ACOBAS*, STEPHANE BLANCHARD*,AzIz EL-AMRAOUI*, FABIEN CROZET*, HERVE PHILIPPE:, MARC ABITBOLt, AND CHRISTINE PETIT*§*Unit6 de Genetique Moleculaire Humaine, Centre National de la Recherche Scientifique Unite de Recherche Associee 1968, Institut Pasteur, 25 rue du DocteurRoux, 75724 Paris Cedex 15, France; tCentre de Recherches Therapeutiques en Ophthalmologie, Laboratoire d'Embryologie Humaine, Faculte de MedecineNecker-Enfants Malades, Universit6 Rene Descartes, 156 rue de Vaugirard, 75015 Paris, France; and tLaboratoire de Biologie Cellulaire, Centre National de laRecherche Scientifique Unite de Recherche Associ6e 1134, Universite Paris-Sud, 91405 Orsay Cedex, France

Communicated by Francois Jacob, Institut Pasteur, Paris, France, December 26, 1995 (received for review November 17, 1995)

ABSTRACT The gene encoding human myosin VIIA isresponsible for Usher syndrome type 1B (USH1B), a diseasewhich associates profound congenital sensorineural deafness,vestibular dysfunction, and retinitis pigmentosa. The recon-stituted cDNA sequence presented here predicts a 2215 aminoacid protein with a typical unconventional myosin structure.This protein is expected to dimerize into a two-headed mol-ecule. The C terminus of its tail shares homology with themembrane-binding domain of the band 4.1 protein superfam-ily. The gene consists of 48 coding exons. It encodes severalalternatively spliced forms. In situ hybridization analysis inhuman embryos demonstrates that the myosin VIIA gene isexpressed in the pigment epithelium and the photoreceptorcells of the retina, thus indicating that both cell types may beinvolved in the USH1B retinal degenerative process. In addi-tion, the gene is expressed in the human embryonic cochlearand vestibular neuroepithelia. We suggest that deafness andvestibular dysfunction in USH1B patients result from a defectin the morphogenesis of the inner ear sensory cell stereocilia.

Usher syndrome (USH) is the most frequent cause of deaf-blindness in humans (1). This dual sensorineural defect affectsboth the organ of Corti and the retina. Three distinct clinicalsubtypes, all recessively transmitted, have been described.USH type 1 (USH1) is the most severe form, characterized byprofound congenital sensorineural hearing loss, constant ves-tibular dysfunction, and prepubertal onset of retinitis pigmen-tosa leading to blindness. Linkage studies in affected familieshave shown that at least four loci (A, B, C, and D) areresponsible for USH1 (2, 3).We have recently identified the gene encoding myosin VIIA

as responsible for USH1B, which accounts for about half of allUSH cases, by identifying nonsense and missense mutationsand small deletions in five affected families (4). Mutations inthe mouse myosin VIIA gene are responsible for the shaker-1phenotype (5), which solely manifests defects of the cochlearand vestibular neuroepithelia without any evidence for animpairment of retinal functions. Histopathological studies inshaker-1 mice have shown that the development of the organof Corti is delayed and that the sensory cells degenerateimmediately after birth (6). However, nothing is yet knownabout the molecular mechanism(s) that lead to the retinaldegeneration in USH1B and to the degeneration of auditorysensory cells in both the human and mouse diseases.We report here on the primary structure of human myosin

VIIA (as deduced from a 7465-nucleotide reconstituted cDNA

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

sequence), its characterization as a member of the unconven-tional myosin family, and evidence for the existence of variousalternatively spliced transcripts. Pathophysiological hypothe-ses for USH1B syndrome will be discussed in light of the resultsobtained from in situ hybridization analysis of the gene ex-pression during embryonic development.

MATERIALS AND METHODScDNA Library Screening. A retinal cDNA library con-

structed in Agtl 1 (Clontech) was screened by using thegenomic A clone 2 already isolated, which contains 10 exons ofthe myosin VIIA gene (4). Three cDNA clones were isolated.An end probe from one of these allowed us to isolate addi-tional clones. A total of four rounds of hybridization wereperformed to obtain the complete coding sequence of themyosin VIIA cDNA.

Sequence Analysis. The sequences were determined on anApplied Biosystems 373ABI DNA sequencer as described (4).1

Yeast Artificial Chromosome (YAC) Library Screening.Two YACs (Centre d'Etude du Polymorphisme Humain),YAC 250B9 and YAC 984B11, hybridizing with the 5' end and3' part of the myosin VIIA cDNA, respectively, were sub-cloned in phage AGEM (Promega). The YAC 250B9 A library[containing the previously described clone 2 covering exons4-13 (4)] was screened with a cDNA probe that extendsbetween nucleotides -267 and 812 of the myosin VIIA cDNA,and three clones were isolated, each containing exon 1, 2, or3. The YAC 984B11 A library was screened with cDNA probescovering position 2000 to the end of the myosin VIIA cDNA,and five additional overlapping recombinant clones, coveringexons 14-48, were thereby isolated.

In Situ Hybridization. Sections. Brain and eye sections wereobtained and treated as previously described (7). Tissues werefrozen with powdered dry ice and stored at -80°C. Cryostatsections (15 iu) were mounted on slides, fixed, and stored at-80°C.Probes. Two antisense oligonucleotide fragments, both de-

rived from parts of the tail in which no splicing out had beendetected, were used as myosin VIIA-specific probes: A, 5'-CTGCAGGCCACCAAGTCCAAGAAGCCAATCATGTT-GCCCGTGACATTCATGGATGGGAC-3' (nucleotides 3745-3803), and B, 5'-AAATTTGCCAAGTTCGCGGCCACCT-ACTTCCAGGGGACAACCACGCACTCCTACACCCGG-3'(nucleotides 2998-3057). The corresponding sense oligonucleo-

Abbreviations: USH, Usher syndrome; YAC, yeast artificial chromo-some.§To whom reprint requests should be addressed (fax 33 1 45676978).¶The sequence reported in this paper has been deposited in theGenBank data base (accession no. U39226).

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tides were used as negative controls (A' and B'). Hybridizationwas performed at 43°C for 20 h. The sections were exposed tox-ray film for 5-10 days, and then to photographic emulsion at 4°Cfor 3-6 weeks. Probes A and A' and B and B' were used onhuman sections and probes A and A' on mouse sections. Thehomologous sequence in mouse presents 96% identity with thehuman sequence (data not shown).

RESULTS AND DISCUSSION

Deduced Primary Structure of Human Myosin VIIA

Eighteen overlapping cDNA clones covering 7465 nucleotideswere selected from a human retinal cDNA library after fourrounds of hybridization screening (see Materials and Methods).The reconstituted cDNA sequence comprises the entire codingregion as well as the complete 3' and part of the 5' noncodingregions. The translation initiation site was identified by thepresence of a strong consensus sequence (8) 14 bp downstreamof an in-frame stop codon. A poly(A) tail was detected 21 bpdownstream of a typical polyadenylylation signal (data notshown). The open reading frame encodes a putative 2215-aaprotein with a predicted molecular mass of 254 kDa (Fig. 1).Sequence comparison with known proteins reveals a typicalunconventional myosin. Unconventional myosins (9) are actin-

Proc. Natl. Acad. Sci. USA 93 (1996) 3233

based motor molecules which transduce the chemical energyderived from the ATP they hydrolyze into the production of aforce enabling them to move along actin filaments. TheirN-terminal head domain, or motor domain, is followed by aneck region (also called regulatory domain) composed of atleast one IQ motif. These motifs of 22-29 aa, with an a-helicalstructure, are expected to bind calmodulin-like chains whichmodulate the myosin mechanical activity (for a review, see ref.10). The tails of unconventional myosins vary dramatically inlength and in sequence from one to another. The tail is thoughtto be membrane-binding, and it is hypothesized that thefunctional specificity of each myosin resides in the bindingspecificity of its tail to a particular membranous compartment,which will thereby be moved relative to the actin filaments.Hence, the name "cellular cargos" attributed to these proteins.Human myosin VIIA consists of a 729-aa motor domain, a

126-aa neck domain, and a 1360-aa tail.Motor Domain. The motor domain harbors a typical ATP-

binding site, GESGAGKTE (residues 158-166), and an actin-binding site, FVRCIKPN (residues 622-629) (11, 12). TheGKTKIFLK C-terminal sequence of this domain is also foundin other unconventional myosins (13, 14). Phylogenetic anal-ysis revealed that the protein closest to myosin VIIA (in termsof genetic distance) is myosin X from Rana catesbeiana, anewly identified and only partially sequenced myosin (Gen-Bank accession no. U14373) (15) (data not shown).

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FIG. 1. Deduced amino acid sequence of myosin VIIA. Numbers indicate the amino acid positions. The conserved binding sites are underlinedand in boldface: the ATP-binding site (residues 158-166) and the actin-binding site (residues 622-629). The IQ motifs of the putativecalmodulin-binding sites are shaded. The exon limits are indicated: + indicating that the splicing occurs between two codons, T indicating that itoccurs after the first base of the preceding codon, and a double-stemmed T indicating that it occurs after the second base of the preceding codon.

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merlin 165 QEELLPKRVINLYQMTPEMWEERITAWYAEHRGRARDEAEMEYLKIAQDLEMYG1111 11 II 11 11 I III I 11 1 II II

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FIG. 2. Homology of the C terminus of the myosin VIIA tail with the membrane-binding domain of the band 4.1 superfamily members: humanmerlin (GenBank accession no. P35240) and mouse talin (GenBank accession no. P26039). Identical amino acids are indicated by a boldface dashand homologous amino acids, by a plain dash.

Regulatory Domain. The neck is composed of an 18-aasegment followed by five consecutive repeats of the IQ motif(16, 17), which classify myosin VIIA among those possessingan extended neck (16). Each repeat is 23 aa long. Repeats 1,2, and 4 match the consensus sequence and are thus likely tobe associated with calmodulin itself (18), while repeats 3 and5 are imperfect repeats which may be associated with calmod-ulin-like proteins.

Tail. Of all the unconventional myosins identified thus far,myosin VIIA possesses the longest tail (1360 aa). It starts witha 78-residue cluster with mixed charges (residues 858-935).Secondary structure predictions by the Lupas program re-vealed that the a-helical segment between positions 864 and935 has a 99% probability of forming a coiled-coil structure(19). This tail segment is therefore expected to lead to thedimerization of the molecule through the formation of acoiled-coil rod, approximately 10.5 nm in length, thus resultingin a two-headed molecule. A similar structure has beendemonstrated for myosins V and VI (13, 20). However, inmyosin VIIA, the dimeric segment would extend for only 71 aaas opposed to 376 and 191 aa in myosins V and VI, respectively.

Self-comparison of the myosin VIIA tail sequence (dotplotcomparison, GCG package) detects two segments of approx-imately 260 aa (residues 1109-1370 and 1749-2017) that areapproximately 28% identical to each other and which, inaddition, show 20% identity to a tail segment (residues 1249 to1505) of the Acanthamoeba high molecular weight myosin(HMWMI or myosin IA) (21) (data not shown).The C-terminal part of some unconventional myosins in-

cludes segments consisting of both positively charged residuesand hydrophobic residues, which have been shown to bindnegatively charged membranous phospholipids (22-24). How-ever, the molecular basis of the specific targeting of each tailto a given membrane is unknown and no ligand of anyunconventional myosin has yet been identified. We investi-gated the potential presence of a basic segment, amphipathic

helix, or hydrophobic domain within the tail of myosin VIIA,which could indicate a capacity to bind membranous phos-pholipids. The calculated pI (isoelectric point) of the tail is8.31, which is less basic than in other myosins, and no amphi-pathic helix was detected. However, the C terminus of the tail(residues 1857-2215) is more basic (pI = 10.4) than the rest ofthe tail and is hydrophilic. Interestingly, the 318 C-terminalamino acids (1898-2215) show homology to the membrane-binding domain of various members of the band 4.1 proteinsuperfamily (25, 26). This superfamily consists of five classes,including band 4.1, talin, and the ERM (ezrin, radixin, moesin,merlin/schwannomin) family (25, 26). These proteins arecharacterized by a 300-aa N-terminal domain which constitutestheir cell membrane-binding domain. From amino acid 2060 tothe end of the protein, myosin VIIA is similar to the C-terminalhalf of this membrane-binding domain and shows 26% identityand 48.5% similarity to talin and about 22% identity and 45%similarity to both merlin/schwannomin and moesin (Fig. 2) (incomparison, the domains of talin and merlin/schwannominshare 21% identity and 48% similarity). The membrane pro-tein ligands of the band 4.1, talin, and ERM family membershave been identified as glycophorin C, integrin, and CD44,respectively (26, 27). Consequently, the homology of the Cterminus of myosin VIIA with the domain defining the band4.1 superfamily strongly suggests that the interaction of myosinVIIA with the membrane(s) is a protein-protein interaction.However, considering the sequence divergence between my-osin VIIA and the band 4.1 superfamily members, myosinVIIA is expected to bind to a specific membrane proteinligand. Fig. 3 schematically represents the deduced structure ofthe myosin VIIA protein.

Gene Structure and Evidence for Alternative Splicing

A total of eight phages from YACs 250B9 and 984B11 wereused to obtain the exon-intron structure of the myosin VIIA

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gene (see Materials and Methods). Exon-intron junctions weredetermined by comparison of genomic DNA sequences to thecDNA sequence. Forty-eight exons covering the entire openreading frame and the 3' untranslated regions were therebycharacterized (G.L., unpublished results). The head is encodedby 17 exons and the tail by 28 exons. The first of three neckexons codes for the 18-aa junction plus the first IQ motif, thesecond for another IQ motif, and the third for the threeremaining IQ repeats. The gene extends over 100 kb, with thefirst 2 introns being by far the largest.Among the 18 independent cDNA clones isolated from a

retinal cDNA library, 6 were found to differ from the recon-stituted cDNA described above (Fig. 4). One clone (R13)contains two deleted sequences within the head; the first one(from nucleotide 850 to nucleotide 1080) corresponds to thesplicing out of exons 8 and 9 and the second (from nucleotide1555 to nucleotide 1690) to that of exon 13. Another clone(R103) contains a deletion within the tail, which results fromthe splicing out of exon 25. Finally, four clones (R210, R206,R185, and R55) carry the deletion of a 114-bp segment (fromnucleotide 4569 to nucleotide 4682), corresponding to the 5'end of exon 34. This deletion correlates with the presence ofa consensus 3' acceptor splice site, YnNCAG, within this exon,which would act as an alternative splice site (28). All of thesealternative splicings conserve the reading frame. For someunconventional myosins, isoforms differing in the number ofIQ domains (29, 30), in the internal sequence of their tail, orin their C termini (31) have been described. The alternativelyspliced retinal cDNA clones analyzed here lead us to anticipatethe existence of various isoforms of the myosin VIIA protein,which would differ in their head and/or tail sequence(s). Theseisoforms might.have a tissue-specific and/or developmentallyregulated distribution. The splicing out of three exons withinthe head of myosin VIIA is an unusual situation, as all themyosin head isoforms described to date are the result of thealternative use of duplicated exons (32). Comparison with thedescribed three-dimensional structure of a conventional my-osin head (11, 12) shows that the "short-headed" myosin VIIAisoform described here would have at least one amino acidknown to interact with ATP, as well as part of the actin-bindingsite, deleted. This most likely defective form may thereforeplay a regulatory role by competing with the "fully active"forms.

In Situ Hybridization Analysis in Human andMouse Embryos

Using reverse transcription followed by the polymerase chainreaction (RT-PCR), we have previously shown that the myosinVIIA mRNA is expressed in several adult mouse and/orhuman tissues, including the kidney, liver, retina, and cochlea(4). We further investigated the gene expression during de-velopment by in situ hybridization on human embryo sections.Taking advantage of the sequence divergence between the tails

of the various unconventional myosins (9), we designed twospecific oligonucleotides from the human myosin VIIA gene(see Materials and Methods). The same hybridization patternwas observed with both oligonucleotides.The myosin VIIA gene was expressed in the otocyst and

olfactory epithelium of 7- and 8-week-old human embryos(Fig. 5 A and B). Within the otocyst, both the cochlear andvestibular neuroepithelia were labeled. Within the olfactoryepithelium the labeling was associated with the developingolfactory receptor sensory neurons. We also investigated theexpression of the gene in the retina. At 5.5 weeks, labeling wasobserved in the outer layer of the optic cup (which laterdifferentiates into the pigment epithelium of the retina). At 7and 8 weeks (Fig. 5 C and D) the labeling in the retina wasrestricted to the retinal pigment epithelium, and it was moreintense at 8 than at 7 weeks. At 19 weeks the expression in thepigment epithelium was barely detectable. In contrast, alabeling of the photoreceptor cells was observed which per-sisted at 24 and 28 weeks (Fig. 5 E and F). In the inner ear ofthe 16.5-day-old mouse embryos only the cochlear and vestib-ular sensory hair cells expressed the myosin VIIA gene. Inaddition, the epithelial cells of the small intestine, hepatocytes,and choroidal plexus were labeled (data not shown).

In recent years considerable attention has been drawn tounconventional myosins in the inner ear sensory hair cells, asthe transduction channel at the tip of the stereocilia of thecochlear and vestibular sensory hair cells is hypothesized to begated by the activity of an unconventional myosin (33, 34).From the human and mouse in situ hybridization data, twomain points emerge. First, all the cell types which have thus farbeen shown to express the myosin VIIA gene, with theexception of the photoreceptor cells, possess microvilli (incochlear and vestibular hair cells they constitute the stereo-cilia). Second, in the human sensory organs, the expression ofthe myosin VIIA gene is concomitant with the appearance ofmicrovilli in the epithelial cells. Considered together, theseresults suggest that myosin VIIA is involved in the develop-ment of the microvillar extensions. In USH1B, both vestibulardysfunction and deafness might result from a defective mor-phogenesis of the stereocilia, the highly specific mechanicalproperties of which are critical for the mechanotransductionprocess.Very little is known about unconventional myosins in the

mammalian retina. All four genes responsible for retinitispigmentosa identified so far are specifically expressed in thephotoreceptor cells; three of them encode proteins involved inthe phototransduction cascade (35). However, the geneticdefect responsible for retinitis pigmentosa in the RCS rat hasbeen shown to be due to a primary pigment epithelial cellanomaly (36). During human embryonic development, themyosin VIIA gene is first expressed by the pigment epithelium,followed by the photoreceptor cells, suggesting that both celltypes might be involved in the retinal degenerative process.However, since the retinitis pigmentosa in USH1B begins to

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FIG. 4. Schematic representation of the myosin VIIA cDNA. Overlapping cDNA clones isolated from a retinal cDNA library are shownunderneath the line representing the complete cDNA (numbering in bp starts from the translation initiation site). The translation initiation siteand the stop codon are indicated, as well as the alternatively spliced exons or part of an exon.

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3236 Developmental Biology: Weil et al.

FIG. 5. In situ hybridization analysis of myosin VIIA gene expression in the human fetus. (A-D) Sections of olfactory epithelium (A), otic vesicle(B), and retina (C and D) at 8 weeks of embryonic development. (E and F) Retinal sections at 28 weeks. A-C and E, antisense probe A; D andF, sense probe A'. A-F are darkfield and C-F are epipolarized light. (A, B, E, and F, x95; C and D, X 190.) In the 8-week-old human fetus, theolfactory epithelium, the otic vesicle, and the retinal pigment epithelium are labeled. In the 28-week-old human fetus the labeling is restricted tothe photoreceptor cells, the pigment epithelium being unlabeled.

develop only at puberty, it will be interesting to determinewhich cell types express the gene in the human eye at this age.In this respect, myosin VIIA has recently been reported to bepresent in the adult rat pigment epithelium but not in thephotoreceptor cells (37); similar localization has been ob-served in the mouse (A.E.-A., unpublished results). Assuminga similar embryonic pattern of expression in mice and humans,this may reflect a biphasic expression of the gene in thepigment cells and a transient expression in the photoreceptorcells. However, alternatively, one must consider that thepattern of expression of the gene in the retina differs from onespecies to another. Such a discrepancy could then account forthe observed presence/absence of a retinal defect associatedwith the myosin VIIA deficiency in humans and mice, respec-tively.

We thank C. Ayer-Le Lievre and G. Couly for their help in analyzingthe in situ hybridization data and P. Kussel for her help in analyzingthe sequence. We are grateful to J.-P. Hardelin and V. Kalatzis forcritical reading of the manuscript. This work was supported by A. andM. Suchert, by the Association Franqaise Retinitis Pigmentosa, and byEuropean Economic Community Grant PL951324.

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