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doi: 10.1242/10.1242/dev.00604

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Alternative splicing affecting a novel domain in the C. elegans EGL-15 FGF receptor confers functional specificity

S. Jay Goodman¹, Catherine S. Branda^2,*, Matthew K. Robinson^2,, Rebecca D. Burdine^1, and Michael J. Stern^2,

¹ Department of Cell Biology, Yale University School of Medicine, I-354 SHM PO Box 208005, New Haven, CT 06520-8005, USA
² Department of Genetics, Yale University School of Medicine, I-354 SHM PO Box 208005, New Haven, CT 06520-8005, USA

View larger version (21K): [in a new window]   Fig. 5. LET-756 can mediate SM chemoattraction. SM distributions in an egl-17(null) background. EGL-17 expressed from its native promoter consistently rescues egl-17(null) at both high and low concentrations. Expression of LET-756 from the egl-17 promoter can rescue the egl-17(null) chemoattraction defect in a subset of lines at a high concentration, but fails to rescue at the lower concentration. The egl-17(null) allele used was n1377. The control array contained only the transformation marker plasmid. The data for lines marked are also represented for comparison in Fig. 6. Asterisks indicate dorsally positioned SMs. % nEgl, percent non-Egl transgenic animals.

View larger version (19K): [in a new window]   Fig. 1. Mutation analysis of egl-15 alleles. Alleles are graphically positioned based on the severity of their phenotype (horizontal axis) and the location of their lesion (vertical axis). The axis representing the level of the essential function of EGL-15 signaling activity is portrayed above by the triangle, with minimal activity corresponding to the Let phenotype and near normal activity corresponding to the Soc phenotype. Nonsense mutations are boxed, splice site mutations are represented in parentheses. n1457 and the other egl-15(Egl) alleles break this allelic series. An additional group of mutations isolated as suppressors of clr-1 (Soc) were characterized only in a clr-1(e1745ts) background: n2217(W633Opal), n2182(A650V), n2189(P876L), n2184(G890E), n2203(G871S) and n2206(W930Amber). The full-length EGL-15(5B) type 1 isoform is 1040 amino acids in length.

View larger version (15K): [in a new window]   Fig. 3. Splice variation of the EGL-15 C terminus. Analysis of egl-15 transcripts revealed multiple splice variants affecting the very C terminus of EGL-15. The C-terminal egl-15 exons are depicted on the left. Thickly edged rectangles represent alternate exons used between the common exons 17 and 19. Shading indicates coding regions. The numbers of cDNAs of each type isolated from each pool are shown on the right. C-terminal alternate forms are represented in approximately equal proportions in both 5A- and 5B-containing isoforms. Two YK ESTs extend to exon 5; both yk36b5 (Type 1) and yk322c4 (Type 3) contain exon 5B. Asterisks indicate stop codons.

View larger version (21K): [in a new window]   Fig. 2. egl-15(5A) mutations result in posteriorly displaced SMs. (A) Distributions of SMs in egl-15(Egl) mutants are represented as box-and-whisker plots (see Materials and Methods). Asterisks indicate dorsally positioned SMs. All four egl-15(Egl) alleles display a similar distribution of posteriorly displaced SMs. The distribution of SMs in the egl-15(Soc) mutant, n1783, is shown for comparison. (B) An extracellular alternate splicing event gives rise to two distinct receptor isoforms. Three out of four egl-15(Egl) alleles contain point mutations that specifically affect the 5A exon. (C) Alternate splicing of exon 5 encoding the EGL-15-specific insert results in two distinct isoforms that specifically differ in this domain. A simple, modular hypothesis for normal signaling specificity is suggested by the phenotypic similarities of mutations affecting the ligands and receptor isoforms. Misexpression of the ligands can allow them to stimulate the heterologous function via the heterologous receptor isoform.

View larger version (23K): [in a new window]   Fig. 4. EGL-15 splice variants mediate different responses. (A) The wild-type egl-15 genomic rescuing fragment [EGL-15(5A+B+)] rescues defects associated with the egl-15 (null) allele, including defects in both the essential role and SM chemoattraction. The essential role is also restored by expression of EGL-15(5A-B+); however, these lines exhibit aberrant SM migration. The EGL-15(5A+B-) transgene failed to restore the essential function in this null mutant, but rescued the chemoattraction defect of the egl-15(Egl) mutant, egl-15(n1458), based on final SM positions. (B) SM distributions for two transgenic lines for each of the constructs shown in A. As these transgenic animals bear extrachromosomal arrays that are mitotically lost at a significant frequency, some of these SMs may have lost the transgene. Complete loss of EGL-15 in the SMs results in centrally dispersed SMs (C.S.B., S.J.G. and M.J.S., unpublished), probably accounting for the outlying SMs.

View larger version (23K): [in a new window]   Fig. 6. LET-756-mediated SM chemoattraction requires EGL-15(5A) activity. The egl-15(5A-5B+) allele n484 was introduced into the background of egl-17(null) rescued transgenic lines from Fig. 5. Removal of EGL-15(5A) in this manner results in the posterior displacement of SMs in all lines tested, demonstrating that this receptor isoform is crucial for SM chemoattraction independent of the ligand used as the chemoattractant.