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An analysis of UNC-52/perlecan domain IV immunoglobulin repeats in myofilament assembly of Caenorhabditis elegans Bush, Jason Allan


The unc-52(II) gene encodes the nematode homologue of perlecan, a mammalian basement membrane (BM) proteoglycan. UNC-52 is essential for proper myofilament assembly and muscle attachment in C. elegans. The longest predicted UNC-52 protein has five structural domains, including a LDL-receptor-like domain (domain II), a laminin-like domain (domain III), an NCAM-like domain (domain IV) and a globular laminin-like domain (domain V). The unc-52 gene consists of 37 exons which, through alternative splicing, generate a number of protein isoforms. These isoforms show both tissue and temporal specificity. The domain structure of unc-52 lends itself to regulation via alternative splicing. In particular, domain IV contains multiple copies of an immunoglobulin-like repeat (Ig-repeat) and many of the repeats are encoded by individual exons. Alternative splicing of exons 16, 17 and 18 alters the number of Ig-repeats within this domain. Null mutations in unc-52 lead to a lethal Pat (paralyzed arrested elongation at two-fold) phenotype (e.g. st549), while mutations in exons 16-18 lead to a viable Unc phenotype. Through the sequence analysis of two new unc-52 mutations, st560 and ra112, I established that domain IV-containing isoforms of unc-52 are critical for myofilament assembly during early development. These two mutations both lead to a Pat phenotype and result in premature truncation of UNC-52 isoforms with domain IV; st560 is a stop codon in exon 13; and rall2 is an deletion removing several Ig-encoding exons within domain IV. Using a Tel excision and double-strand break repair scheme to vary the number of Ig repeats within an isoform, I addressed the functional significance of domain IV in establishing proper muscle assembly. I sought to determine the minimal number of Ig repeats necessary within domain IV to allow proper myofilament assembly and to determine the role of combinatorial Ig repeats in assembly. I characterized eight in-frame deletion alleles created by Tel excision which eliminate from 1 to 4 Ig repeats. Animals homozygous for these deletions appear wild-type in movement and muscle structure. The mec-8 gene encodes a putative RNA-binding protein that is required for some of the alternative splicing of unc-52 (Lundquist et al., 1996). In the absence of MEC-8 function, otherwise viable mutations in the alternatively-spliced region of unc-52 are lethal because the affected exons are no longer spliced out of embryonic unc-52 pre-mRNA (Lundquist et al., 1996). I constructed several mec-8; unc-52(deletion) double mutants to ask whether MEC-8 function was still required in the absence of its splicing target. I found that mec-8(null); unc-52(viable deletion) combinations are phenotypically wild-type, suggesting a complete independence from mec-8.

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