Complex Patterns of Alternative Splicing Mediate the Spatial and Temporal Distribution of Perlecan/UNC-52 inCaenorhabditis elegans

Abstract

The unc-52 gene encodes the nematode homologue of mammalian perlecan, the major heparan sulfate proteoglycan of the extracellular matrix. This is a large complex protein with regions similar to low-density lipoprotein receptors, laminin, and neural cell adhesion molecules (NCAMs). In this study, we extend our earlier work and demonstrate that a number of complex isoforms of this protein are expressed through alternative splicing. We identified three major classes of perlecan isoforms: a short form lacking the NCAM region and the C-terminal agrin-like region; a medium form containing the NCAM region, but still lacking the agrin-like region; and a newly identified long form that contains all five domains present in mammalian perlecan.Using region-specific antibodies and unc-52 mutants, we reveal a complex spatial and temporal expression pattern for these UNC-52 isoforms. As well, using a series of mutations affecting different regions and thus different isoforms of UNC-52, we demonstrate that the medium NCAM-containing isoforms are sufficient for myofilament lattice assembly in developing nematode body-wall muscle. Neither short isoforms nor isoforms containing the C-terminal agrin-like region are essential for sarcomere assembly or muscle cell attachment, and their role in development remains unclear. INTRODUCTIONBasement membranes are specialized regions of extracellular matrix (ECM) that have important roles in many fundamental developmental and regenerative processes, including cell adhesion and migration, signal transduction, and even gene regulation (Martin and Timpl, 1987;Yurchenco and Schittny, 1990). Many of these processes are mediated by specific interactions between basement membrane components and transmembrane receptors such as integrin (Hynes, 1992). Basement membranes contain a large number of different components, including laminin, collagen, nidogen, and heparan sulfate proteoglycans (Yurchenco and O'Rear, 1994;Timpl and Brown, 1996). Homologues of these proteins have been identified in the nematode Caenorhabditis elegans (reviewed in Kramer, 1997), and mutations are associated with several of these components (Guo et al., 1991;Ishii et al., 1992;Rogalski et al., 1993;Sibley et al., 1993). This genetic approach is helping to reveal the function of these basement membrane proteins during morphogenesis. In this study, we focus on perlecan, the major basement membrane heparan sulfate proteoglycan, and its role in muscle development in C. elegans.In C. elegans, a specialized basement membrane underlies the body-wall muscles and anchors the myofilament lattice through integrin-containing adhesion complexes (reviewed in Moerman and Fire, 1997). In adult animals, there are 95 body-wall muscle cells arranged in four quadrants, two dorsal and two ventral, beneath the hypodermis (reviewed in . Each quadrant runs the length of the animal and consists of a double row of spindle-shaped cells. Within each muscle cell, the thin and thick filaments of the myofilament lattice lie just beneath the plasma membrane ...

Selective assembly of fibulin‐1 splice variants reveals distinct extracellular matrix networks and novel functions for perlecan/UNC‐52 splice variants

“…In late larval development, an MEC-8 -dependent alternate splice of IG8-IG10 within the M isoform is required for myofilament organization and function (Lundquist et al, 1996;Mullen et al, 1999). Our data suggest that this splice variant is also required late in larval development for assembly of fibulin-1D and possibly other ECM proteins at the uterine attachment.…”

“…1). A region of thick extracellular matrix assembles between utse and the seam cells that contains several basement membrane components that are conserved in all metazoans, including perlecan, hemicentin, and fibulins-1C and -1D (White, 1988;Mullen et al, 1999;Vogel and Hedgecock, 2001;Muriel et al, 2005).…”

“…We think that this possibility is less likely for multiple reasons. First, perlecan null alleles are recessive [i.e., reducing perlecan gene dosage by 50% has no effect on phenotype (Williams and Waterston, 1994;Mullen et al, 1999)]. In addition, nonsense mutations within this alternately spliced region [e.g., unc-52 (e444)] disrupt accumulation of perlecan in adult body wall muscle basement membranes, but accumulation of perlecan at uterine attachments and pharynx are relatively unaffected (Mullen et al, 1999).…”

“…Nonsense mutations within alternately spliced exons affect a subset of unc-52 gene products and result in animals that survive into adulthood, but become progressively paralyzed. In these mutants, embryonic and larval accumulation of perlecan in body wall muscle basement membranes is relatively normal, although perlecan accumulation in adult body wall muscle is noticeably decreased (Mullen et al, 1999;Rogalski et al, 2001). Alternate splicing of perlecan is also found in mouse, including long, medium, and short forms with potential splicing within these domains that is reminiscent of the nematode splice pattern (Noonan and Hassell, 1993;Joseph et al, 1996).…”

“…In addition to nidogen, which interacts with vertebrate fibulin-1C at a 30-fold higher affinity than with fibulin-1D (Sasaki et al, 1995), a candidate to interact with fibulin is perlecan, the product of the unc-52 gene in C. elegans. Like fibulin-1, perlecan is found at hemidesmosome-mediated uterine attachments, although a function there has not been described (Mullen et al, 1999). In C. elegans, perlecan/UNC-52 is found in the basement membrane between body wall muscle and hypodermis and is required for muscle cell polarity and myofilament assembly (Rogalski et al, 1993).…”