Cloning of the murine unconventional myosin gene Myo9b and identification of alternative splicing

Abstract

We report the cloning of a cDNA for the mouse unconventional myosin Myo9b, the orthologue of the rat myr5 and human MYOIXb genes. A full-length spleen cDNA of 7087 bp encoding a protein of 1961 amino acids was isolated. By RT–PCR, we show that Myo9b is expressed in a wide range of tissues, including heart, brain, muscle and inner ear. In addition, we have identified two alternatively spliced exons. Equivalent exons have not been previously reported for either the human or rat homologues. These exons are located in the Myo9b specific actin-binding site insert of the head domain and in the tail region. A third splice form utilizing an alternative reading frame within the 3′UTR is also described. Several polymorphisms within the coding region were identified; of interest is an in-frame 33 bp imperfect duplication within the tail region that was observed only in the C57Bl/6 strain. Myo9b has been previously mapped to mouse chromosome 8 and is a candidate for the mouse mutations myodystrophy and quinky.

Introduction

Myosins are molecular motors that convert chemical energy, in the form of ATP, to mechanical force exerted against actin. Since the description of the first class of myosins in 1987, 14 additional classes, based on phylogenetic comparisons of the motor domains and features of the tail, have been identified (Sellers et al., 1996, Wang et al., 1998). These classes have been numbered chronologically. Unconventional myosins (i.e. anything not resembling the two-headed, filament forming conventional myosins of class-II) are thought to play important roles in cell migration, endocytosis and intracellular transport (Mermall et al., 1998, Mooseker and Cheney, 1995, Titus, 1997). The particular importance of myosins in a number of sensory systems, including hearing, balance and vision, has been well documented (Hasson and Mooseker, 1997). In fact, of the four mapped myosins associated with diseases, three have been implicated in human and/or murine deafness; myosin VI in the Snell's waltzer mouse mutation (Avraham et al., 1995), myosin VIIa in human Usher syndrome and mouse shaker-1 (Gibson et al., 1995, Weil et al., 1995), and MYO15 in human nonsyndromic deafness DFNB3 and mouse shaker-2 (Probst et al., 1998, Wang et al., 1998).

Human MYOXIb belongs to the same class as the fifth unconventional myosin identified from rat, myr5 (Reinhard et al., 1995, Wirth et al., 1996). Phylogenetically, myr5 is the most distant relative of conventional myosin (myosin-II). A closely related gene myr7 has recently been cloned from rat and human (Chieregatti et al., 1998). The MYR5/MYOIXb protein contains several functional domains not found previously in myosins, including a novel N-terminal domain of 140 amino acids and a 126-amino-acid insertion in the putative-actin binding domain, which has been proposed to interact with F-actin in a standard, ATP-sensitive fashion (Reinhard et al., 1995). In addition, the tail domain contains a putative GTPase-activating protein (GAP) domain of the rho/rac family of ras-like G-proteins (Reinhard et al., 1995, Wirth et al., 1996). It has been proposed that myr5 could be involved in the regulation of the organization of the actin filaments along which it might produce force and movement (Reinhard et al., 1995). Northern blot analysis of both myr5 and MYOIXb has shown that these myosins are widely expressed but show considerable variability in expression level, being especially concentrated in peripheral blood leukocytes (Reinhard et al., 1995, Wirth et al., 1996). Using anti-myr5 antibodies in immunofluorescence, Wells et al. (1997) showed Myo9b, along with several other members of the myosin superfamily, to be expressed at higher levels in murine myoblasts than in mature myotubes.

To date, myosin IXb cDNAs have only been cloned from human (Bement et al., 1994, Wirth et al., 1996) and rat (Reinhard et al., 1995). Here, we report a full-length cDNA for the mouse. Several alternative splice forms of murine Myo9b are described. In addition, an unusual polymorphism consisting of an in-frame imperfect duplication within the coding region of the gene has been identified.