Studies on the molecular and functional analysis of tail-anchored membrane associated protein (SLMAP) in Drosophila melanogaster

Abstract

Previous work in our lab identified a novel gene encoding a unique family of tail-anchored alpha-helical coiled-coil proteins termed SLMAPs (sarcolemmal membrane associated proteins) that were highly conserved in rodents and humans. Alternative splicing yields three SLMAP isoforms: a ubiquitously expressed isoform (SLMAP3), and two tissue-specific variants (SLMAP1 and SLMAP2). The salient features of SLMAP are a hydrophobic C-terminal sequence, which targets membranes, a central coiled-coil structure and N-terminal fork head associated domain (FHA). A sequence in fruit flies (corresponding to CG17494 in flybase) was identified that shares 28.6% identity with the full-length mammalian SLMAP3 amino acid sequence. However, a 55.9% identity was identified at the fork head associated domain of Drosophila and SLMAP3 isoform, whereas a 25% identity was shared at the C-terminal transmembrane domain. The rest of the protein was predicted to form a coiled-coil structure. In view of these similarities with the mammalian SLMAP3 isoform, this Drosophila gene was designated Dslmap. In situ hybridization on whole mount embryos revealed a ubiquitous expression of Dslmap mRNA throughout various stages of embryonic development. To study the function of Dslmap during embryonic development, a GAL4-driven hairpin-induced RNA interference (RNAi), as well as overexpression experiments were performed. The Gal4/UAS binary system was used to allow hairpin RNA to conditionally silence Dslmap expression in Drosophila. A pan-neuronal driver (Elav-Gal4) that expresses GAL4 in every cell of the nervous system from embryogenesis onward was mated to transgenic flies carrying the activatable Dslmap RNAi vectors. The resulting phenotypes revealed dramatic defects in the development of axon scaffold in the fly central nervous system (CNS) with particular structural defects within the longitudinal connectives and commissures. An overexpression construct was also made using pUAST vector carrying the full length Dslmap cDNA in frame with a yeast upstream activator sequence (UAS) to generate transgenic lines. Overexpression in the transgenic lines was activated by mating with the CNS Gal4 lines, which resulted in phenotypes that revealed similar defects in the CNS to those observed in the RNAi lines. These data revealed that the Drosophila genome contains a SLMAP homologue, which is ubiquitously expressed and plays a critical role in developing neurons and glia.