Structure-functional analysis of the extracellular domain of the roughest cell adhesion molecule in Drosophila melanogaster using the CRISPR/Cas9 methodology

Abstract

The roughest (rst) gene shows pleiotropic behaviour and its role in several processes during the development of Drosophila melanogaster has been described, such as axonal pathfinding, larval salivary gland histolysis, myoblast fusion during the formation of embryonic musculature and differentiation of pigment cells during compound eye development. The gene encodes a single-pass transmembrane protein containing five immunoglobulin domains in its extracellular portion (509 amino acids) and a cytoplasmic tail of 208 amino acids, rich in serine and threonine residues, besides functionally important subdomains - like the glutamine rich portion known as opa-like domain and a PDZ domain at the carboxyl-terminus of the protein. Overexpression of constructs where the intracellular portion of the protein was completely or partially deleted lead to salivary gland histolysis and compound eye development defects, interruption of blastoderm cellularization and disruption ofembryonic central nervous system midline formation. However, the phenotypes derived from complete or partial deletion of the Rst extracellular domains have not been investigated. To explore the functional role of these domains two constructions will be generated by CRISPR/Cas9 technology: one deleting 441 aa of the extracellular domain and another that partially deletes only the two Ig-domains nearest to the N-terminal region, keeping the glycine spacer sequence and removing 149 amino acids. Both deletions begin 60 amino acids after the start codon and retain the transmembrane region and the signal peptide. Thus, it will be possible to assess whether these deletions are responsible for generating new phenotypes or phenotypes that are similar to ones already observed in deletions of different regions of the Roughest protein.