Cloning and functional studies of a novel gene aberrantly expressed in RB-deficient embryos

The tumor suppressor RE regulates diverse cellular processes such as G1/S transition, cell differentiation, and cell survival. Indeed, Rb-knockout mice exhibit phenotypes including ectopic mitosis, defective differentiation, and extensive apoptosis in the neurons. Using differential display, a novel gene, Rig-1, was isolated based on its elevated expression in the hindbrain and spinal cord of Rb-knockout embryos. The longest open reading frame of Rig-1 encoded a polypeptide that consists of a putative extracellular segment with five immunoglobulin-like domains and three fibronectin III-like domains, a putative transmembrane domain, and a distinct intracellular segment. The Rig-1 sequence was 40% identical to the recently identified roundabout protein. Consistent with the predicted transmembrane nature of the protein, Rig-i protein was present in the membranous fraction. Antisera raised against the putative extracellular and intracellular segments of Rig-i reacted with an similar to 210-kDa protein in mouse embryonic CNS. Rig-i mRNA was transiently expressed in the embryonic hindbrain and spinal cord. Elevated levels of Rig-i mRNA and protein were found in Rb-/- embryos. Ectopic expression of a transmembrane form of Rig-1, but not the secreted form, promoted neuronal cell entrance to S phase and repressed the expression of a marker of differentiated neuron, T alpha 1 tubulin. Thus Rig-1, a possible distant relative of roundabout, may mediate some of the pleiotropic roles of RE in the developing neurons. (C) 1999 Academic Press.