Cloning and Functional Studies of a Novel Gene Aberrantly Expressed in RB-Deficient Embryos

Abstract

The tumor suppressor RB 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 ofRb-knockout embryos. The longest open reading frame ofRig-1encoded 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-1 protein was present in the membranous fraction. Antisera raised against the putative extracellular and intracellular segments of Rig-1 reacted with an ∼210-kDa protein in mouse embryonic CNS. Rig-1 mRNA was transiently expressed in the embryonic hindbrain and spinal cord. Elevated levels of Rig-1 mRNA and protein were found inRb^−/−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, Tα1 tubulin. ThusRig-1,a possible distant relative ofroundabout,may mediate some of the pleiotropic roles of RB in the developing neurons.