Callosal axon guidance defects in p35(-/-) mice.

Mice lacking p35, an activator of cdk5 in the central nervous system (CNS), exhibit defects in a variety of CNS structures, most prominently characterized by a disruption in the laminar structure of the neocortex (Chae et al., 1997). In addition, alterations of certain axonal fiber tracts are found in the cortex of p35 mutant mice. Notably, the corpus callosum appears bundled at the midline, but dispersed lateral to the midline. Tracer injection experiments in adult p35 mutant mice reveal that projecting cortical axons fail to assimilate into the corpus callosum, and take oblique paths to the midline. After crossing the midline, cortical axons defasciculate prematurely from the corpus callosum and take similarly oblique paths through the cortex. This callosal phenotype is not detected in reeler mice, which also exhibit defects in cortical lamination, suggesting that the lack of fasciculation of callosal axons is not an inherent manifestation of a disruption of cortical lamination. The embryonic callosal axon tract is defasciculated before crossing the midline, suggesting that axon guidance may be affected during embryonic development of the corpus callosum. In addition, embryonic thalamocortical afferents also exhibit a defasciculated phenotype. These results suggest that defective axonal fasciculation and guidance may be primary responses to the loss of p35 in the cortex. Furthermore, this study postulates a role for the p35/cdk5 kinase in molecular signaling pathways necessary for proper guidance of selective axons during embryonic development.

Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue. We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2. The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouse Ate1 gene. Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster) Ate1 genes encode a single form of ATE1p. A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p-GFP is exclusively cytosolic. Mouse ATE1-1p and ATE1-2p were examined by expressing them in ate1Delta Saccharomyces cerevisiae in the presence of test substrates that included Asp-betagal (beta-galactosidase) and Cys-betagal. Both forms of the mouse R-transferase conferred instability on Asp-betagal (but not on Cys-betagal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p. The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is approximately 0.1 in the skeletal muscle, approximately 0.25 in the spleen, approximately 3.3 in the liver and brain, and approximately 10 in the testis, suggesting that the two R-transferases are functionally distinct.

The mouse and human genes encoding the recognition component of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3alpha. Mouse UBR1p (E3alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans approximately 120 kilobases of genomic DNA and contains approximately 50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.