ABSTRACT
The Ran-binding protein 2 (RanBP2) is a vertebrate mosaic protein composed of four interspersed RanGTPase binding domains (RBDs), a variable and species-specific zinc finger cluster domain, leucine-rich, cyclophilin, and cyclophilin-like (CLD) domains. Functional mapping of RanBP2 showed that the domains, zinc finger and CLD, between RBD1 and RBD2, and RBD3 and RBD4, respectively, associate specifically with the nuclear export receptor, CRM1/exportin-1, and components of the 19 S regulatory particle of the 26 S proteasome. Now, we report the mapping of a novel RanBP2 domain located between RBD2 and RBD3, which is also conserved in the partially duplicated isoform RanBP2L1. Yet, this domain leads to the neuronal association of only RanBP2 with two kinesin microtubule-based motor proteins, KIF5B and KIF5C. These kinesins associate directly in vitro and in vivo with RanBP2. Moreover, the kinesin light chain and RanGTPase are part of this RanBP2 macroassembly complex. These data provide evidence of a specific docking site in RanBP2 for KIF5B and KIF5C. A model emerges whereby RanBP2 acts as a selective signal integrator of nuclear and cytoplasmic trafficking pathways in neurons.
Subject(s)
Microtubule-Associated Proteins/metabolism , Nuclear Pore Complex Proteins/metabolism , Animals , Binding Sites , Biological Transport , Brain Chemistry , Cattle , Microtubule-Associated Proteins/chemistry , Molecular Chaperones , Molecular Weight , Nuclear Pore Complex Proteins/chemistry , Rabbits , Retina/chemistryABSTRACT
The Ran-binding protein 2 (RanBP2) is a giant scaffold and mosaic cyclophilin-related nucleoporin implicated in the Ran-GTPase cycle. There are no orthologs of the RanBP2 gene in yeast and Drosophila genomes. In humans, this bona fide gene is partially duplicated in a RanBP2 gene cluster and lies in a hot spot for recombination on Chromosome (Chr) 2q. This genetic heterogeneity renders further significance of this genomic region in human disease due to its possible involvement in genetically linked disorders such as juvenile nephronophthisis, congenital hepatic fibrosis, and chorioretinal dysplasia. Structure-function studies on bovine RanBP2 indicate that this protein is involved in integrating nucleocytoplasmic transport pathways with protein biogenesis such as production of functional opsin. To gain further insight into the complex functions of RanBP2 in the development and function of the neuroretina and other tissues, and proceed towards the functional analysis of RanBP2 and its molecular partners in vivo, we have determined the complete genomic organization of the murine RanBP2 gene. The gene consists of 29 exons spread over 50 kb and contains a mega-exon of 4663 bp that encompasses the variable Zn-finger-rich domain of RanBP2. This may account, in part, for a predisposition of recombination of this locus and variability of the number of Zn-fingers across mammalian species. The RanBP2 promoter contains tissue-specific elements. A CpG island encompasses this region up to the first intron, making RanBP2 gene expression susceptible of epigenetic regulation. This murine RanBP2 transcript has a tissue-restricted expression profile, and the conceptual protein is 82% identical to human RanBP2. The gene maps to mouse Chr 10, 30 cM proximal of the centromere.
Subject(s)
DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Nuclear Pore Complex Proteins , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Animals , Base Sequence , Blotting, Southern , Cell Nucleus/metabolism , Chromosome Mapping , Chromosomes, Human, Pair 2 , CpG Islands , Cytoplasm/metabolism , DNA, Complementary/metabolism , DNA-Binding Proteins/chemistry , Exons , Female , Gene Library , Humans , Introns , Male , Mice , Mice, Inbred C57BL , Models, Genetic , Molecular Chaperones , Molecular Sequence Data , Nuclear Proteins/chemistry , Physical Chromosome Mapping , Protein Binding , Protein Structure, Tertiary , Recombination, Genetic , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Tissue DistributionSubject(s)
Aneurysm/complications , Celiac Artery , Hepatic Artery , Splenic Artery , Thrombosis/complications , Acute Disease , Aneurysm/surgery , Female , Hepatic Artery/surgery , Humans , Male , Middle Aged , Splenectomy , Splenic Artery/surgery , Splenic Infarction/etiology , Splenic Infarction/surgery , Thrombosis/surgeryABSTRACT
Due to the complete absence of ribosomal DNA (genetic symbol bb-), the Xbb- chromosome of Drosophila is lethal both in homozygous conditions and in compound with the Xbb- chromosome. However, in the cross between the C(1)RM/Ybb- females and the Xbb-/BSYbb+ males, characterized by the development of lethal Xbb-/Ybb- zygotes, two fertile males were detected. These males possessed all the markers of the Xbb- chromosome but lacked the Y chromosome BS marker. Genetic analysis of their progeny showed that genes responsible for restoration of viability and fertility of these exceptional males were associated with the X chromosome. The crossover tests showed that in one case these genes were tightly linked to the w locus (the bbAM1 allele), and in the second case they were located 12.6 map units to the right of the Tu locus (the bbAM7 allele). It has also been shown that the bb locus was transposed to the X chromosome within the short arm of Y chromosome. Transposition of the BSYbb+ chromosome-specific rDNA sequences to the X chromosome was confirmed by means of Southern blotting. These data indicate that replacement of the bb locus is realized by transposition rather than recombination.
Subject(s)
DNA Transposable Elements , Drosophila melanogaster/genetics , Animals , Blotting, Southern , Crossing Over, Genetic , DNA, Ribosomal , Drosophila melanogaster/enzymology , Female , Homozygote , Male , X Chromosome , Y ChromosomeABSTRACT
Molecular analysis of several mutations of the white locus was performed. The mutations were obtained as a result of mutagenesis in the unstable white-starka locus described previously. A large insert (approximately 15 kb in length) downstream of the first exon of the white gene was shown to be partly excised. Simultaneous excisions of various regions from the natural regulatory region of the white locus were also observed. Restriction enzyme analysis showed that the observed mutations and reversions resulted from large deletions from the regulatory gene region. Southern and Northern blot hybridization showed the presence of unknown regulatory regions within and outside the insert.
Subject(s)
ATP-Binding Cassette Transporters , Alleles , Drosophila Proteins , Drosophila/genetics , Eye Proteins , Insect Proteins/genetics , Mutation , Animals , Blotting, Northern , Blotting, Southern , Chromosome Mapping , Regulatory Sequences, Nucleic Acid , Sequence DeletionABSTRACT
In 1992, Bashkirov et al. described the case of occurrence in line C(1)RM, yw/0; Dp(1; 3) wvco/+ of a unique female with light, uniformly colored eyes. This trait was not inherited together with Dp(1; 3) wvco but was linked to the X chromosome. We denoted this new allele as white-starka (wstr). It proved to be unstable, demonstrating mutational transitions to other allelic states with frequencies of 10(-3) to 10(-5). Southern blot analysis showed that the wstr mutation was induced by an insertion of an unknown sequence with a length approximately evaluated by the authors in 1992 as 7 kb. Using a wider spectrum of restriction endonucleases and a prolonged gel running, we were able to estimate the size of this insertion more precisely. According to our data, in the case of wstr we are dealing with an insertion in the locus white of a sequence 15.1 kb long. As seen from our restriction map of the allele wstr, this insertion does not resemble retroposons with long terminal repeats, but is possibly similar to LINE mobile elements of Het-A and TART types, characteristic for heterochromatin.