Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry

Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis

Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358

Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. This interaction is stimulated by RanGTP but remains of a relatively low affinity. In order to identify strong signals, we screened a 15-mer random peptide library for CRM1 binding, both in the presence and absence of RanGTP. Under each condition, strikingly similar signals were enriched, conforming to the NES consensus sequence. A derivative of an NES selected in the absence of RanGTP exhibits very high affinity for CRM1 in vitro and stably binds without the requirement of RanGTP. Localisation studies and RNA interference demonstrate inefficient CRM1-mediated export and accumulation of CRM1 complexed with the high-affinity NES at nucleoporin Nup358. These results provide in vivo evidence for a nuclear export reaction intermediate. They suggest that NESs have evolved to maintain low affinity for CRM1 to allow efficient export complex disassembly and release from Nup358

Comprehensive Detection of Genomic Duplications and Deletions in the DMD Gene, by Use of Multiplex Amplifiable Probe Hybridization

Duplications and deletions are known to cause a number of genetic disorders, yet technical difficulties and financial considerations mean that screening for these mutations, especially duplications, is often not performed. We have adapted multiplex amplifiable probe hybridization (MAPH) for the screening of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. MAPH involves the quantitative recovery of specifically designed probes following hybridization to immobilized genomic DNA. We have engineered probes for each of the 79 exons of the DMD gene, and we analyzed them by using a 96-capillary sequencer. We screened 24 control individuals, 102 patients, and 23 potential carriers and detected a large number of novel rearrangements, especially small, one- and two-exon duplications. A duplication of exon 2 alone was the most frequently occurring mutation identified. Our analysis indicates that duplications occur in 6% of patients with DMD. The MAPH technique as modified here is simple, quick, and accurate; furthermore, it is based on existing technology (i.e., hybridization, PCR, and electrophoresis) and should not require new equipment. Together, these features should allow easy implementation in routine diagnostic laboratories. Furthermore, the methodology should be applicable to any genetic disease, it should be easily expandable to cover >200 probes, and its characteristics should facilitate high-throughput screening

A Supraphysiological Nuclear Export Signal Is Required for Parvovirus Nuclear Export

CRM1 exports proteins that carry a short leucine-rich peptide signal, the nuclear export signal (NES), from the nucleus. Regular NESs must have low affinity for CRM1 to function optimally. We previously generated artificial NESs with higher affinities for CRM1, termed supraphysiological NESs. Here we identify a supraphysiological NES in an endogenous protein, the NS2 protein of parvovirus Minute Virus of Mice (MVM). NS2 interacts with CRM1 without the requirement of RanGTP, whereas addition of RanGTP renders the complex highly stable. Mutation of a single hydrophobic residue that inactivates regular NESs lowers the affinity of the NS2 NES for CRM1 from supraphysiological to regular. Mutant MVM harboring this regular NES is compromised in viral nuclear export and productivity. In virus-infected mouse fibroblasts we observe colocalization of NS2, CRM1 and mature virions, which is dependent on the supraphysiological NS2 NES. We conclude that supraphysiological NESs exist in nature and that the supraphysiological NS2 NES has a critical role in active nuclear export of mature MVM particles before cell lysis