Research objectives and content
To study nuclear RNA export, an in vitro approach is proposed based on the assembly of synthetic nuclei in Xenopus egg extracts. These nuclei are functional for protein import, they transcribe the intranuclear U1 snRNA reporter gene and they actively export the RNA. The aim is to elucidate the mechanism by which the cap binding complex (CBC) mediates U snRNA export and to identify putative other factors involved. Affinity purified antibodies raised against CBC will be used in this system to inhibit export directly and to immunodeplete extracts for CBC. Mutant versions of recombinant CBC will be added to the depleted extracts in an attempt to determine the region of CBC that is required for export. Additionally, extracts will be biochemically fractionated in order to identify other factors involved in snRNA export. A similar approach will be used to study nuclear export of different classes of RNA.
Training content (objective, benefit and expected impact)
In Dr. Mattaj's lab there is a lot of experience and knowledge about nucleocytoplasmic transport and many recombinant (mutant) transport factors are available. With the described project I could profit from this and learn new techniques (e.g. expression/purification of recombinant proteins, chromatography) and extend my knowledge in the field.