The localization within oocytes of certain mRNAs encoding transcription and growth factors is among the earliest observed steps in the acquisition of positional information specifying body plan. The mechanism by which mRNAs are targeted to specific cytoplasmic regions is unknown. I propose to investigate the mechanism of mRNA localization by applying current biochemical and molecular biological techniques to this problem utilizing Xenopus oocytes as an experimental system. Vg1 is a TGF-Beta-like growth factor which induces dorsal mesoderm in the Xenopus blastula. Proper function of Vg1 depends on localization of it's mRNA to the vegetal cortex of the oocyte. This localization is directed by a 340 nt localization element (LE) in the 3'UTR of the transcript. I propose to map specific functional sequences within this element using in vitro mutagenesis of the Vg1 LE and an in vivo assay for localization. Localization of reporter sequences fused to mutant Vg1 elements will be monitored by a rapid whole mount in situ hybridization method. Once important sequences, and/or point mutants have been defined in the Vg1 LE which block localization, immobilized transcripts of the wildtype LE will be used to affinity purify components of the RNA localization machinery from oocyte extracts using the mutant localization element transcripts as negative controls. Using 2-D gel electrophoresis to compare protein populations bound to wild type and mutant Vg1 LE's will allow identification of specifically binding proteins. These will be cloned-and characterized as candidate localization factors. Function of these various proteins will be tested in both in vivo through examination of disruption of their expression. Function will also be tested in vitro by cosedimentation assays with purified cytoskeletal filaments and by reconstitution of RNA localization activities.
| Arn, Eric A; Cha, Byeong J; Theurkauf, William E et al. (2003) Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins. Dev Cell 4:41-51 |
