The characterization of two proteins that bind to the localization element of Xenopus Vg1 mRNA.

摘要

Vg1 is a maternal mRNA that is localized to the vegetal pole during the middle stages of oogenesis and encodes a member of the transforming growth factor-beta (TGF-beta) family of proteins. A 340-nt region, the Vg1 localization element (VLE), within 3'UTR of this mRNA is sufficient for the proper localization. Two clones that encode proteins that bind to this region have been isolated. One of the proteins has two RNP motifs at the N-terminal and a proline-rich domain at the C-terminal and is named proline-rich RNA-binding protein (Prrp). Mobility shift assays show that Prrp binds with high affinity and specificity to VLE RNA in vitro. An immunoprecipitation assay determined that, in oocytes, Prrp is associated with Vg1 mRNA as well as VegT mRNA, which like Vg1 mRNA uses the late localization pathway, but not with Xcat-2 or Xwnt-11 mRNAs, which use the early pathway. Surprisingly, the immunoprecipitation assay also showed that Prrp binds to An1 and An3 mRNAs, which are localized to the animal pole, indicating that Prrp does not determine the direction of RNA movement. Prrp and Vg1 mRNA were shown to colocalize to the vegetal cortex in stage IV oocytes using fluorescence in situ hybridization and immunocytochemistry with confocal microscopy. Furthermore, a blot overlay assay revealed that the proline-rich domain of Prrp interacts with profilin, which is an actin-associated protein that regulates both the distribution and dynamics of microfilament assembly. This result suggests that Prrp may play a role in building connections between Vg1 mRNA and actin microfilaments.;The second clone is a Xenopus homologue of a KH-type splicing regulatory protein (KSRP) and a transcription factor, FUSE binding protein 2 (FBP2), which contains four K homology RNA-binding domains. FBP2/KSRP also binds with high affinity to VLE RNA in vitro. However, immunoprecipitation assays show that FBP2/KSRP is associated with not only Vg1 and VegT, but also Xcat-2 and Xwnt-11, indicating that FBP2/KSRP is either a general factor involved in RNA localization or involved in other types of RNA processing.