Biochemical characterization of a cellular structure retaining vegetally localized RNAs in Xenopus late stage oocytes

Two pathways operate during Xenopus oogenesis to localize a small number of RNAs to the vegetal cortex. Correct localization of these RNAs is essentia l to normal development as the proteins they encode are involved in specify ing cell type and in patterning the early embryo. Binding these RNAs to the vegetal cortex and thus preserving their localized condition is a critical step, although little is known about how this is achieved. In this study, we have used a biochemical approach to examine the anchoring step. Xlsirts, an abundant localized RNA (locRNA), was selectively enriched in a detergen t-insoluble fraction (DIF) prepared from oocytes that had completed the RNA localization process. These putative RNA-anchoring complexes were analyzed by density gradient centrifugation and in RNA-protein binding assays. Cort ical Xlsirts and other localized RNAs are specifically found in the heavy r egion of sucrose gradients and in the pellet, quite different from other ce llular RNPs. Four proteins were identified by UV-crosslinking that bound th e Xlsirts localization signal in the carter, but not in the soluble fractio n. These are likely members of the anchoring complex and appear to include vera, a characterized Vg1 RNA binding protein. Vera was found to co-sedimen t with other locRNAs found in the vegetal cortex, suggesting that it is a c ommon component of locRNPs. Finally, we found that locRNPs extracted into t he soluble fraction had the same buoyant density as typical ooplasmic RNPs. We propose that locRNAs are organized and anchored in the cortex as typica l RNPs. J. Cell. Biochem. 80:560-570, 2001. (C) 2001 Wiley-Liss, Inc.