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 essential to normal development as the proteins they encode are involved in specifying 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 detergent-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. Cortical Xlsirts and other localized RNAs are specifically found in the heavy region of sucrose gradients and in the pellet, quite different from other cellular RNPs. Four proteins were identified by UV-crosslinking that bound the Xlsirts localization signal in the cortex, but not in the soluble fraction. These are likely members of the anchoring complex and appear to include vera, a characterized Vg1 RNA binding protein. Vera was found to co-sediment with other locRNAs found in the vegetal cortex, suggesting that it is a common component of locRNPs. Finally, we found that locRNPs extracted into the soluble fraction had the same buoyant density as typical ooplasmic RNPs. We propose that locRNAs are organized and anchored in the cortex as typical RNPs.
Bubunenko
M
M
Department of Cell Biology and Anatomy, University of Miami School of Medicine, 1011 NW 15th Street, Miami, FL 33136, USA.
King
M L
ML
D000818:Animals; D002454:Cell Differentiation; D002499:Centrifugation, Density Gradient; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D007633:Keratins / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism; D016212:Transforming Growth Factor beta; D014466:Ultraviolet Rays; D014981:Xenopus / Q000196:embryology*; D029867:Xenopus Proteins
D002712:Chlorides; C513103:GDF1 protein, Xenopus; D006023:Glycoproteins; D016212:Transforming Growth Factor beta; D029867:Xenopus Proteins; D002586:Cesium; D013395:Sucrose; D012313:RNA; D007633:Keratins; C025407:cesium sulfate; C028019:cesium chloride