A perinucleolar compartment contains several RNA polymerase III transcripts as well as the polypyrimidine tract-binding protein, hnRNP I.

We have investigated the subcellular organization of the four human Y RNAs. These RNAs, which are transcribed by RNA polymerase III, are usually found complexed with the Ro autoantigen, a 60-kD protein. We designed 2'-OMe oligoribonucleotides that were complementary to accessible single-stranded regions of Y RNAs within Ro RNPs and used them in fluorescence in situ hybridization. Although all four Y RNAs were primarily cytoplasmic, oligonucleotides directed against three of the RNAs hybridized to discrete structures near the nucleolar rim. We have termed these structures "perinucleolar compartments" (PNCs). Double labeling experiments with appropriate antisera revealed that PNCs are distinct from coiled bodies and fibrillar centers. Co-hybridization with a genomic DNA clone spanning the human Y1 and Y3 genes showed that PNCs are not stably associated with the transcription site for these Y RNAs. Although 5S rDNA was often located near the nucleolar periphery, PNCs are not associated with 5S gene loci. Two additional pol III transcripts, the RNA components of RNase P and RNase MRP, did colocalize within PNCs. Most interestingly, the polypyrimidine tract-binding protein hnRNP I/PTB was also concentrated in this compartment. Possible roles for this novel nuclear subdomain in macromolecular assembly and/or nucleocytoplasmic shuttling of these five pol III transcripts, along with hnRNP I/PTB, are discussed.

Xenopus Ro ribonucleoproteins: members of an evolutionarily conserved class of cytoplasmic ribonucleoproteins.

Ro small ribonucleoproteins consist of a 60-kDa protein and possibly additional proteins complexed with several small RNA molecules. The RNA components of these particles, designated Y RNAs, are about 100 nt long. Although these small ribonucleoproteins are abundant components of a variety of vertebrate species and cell types, their subcellular location is controversial, and their function is completely unknown. We have identified and characterized the Ro RNPs of Xenopus laevis. Three of the four distinct Xenopus Y RNAs appear to be related to the previously sequenced human hY3, hY4, and hY5 RNAs. The fourth Xenopus Y RNA, xY alpha, does not appear to be a homologue of any of the human Y RNAs. Each of the human and Xenopus Y RNAs possesses a conserved stem that contains the binding site for the 60-kDa Ro protein. Xenopus and human 60-kDa Ro proteins are 78% identical in amino acid sequence, with the conservation extending throughout the entire protein. When human hY3 RNA is mixed with Xenopus egg extracts, the human RNA assembles with the Xenopus Ro protein to form chimeric Ro ribonucleoproteins. By analyzing RNA extracted from manually enucleated oocytes and germinal vesicles, we have determined that Y RNAs are located in the oocyte cytoplasm. By examining the distribution of mouse Ro ribonucleoproteins in cytoplast and karyoplast fractions derived from L-929 cells, we have determined that Ro ribonucleoprotein particles also primarily reside in the cytoplasm of mammalian cells.

Provisional bilateral symmetry in Xenopus eggs is established during maturation.

Dorsal-ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30 degree rotation of the subcortical yolk mass relative to the egg cortex. This 'rotation of symmetrisation' is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15 degrees from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90 degrees from upright during maturation in vitro, the maturation spot developed 15 degrees or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.