Centrosomes are the major microtubule-organizing center in animal cells. They are composed of a pair of [9(3) ؉ 0] centrioles surrounded by a relatively ill-defined pericentriolar matrix, provide the ciliary centriole-kinetosome (basal body) progenitor, and organize the assembly of microtubules into the mitotic spindle during cell division. Despite >100 years of microscopic observation and their obvious significance, our understanding of centrosome composition, dynamic organization, and mechanism of action is limited when compared with that of other cellular organelles. Centrosomes duplicate only once per cell cycle to ensure development of a normal bipolar spindle. The initial event in centrosome duplication is centriole replication, which is generative, semiconservative, and independent of the nucleus. Such observations led to the proposal that centrosomes contain their own complement of nucleic acids, possibly representative of an organellar genome comparable with those described for mitochondria and chloroplasts. The consensus in the field is that centrosomes lack DNA but may contain RNA. We isolated centrosomes from oocytes of the surf clam, Spisula solidissima, and purified from them a unique set of RNAs. We show here by biochemical means and subcellular in situ hybridization that the first transcript we analyzed is intimately associated with centrosomes. Sequence analysis reveals that this centrosome-associated RNA encodes a conserved RNA-directed polymerase domain. The hypothesis that centrosomes contain an intrinsic complement of specific RNAs suggests new opportunities to address the century-old problem of centrosome function, heredity, and evolution.centriole ͉ centrosomal RNA ͉ microtubule-organizing center T he process of centrosome duplication is tightly regulated and under the control of both cytoplasmic and intrinsic factors (1, 2) to ensure that a normal bipolar spindle is formed during mitosis and that the genome is divided equally between daughter cells. The first observable event in centrosome duplication is centriole replication. Like mitochondria and chloroplasts, centriole replication in embryos is independent of the nucleus, but unlike the former two classes of organelles, centrioles replicate not by fission but in a semiconservative, generative manner. These and other observations have led some investigators to propose that the centrosome (or more specifically, the centriole) is of endosymbiotic evolutionary origin and, like mitochondria and chloroplasts, may contain its own genome (3).The question of whether centrosomes or centrioles contain nucleic acids has remained unresolved since biochemical studies of the mitotic apparatus in the 1950s revealed ''pentosenucleic acid'' in the polar regions (4-8). The consensus, expressed in the most recent review on the subject, concludes that evidence for DNA in centrosomes is lacking and that significant evidence exists to the contrary (9). The existence of RNA in these organelles, however, remains an open question, although much of the historica...

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Pigpen is a 67-kDa Sepharose-binding molecule isolated from mammalian endothelial and retinal pigmented epithelial cells. The protein is distributed nonhomogeneously in the nucleus, exhibiting diffuse staining throughout (excluding nucleoli), together with a small number of intensely stained focal points, or granules, and punctate staining along the nuclear envelope. Pigpen was absent or greatly attenuated in the nonepithelial cell types we examined, including fibroblasts, myeloma, and astroglia. cDNA sequence analysis revealed a positively charged molecule with an RNP-CS RNA-binding domain, 19 RGG repeats, and a consensus tyrosine phosphorylation site in the C-terminus. The amino terminal portion of the molecule is characterized by 7 glutamine-rich hexapeptide repeats similar to those found in the transactivation domain of known transcription activators. Pigpen has a high level of identity with the FUS gene product, TLS (Translocated in Liposarcoma; Crozat et al, 1993; Rabbits et al., 1993), a new member of the EWS family of proteins. Expression of pigpen is regulated during the transition between active and quiescent endothelial cell phenotypes. Both mRNA and overall protein levels are maintained at a steady level in actively growing cells. The number of nuclear granules increases as cultures approach confluency. When cells reach confluency, overall expression is sharply reduced and the number of nuclear focal points declines gradually. We observed that reactivation of endothelial cells locally by wounding of confluent cultures resulted in a spatially restricted reactivation of pigpen expression. This pattern of expression, taken together with structural data, suggests that pigpen may function in the growth and differentiation of endothelial cells during angiogenesis.