Abstract. The XIST gene is implicated in X chromosome inactivation, yet the RNA contains no apparent open reading frame. An accumulation of XIST RNA is observed near its site of transcription, the inactive X chromosome (Xi). A series of molecular cytogenetic studies comparing properties of XIST RNA to other protein coding RNAs, support a critical distinction for XIST RNA; XIST does not concentrate at Xi simply because it is transcribed and processed there. Most notably, morphometric and 3-D analysis reveals that XIST RNA and Xi are coincident in 2-and 3-D space; hence, the XIST RNA essentially paints Xi. Several resuits indicate that the XIST RNA accumulation has two components, a minor one associated with transcription and processing, and a spliced major component, which stably associates with Xi. Upon transcriptional inhibition the major spliced component remains in the nucleus and often encircles the extra-prominent heterochromatic Barr body. The continually transcribed XIST gene and its polyadenylated RNA consistently localize to a nuclear region devoid of splicing factor/poly A RNA rich domains. XIST RNA remains with the nuclear matrix fraction after removal of chromosomal DNA. XIST RNA is released from its association with Xi during mitosis, but shows a unique highly particulate distribution. Collective results indicate that XIST RNA may be an architectural element of the interphase chromosome territory, possibly a component of nonchromatin nuclear structure that specifically associates with Xi. XIST RNA is a novel nuclear RNA which potentially provides a specific precedent for RNA involvement in nuclear structure and cis-limited gene regulation via higher-order chromatin packaging. INACTIVATION of the X chromosome in mammalian females as a means to achieve gene dosage compensation was hypothesized over 30 years ago (Lyon, 1961). Because of its enormous biological and clinical importance, this process has been studied extensively and many varying models have been proposed. However, the molecular mechanisms involved remain largely undefined (reviewed in Gartler and Riggs, 1983;Gartler et al., 1992;Rastan, 1994). It is generally agreed that the X inactivation process involves at least three stages: (a) an initiation event which culminates in distinguishing the active X chromosome; (b) propagation of inactivation in cis throughout the X chromosome; and (c) maintenance of inactivation throughout the cell cycle and cell division. Inactive genes on the X chromosome are methylated, however some evidence indicates that methylation appears to follow rather than precede or be precisely concomitant with inactivation (Lock et al., 1987 body, formed by heterochromatinization of all or part of the inactive X chromosome (Barr and Carr, 1962), indicates that a change in higher-level chromatin packaging is involved in the global transcriptional silencing in cis. Because of the exceptional stability of X inactivation it has been postulated that multiple levels of control are involved to assure its maintenance .A potential b...

Convergent findings from our behavioral screen for memory mutants and DNA microarray analysis of transcriptional responses during memory formation in normal animals suggest the involvement of the pumilio/staufen pathway in memory. Behavioral experiments confirm a role for this pathway and suggest a molecular mechanism for synapse-specific modification.

Abstract. This work demonstrates a highly nonrandom distribution of specific genes relative to nuclear domains enriched in splicing factors and poly(A) ÷ RNA, and provides evidence for the direct involvement of these in pre-mRNA metabolism. As investigated in hundreds of diploid fibroblasts, human collagen Ietl and 13-actin DNA/RNA showed a very high degree of spatial association with SC-35 domains, whereas three nontranscribed genes, myosin heavy chain, neurotensin, and albumin, showed no such preferential association. Collagen Itxl RNA accumulates within the more central region of the domain, whereas [3-actin RNA localizes at the periphery. A novel approach revealed that collagen RNA tracks are polarized, with the entire gene at one end, on the edge of the domain, and the RNA extending into the domain. Intron 26 is spliced within the RNA track at the domain periphery. Transcriptional inhibition studies show both the structure of the domain and the gene's relationship to it are not dependent upon the continued presence of accumulated collagen RNA, and that domains remaining after inhibition are not just storage sites. Results support a model reconciling light and electron microscopic observations which proposes that transcription of some specific genes occurs at the border of domains, which may also function in the assembly or distribution of RNA metabolic components. In contrast to the apparently random dispersal of total undefined hnRNA synthesis through interdomain space, transcription and splicing for some genes occurs preferentially at specific sites, and a high degree of individual pre-mRNA metabolism is compartmentalized with discrete SC-35 domains.ESPITE the remarkable complexity of critical functions the nucleus performs, a simplified view of the extra-nucleolar nucleoplasm has persisted for many years. The idea that there exists some higher-level organization of the nucleoplasm that facilitates basic nuclear functions has been proposed for some time (see for example Comings, 1980;Blobel, 1985;Jackson, 1991;Lawrence et al., 1993), but the extent to which it exists is still largely unknown. It is known that the distributions of heterochromatin and satellite sequences are cell type specific (Manuelidis, 1984), that chromosomes occupy discrete nuclear "territories" (Cremer, 1982;Lichter, 1988;Pinkel et al., 1988), and that "chromosome position effects" can impact the expression of transgenes for largely unknown reasons (for example AI-Shawi et al., 1990). Relative to overall nuclear space, individual genes do not localize to precise coordinates, but distribute within preferred nuclear regions (Ward, W. S., J. A. McNeil, and J. B. Lawrence, manuscript submitted for publication; Lawrence et al., 1993). However, as explored in this work, greater orAddress all correspondence to Jeanne Bentley Lawrence, Dept. of Cell Biology, University of Massachusetts Medical Center, 55 Lake Ave. North, Worcester, MA 01655.Drs. Xing and Johnson contributed equally to this work.der may become apparent when sequences are local...

A quantitative three-dimensional analysis of nuclear components involved in precursor messenger RNA metabolism was performed with a combination of fluorescence hybridization, immunofluorescence, and digital imaging microscopy. Polyadenylate [poly(A)] RNA-rich transcript domains were discrete, internal nuclear regions that formed a ventrally positioned horizontal array in monolayer cells. A dimmer, sometimes strand-like, poly(A) RNA signal was dispersed throughout the nucleoplasm. Spliceosome assembly factor SC-35 localized within the center of individual domains. These data support a nuclear model in which there is a specific topological arrangement of noncontiguous centers involved in precursor messenger RNA metabolism, from which RNA transport toward the nuclear envelope radiates.

We investigated whether genes escape X chromosome inactivation by positioning outside of the territory defined by XIST RNA. Results reveal an unanticipated higher order organization of genes and noncoding sequences. All 15 X-linked genes, regardless of activity, position on the border of the XIST RNA territory, which resides outside of the DAPI-dense Barr body. Although more strictly delineated on the inactive X chromosome (Xi), all genes localized predominantly to the outer rim of the Xi and active X chromosome. This outer rim is decorated only by X chromosome DNA paints and is excluded from both the XIST RNA and dense DAPI staining. The only DNA found well within the Barr body and XIST RNA territory was centromeric and Cot-1 DNA; hence, the core of the X chromosome essentially excludes genes and is composed primarily of noncoding repeat-rich DNA. Moreover, we show that this core of repetitive sequences is expressed throughout the nucleus yet is silenced throughout Xi, providing direct evidence for chromosomewide regulation of ''junk'' DNA transcription. Collective results suggest that the Barr body, long presumed to be the physical manifestation of silenced genes, is in fact composed of a core of silenced noncoding DNA. Instead of acting at a local gene level, XIST RNA appears to interact with and silence core architectural elements to effectively condense and shut down the Xi.Barr body ͉ chromosome territory ͉ nuclear organization ͉ XIST ͉ noncoding RNA X inactivation in mammalian females is a prominent example of the formation of facultative heterochromatin during early development, which prevents the deleterious effects of overexpression of X-linked genes. In interphase, the inactive X chromosome (Xi) is found as a condensed heterochromatic Barr body, usually positioned at the nuclear or nucleolar periphery (1-4). Because many genes have been identified that escape X inactivation, packaging differences of sequences at some level within the Xi is presumed (5, 6). Although it is generally assumed that the Barr body is condensed DNA comprising genes normally expressed on the active X chromosome (Xa), the specific makeup of the Barr body has not been investigated.X inactivation is a multistep process initiated by XIST (7-9). Just after XIST RNA sweeps across the chromosome, a defined pattern of chromatin changes including histone modifications, recruitment of macroH2A, and methylation occurs (for review see refs. 10 and 11). We have shown that XIST RNA remains in the nucleus, functionally associated with the inactive chromatin, forming an interphase territory coincident with Xi (12, 13). We incorporated our results into two models for the higher-level organization of the inactive X chromosome (12). In each alternate view, XIST RNA would induce differences in the packaging of the chromatin to facilitate inactivation. In the first model, all genes, regardless of whether they escape from or are subject to X inactivation, would be interspersed throughout the chromosome territory and would not be cytologically disti...