Nonrandom gene organization: structural arrangements of specific pre-mRNA transcription and splicing with SC-35 domains.

Abstract

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...

The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences

“…The genes are found outside of the XIST RNA (yellow), which extends beyond the Barr body, the visible manifestation of the heterochromatin. 49,50,58,59) and splicing and transcription factors localize (22,49,51,60,61). The act of silencing and condensing the inner core of Xi may make the existing concentration of protein-coding genes in the peripheral region more pronounced and observable.…”

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

“…This RNA movement persists in transcriptionally inhibited cells. The movement of endogenous pre-mRNA to the speckles has been demonstrated in both transcriptionally active and silenced cells (Xing et al, 1995;Dirks et al, 1997;Ishov et al, 1997;Smith et al, 1999;Snaar et al, 1999;Johnson et al, 2000;Melčák et al, 2000). In addition, it has been shown that microinjected intron-containing RNA is within the cell nucleus processed into functional mRNA (Graessmann and Graessmann, 1982) and that several microinjected RNAs transcribed by RNA polymerase II and III localized at specific nuclear sites at which their corresponding endogenous counterparts were present in the steady-state distribution as shown by in situ hybridization (Jacobson et al, 1995(Jacobson et al, , 1997 Jacobson and Pederson, 1998a,b;Pederson, 1999).…”

“…Primary transcripts of certain genes as well as the spliced RNAs are mapped at sites of active transcription and outside of speckles (Zhang et al, 1994;Smith et al, 1999). On the other hand, pre-mRNAs from some other genes are shown to be associated with nuclear speckles (Xing et al, 1993(Xing et al, , 1995Huang and Spector, 1996;Ishov et al, 1997;Smith et al, 1999;Snaar et al, 1999;Johnson et al, 2000;Melčák et al, 2000). It thus remains unclear as to whether the nuclear speckles reflect localized accumulations of active splicing factors and whether the nucleus is compartmentalized with respect to splicing.…”

Human origin recognition complex is essential for HP1 binding to chromatin and heterochromatin organization

“…HeLa cells were preextracted in cytoskeletal buffer (CSK: 100 mM NaCl, 300 mM sucrose, 3 mM MgCl 2, 10 mM Pipes at pH 6.8) containing 0.5% Triton-X 100 for 5 min on ice and fixed with 3.7% freshly prepared formaldehyde for 15 min at room temperature. The cells were washed in 1× PBS (pH 7.2) and heat denatured in 70% formamide and 2× SSC at 72°C for 5 min followed by hybridization with labeled chromosome 9-specific satellite probe (Q-biogene) in 2× SSC, 50% formamide, 10% dextran sulfate, yeast tRNA, and Cot-1 DNA overnight at 37°C as previously described (63).…”