Molecular anatomy of a speckle

Abstract

Direct localization of specific genes, RNAs, and proteins has allowed the dissection of individual nuclear speckles in relation to the molecular biology of gene expression. Nuclear speckles (aka SC35 domains) are essentially ubiquitous structures enriched for most pre-mRNA metabolic factors, yet their relationship to gene expression has been poorly understood. Analyses of specific genes and their spliced or mature mRNA strongly support that SC35 domains are hubs of activity, not stores of inert factors detached from gene expression. We propose that SC35 domains are hubs that spatially link expression of specific pre-mRNAs to rapid recycling of copious RNA metabolic complexes, thereby facilitating expression of many highly active genes. In addition to increasing the efficiency of each step, sequential steps in gene expression are structurally integrated at each SC35 domain, consistent with other evidence that the biochemical machineries for transcription, splicing, and mRNA export are coupled. Transcription and splicing are subcompartmentalized at the periphery, with largely spliced mRNA entering the domain prior to export. In addition, new findings presented here begin to illuminate the structural underpinnings of a speckle by defining specific perturbations of phosphorylation that promote disassembly or assembly of an SC35 domain in relation to other components. Results thus far are consistent with the SC35 spliceosome assembly factor as an integral structural component. Conditions that disperse SC35 also disperse poly(A) RNA, whereas the splicing factor ASF/SF2 can be dispersed under conditions in which SC35 or SRm300 remain as intact components of a core domain.

Figure 1. Overview: The Anatomy of an SC35 Domain as Relates to the Molecular Biology of Gene Expression

1. A) Poly(A) RNA (red) is concentrated in all SC35 domains (green).

2. B) Collagen 1A1 RNA (red) is consistently seen within an SC35 domain (green) (from: (Johnson et al., 2000))

3. C) Collagen 1A1 RNA (green) and collagen 1A2 RNA (red) can localize within a common SC35 (blue) (from: (Shopland et al., 2003))

4. D) Collagen 1A1 DNA hybridization (red) positions at the periphery of an SC35 domain (green).

5. E) Collagen 1A1 (green) and Col 1A2 (red) DNA hybridization shows multiple genes at the periphery of an SC35 domain (blue).

6. F) Dystrophin nuclear RNA accumulation (green) detected here using a cDNA probe to exons 1-11 (L. Kunkel Harvard School of Medicine, Boston, MA) is never observed to colocalize with SC35 domains (red) (Smith et al., 1999).

7. G) Exon suppression RNA hybridization with a labeled Collagen genomic probe (red) competed with excess cDNA shows that most introns are removed at the periphery of the SC35 domain (green) (from: (Johnson et al., 2000))

8. H) Comparison of collagen intron 24 and intron 26 shows that intron 24 is spliced later, and is not restricted to the periphery of domain or RNA track (green) (from: (Johnson et al., 2000))

9. I) In Osteogenesis Imperfecta, collagen mRNA from the mutant allele (lower signal and insets) retains intron 26 (green) throughout the mRNA track (red) and thus the Sc35 domain. The intron is removed from mRNA of the normal allele (upper signal) (from: (Johnson et al., 2000))

10. J) Visualization of three different mRNAs(beta actin, green, and collagen 1A1 and collagen 1A2, red) within Sc35 domains (blue), illustrates that half of the domains in this cell label with probes to just three genes. (from: Shopland, 2002])

Figure 2. Relationship of Poly(A) containing RNA to SC35

(A) Three color images of Poly(A) RNA (red) detected by hybridization with an oligo dT probe and SC35 (green), detected with an anti-SC35 monoclonal antibody (Sigma) in human fibroblasts. DNA (blue) is detected by DAPI stain. Grayscale images of each color are represented in B, C and D. (E) Blowup of a single SC35 domain with poly(A). (F) The image in E was masked by color thresholding of red and green signals to more clearly show that poly(A) signal extends beyond the borders of the SC35 signal. (G) Histogram of signal intensities along a line shown in (A) demonstrates that poly(A) (red line) defines regions larger than those seen with SC35 staining (green line) and that these domains lie within regions of lower DNA density (blue line).

Figure 3. A Model of SC35 Domain Function

(A) Multiple genes (blue) associate with a single domain. Transcription occurs at the outer edge of the domain, with all or almost all introns removed in the periphery at or near the gene. Essentially spliced mRNA distributes throughout the domain. (B) Model suggesting a functional rationale for coupling the completion of mRNA maturation and release for mRNA export with the recycling/preassembly of factors within SC35 domains. Because the RNA metabolic machinery requires interaction of such a large number of different factors, their concentration at a site would facilitate recycling and a rapid rate of reuse for expression of adjacent genes. The closer the completion of maturation (and release for export) occurs to putative sites of recycling, the more efficient the total process. In this model, mRNAs enter the domains, where splicing complex recycling and binding of export factors occurs. If mRNAs are not properly processed, transit through the domain may be impeded, thus domains may also be the site of mRNA “screening” functions.

Figure 4. Perturbation of phosphorylation provides a means to promote assembly or disassembly of SC35 domains and investigate their structural underpinnings

A) Cantharidin (PP2A inhibitor) treatment of TIG-1 cells results in the accumulation of SC35 (red) into larger domains. Compare to untreated cells in Fig 1 and 2. B) Tautomycin (PP1 inhibitor) treatment results in the dispersion of SC35 (green) from domains into the nucleoplasm as small punctuate dots. C) Poly(A) RNA (red) localization to domains is similarly affected by tautomycin treatment, following the pattern of SC35 ( green). D) Poly(A) RNA (green) accumulates into the larger domains along with the SC35 (red) after cantharidin treatment. E) When the SC35 (green) domains break down, collagen RNA (red) is no longer found accumulated in tracks or domains. F) Collagen RNA (green) is found in very large tracks in the large SC35 (red) domains found in cantharidin treated TIG-1 cells. G) Collagen mRNA appears to be efficiently spliced in cantharidin treated cells because the signal for intron 26 (green) is as small as those seen in control cells and intron-containing RNA does not accumulate in SC35 domains (red).

Figure 5. Tautamycin treatment (phosphatase 1A inhibition) results in dispersal of ASF/SF2 from the SC35 domain before SRM-300

A) In untreated TIG-1 cells ASF/SF2 (green) is present throughout the nucleoplasm but somewhat more concentrated within the SC35 domain (speckle), defined by SRm300 staining (red) B) Upon cantharidin treatment, the ASF/SF2 protein (green) appears to increase its presence within the SRm300 (red) domains. C) In low tautomycin concentrations, where SC35 domains have yet to break down and SRm300 (red) is still concentrated in domains, ASF/SF2 (green) is observed to disperse from the speckle (SRm300 - red).