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Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific.

Smith KP, Moen PT, Wydner KL, Coleman JR, Lawrence JB - J. Cell Biol. (1999)

Bottom Line: These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space.The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors.This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

ABSTRACT
Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript "trees" of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional "tracks" of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism.

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Frequency of LMNA, E2F4, LBR, and  LMNB1 gene/RNA associations with SC-35 domains. Nuclei were scored for association of each  of the four gene/RNAs with SC-35 domains. Signals were scored as associated if they either overlapped SC-35 rich regions or if no space was visible between the focus and the domain. At least  two investigators scored each experiment. N =  number of signals scored.
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Figure 8: Frequency of LMNA, E2F4, LBR, and LMNB1 gene/RNA associations with SC-35 domains. Nuclei were scored for association of each of the four gene/RNAs with SC-35 domains. Signals were scored as associated if they either overlapped SC-35 rich regions or if no space was visible between the focus and the domain. At least two investigators scored each experiment. N = number of signals scored.

Mentions: For all four of these genes, the nuclear RNA accumulations generally produced small spot-like signals, smaller than either the dystrophin or MyHC nuclear RNA signals. This suggests that the signals largely comprise nascent transcripts on the gene (although there was sometimes a suggestion of RNA tracks for LMNA). Analysis of these genes/RNAs relative to SC-35 in fibroblasts demonstrated striking differences in levels of association with the prominent SC-35 domains, as seen in Figs. 7 and 8. In the vast majority of cells, LMNA and E2F4 were not visibly separate from the SC-35 domains, whereas the LMNB1 and LBR genes/RNAs remained spatially separate, even when they were in the vicinity of a domain. The low levels of apparent association of these two sequences are similar to that previously seen for inactive genes and are consistent with random expectations within the limits of microscopic resolution (Xing et al., 1995). As viewed by light microscopy, LMNA and E2F4 contacted SC-35 domains four- to fivefold more often than LMNB1 and LBR (Fig. 8). This represents a significant level of association for genes that are not nearly as highly expressed as MyHC in muscle cells, and is higher than the level of association reported for fibronectin with SC-35 (Xing et al., 1993). It should be noted that these sequences were studied in a cycling fibroblast population, which may introduce some variability between cells; e.g., E2F4 expression varies during the cell cycle (Sardet et al., 1995). The nonrandom association of a regulatory gene such as E2F4 establishes that the spatial association with domains is not restricted to genes for the most abundant structural proteins. Collectively, our results provide strong evidence that such organization is common and involves a substantial subset of protein coding genes. These results lead us to conclude that association with SC-35 domains is locus specific and not solely dependent upon gene size, complexity or expression level. They further indicate that within the constraints of nuclear structure, active loci have differential access to the immediate and copious supply of RNA metabolic factors in the interchromatin domains, visualized here by immunofluorescence to assembly factor SC-35. Further implications of these results are considered in the Discussion.


Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific.

Smith KP, Moen PT, Wydner KL, Coleman JR, Lawrence JB - J. Cell Biol. (1999)

Frequency of LMNA, E2F4, LBR, and  LMNB1 gene/RNA associations with SC-35 domains. Nuclei were scored for association of each  of the four gene/RNAs with SC-35 domains. Signals were scored as associated if they either overlapped SC-35 rich regions or if no space was visible between the focus and the domain. At least  two investigators scored each experiment. N =  number of signals scored.
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Related In: Results  -  Collection

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Figure 8: Frequency of LMNA, E2F4, LBR, and LMNB1 gene/RNA associations with SC-35 domains. Nuclei were scored for association of each of the four gene/RNAs with SC-35 domains. Signals were scored as associated if they either overlapped SC-35 rich regions or if no space was visible between the focus and the domain. At least two investigators scored each experiment. N = number of signals scored.
Mentions: For all four of these genes, the nuclear RNA accumulations generally produced small spot-like signals, smaller than either the dystrophin or MyHC nuclear RNA signals. This suggests that the signals largely comprise nascent transcripts on the gene (although there was sometimes a suggestion of RNA tracks for LMNA). Analysis of these genes/RNAs relative to SC-35 in fibroblasts demonstrated striking differences in levels of association with the prominent SC-35 domains, as seen in Figs. 7 and 8. In the vast majority of cells, LMNA and E2F4 were not visibly separate from the SC-35 domains, whereas the LMNB1 and LBR genes/RNAs remained spatially separate, even when they were in the vicinity of a domain. The low levels of apparent association of these two sequences are similar to that previously seen for inactive genes and are consistent with random expectations within the limits of microscopic resolution (Xing et al., 1995). As viewed by light microscopy, LMNA and E2F4 contacted SC-35 domains four- to fivefold more often than LMNB1 and LBR (Fig. 8). This represents a significant level of association for genes that are not nearly as highly expressed as MyHC in muscle cells, and is higher than the level of association reported for fibronectin with SC-35 (Xing et al., 1993). It should be noted that these sequences were studied in a cycling fibroblast population, which may introduce some variability between cells; e.g., E2F4 expression varies during the cell cycle (Sardet et al., 1995). The nonrandom association of a regulatory gene such as E2F4 establishes that the spatial association with domains is not restricted to genes for the most abundant structural proteins. Collectively, our results provide strong evidence that such organization is common and involves a substantial subset of protein coding genes. These results lead us to conclude that association with SC-35 domains is locus specific and not solely dependent upon gene size, complexity or expression level. They further indicate that within the constraints of nuclear structure, active loci have differential access to the immediate and copious supply of RNA metabolic factors in the interchromatin domains, visualized here by immunofluorescence to assembly factor SC-35. Further implications of these results are considered in the Discussion.

Bottom Line: These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space.The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors.This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

ABSTRACT
Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript "trees" of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional "tracks" of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism.

Show MeSH