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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 association of muscle-specific RNAs with SC-35 defined domains.  MyHC and dystrophin RNA signals were scored  as coincident with, or completely separate from  SC-35 domains. Three different dystrophin  probes, midgenomic, mid-cDNA, and 5′ cDNA  were used. At least two investigators scored each  experiment. N = number of signals scored.
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Figure 3: Frequency of association of muscle-specific RNAs with SC-35 defined domains. MyHC and dystrophin RNA signals were scored as coincident with, or completely separate from SC-35 domains. Three different dystrophin probes, midgenomic, mid-cDNA, and 5′ cDNA were used. At least two investigators scored each experiment. N = number of signals scored.

Mentions: To determine the distribution of MyHC and dystrophin nuclear RNA foci relative to SC-35 domains, cultures containing myotubes were probed for either MyHC or dystrophin nuclear transcripts and subsequently stained with antibodies against the spliceosome assembly factor SC-35 (Fig. 2, A–F). Using a very narrow depth of field objective (NA = 1.4) and focusing through the RNA and SC-35 signals, it was apparent if these signals occupied the same space. In essentially all of the 50 cells analyzed, the MyHC RNA foci colocalized with a prominent SC-35 domain (Fig. 2, A–C and Fig. 3). Most commonly, the MyHC RNA accumulation detected with the genomic probe did not fill the entire ∼1–3-μ domain and had a different contour. Unlike nuclear accumulations of fibronectin and actin mRNAs which localized to the domain periphery (Xing et al., 1993, 1995), the MyHC RNA was also clearly detected within the inner region of the domain, as previously shown for collagen 1α1 RNA. The accumulation of MyHC RNA within SC-35 domains provides further evidence for the presence of pre-mRNA in these regions, contrary to the expectation that large factor-rich domains are storage sites, but consistent with the uniform presence of poly A RNA in them (Carter et al., 1991). These results also suggest that MyHC transcription and splicing is associated with the SC-35 domain, as directly demonstrated for fibronectin (Xing et al., 1993) and collagen 1α1 RNA (Xing et al., 1995). In contrast to the similarity in size and appearance of their nuclear RNA accumulations, MyHC and dystrophin RNA showed completely different distributions relative to the SC-35 domains. The dystrophin RNA accumulations were never within the domains, whereas the MyHC RNA foci consistently were. In fact, as illustrated in Fig. 2, D–F, with the exception of a small fraction of cells, there was no discernible increase in SC-35 concentration coincident with the large, bright dystrophin RNA accumulation. Since the linear dimensions of this enormous gene can extend 1–2 μ or more at interphase (Lawrence et al., 1990), this analysis was repeated with cDNA and genomic probes to different regions of the primary transcript. These results confirmed that no part of the dystrophin RNA focus was preferentially associated with SC-35 domains (Fig. 3). At most, the edge of the dystrophin RNA accumulation occasionally appeared to touch an SC-35 domain, but the body of the RNA accumulation did not ever closely associate with domains (Fig. 2, D–F and Fig. 3).


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 association of muscle-specific RNAs with SC-35 defined domains.  MyHC and dystrophin RNA signals were scored  as coincident with, or completely separate from  SC-35 domains. Three different dystrophin  probes, midgenomic, mid-cDNA, and 5′ cDNA  were used. At least two investigators scored each  experiment. N = number of signals scored.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132926&req=5

Figure 3: Frequency of association of muscle-specific RNAs with SC-35 defined domains. MyHC and dystrophin RNA signals were scored as coincident with, or completely separate from SC-35 domains. Three different dystrophin probes, midgenomic, mid-cDNA, and 5′ cDNA were used. At least two investigators scored each experiment. N = number of signals scored.
Mentions: To determine the distribution of MyHC and dystrophin nuclear RNA foci relative to SC-35 domains, cultures containing myotubes were probed for either MyHC or dystrophin nuclear transcripts and subsequently stained with antibodies against the spliceosome assembly factor SC-35 (Fig. 2, A–F). Using a very narrow depth of field objective (NA = 1.4) and focusing through the RNA and SC-35 signals, it was apparent if these signals occupied the same space. In essentially all of the 50 cells analyzed, the MyHC RNA foci colocalized with a prominent SC-35 domain (Fig. 2, A–C and Fig. 3). Most commonly, the MyHC RNA accumulation detected with the genomic probe did not fill the entire ∼1–3-μ domain and had a different contour. Unlike nuclear accumulations of fibronectin and actin mRNAs which localized to the domain periphery (Xing et al., 1993, 1995), the MyHC RNA was also clearly detected within the inner region of the domain, as previously shown for collagen 1α1 RNA. The accumulation of MyHC RNA within SC-35 domains provides further evidence for the presence of pre-mRNA in these regions, contrary to the expectation that large factor-rich domains are storage sites, but consistent with the uniform presence of poly A RNA in them (Carter et al., 1991). These results also suggest that MyHC transcription and splicing is associated with the SC-35 domain, as directly demonstrated for fibronectin (Xing et al., 1993) and collagen 1α1 RNA (Xing et al., 1995). In contrast to the similarity in size and appearance of their nuclear RNA accumulations, MyHC and dystrophin RNA showed completely different distributions relative to the SC-35 domains. The dystrophin RNA accumulations were never within the domains, whereas the MyHC RNA foci consistently were. In fact, as illustrated in Fig. 2, D–F, with the exception of a small fraction of cells, there was no discernible increase in SC-35 concentration coincident with the large, bright dystrophin RNA accumulation. Since the linear dimensions of this enormous gene can extend 1–2 μ or more at interphase (Lawrence et al., 1990), this analysis was repeated with cDNA and genomic probes to different regions of the primary transcript. These results confirmed that no part of the dystrophin RNA focus was preferentially associated with SC-35 domains (Fig. 3). At most, the edge of the dystrophin RNA accumulation occasionally appeared to touch an SC-35 domain, but the body of the RNA accumulation did not ever closely associate with domains (Fig. 2, D–F and Fig. 3).

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