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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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Types and frequencies of RNA and gene associations with SC-35 domains.  (A) RNA foci of genes examined to date exhibit three  distributions relative to SC-35 domains. Type I associations are defined by an RNA  that overlaps the SC-35 domain. This type of association is evident in MyHC and  collagen RNAs. In Type II  associations demonstrated by  LMNA and E2F4, the RNA  signal is seen at the periphery  of the SC-35 domain. Nonassociated RNA signals such  as dystrophin, LMNB1, and  LBR are visibly separate  from the SC-35–rich domains. Inactive genes are  also not associated with SC-35 domains. (B) Frequency  of association with SC-35 of  genes and RNAs studied to  date. The five known associated genes/RNAs (all active,  green) contact SC-35 domains at least 70% of the  time with the two Type I  genes collagen and MyHC  approaching 100%. The four  active (green) nonassociated  genes/RNAs are seen with  SC-35 domains less than 20%  of the time, at a level comparable to inactive genes (red).  Association refers to overlap  or contact with SC-35 domain. XIST is an intron-containing gene that does not  code for a protein (Clemson  et al., 1996). #, from Xing et  al. (1993); *, from Xing et al.  (1995).
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Figure 9: Types and frequencies of RNA and gene associations with SC-35 domains. (A) RNA foci of genes examined to date exhibit three distributions relative to SC-35 domains. Type I associations are defined by an RNA that overlaps the SC-35 domain. This type of association is evident in MyHC and collagen RNAs. In Type II associations demonstrated by LMNA and E2F4, the RNA signal is seen at the periphery of the SC-35 domain. Nonassociated RNA signals such as dystrophin, LMNB1, and LBR are visibly separate from the SC-35–rich domains. Inactive genes are also not associated with SC-35 domains. (B) Frequency of association with SC-35 of genes and RNAs studied to date. The five known associated genes/RNAs (all active, green) contact SC-35 domains at least 70% of the time with the two Type I genes collagen and MyHC approaching 100%. The four active (green) nonassociated genes/RNAs are seen with SC-35 domains less than 20% of the time, at a level comparable to inactive genes (red). Association refers to overlap or contact with SC-35 domain. XIST is an intron-containing gene that does not code for a protein (Clemson et al., 1996). #, from Xing et al. (1993); *, from Xing et al. (1995).

Mentions: As suggested here for MyHC (see Fig. 9 B), other results directly demonstrate that, for genes that associate with domains, association is linked to cell type–specific expression (Moen et al., manuscript in preparation). For MyHC and dystrophin, the simplest interpretation might a priori be that the difference in SC-35 association merely reflects the amount of splicing factors bound to each of their pre-mRNAs. However, results show that it is not that simple, and that the domain associated with MyHC is not formed merely by splicing factors bound to unspliced MyHC transcripts. Although the RNA foci for MyHC and dystrophin are comparable in size and intensity when probes of similar size are used, suggesting similar numbers of transcripts, the difference in localization with SC-35 is striking. Clearly, the lower level factors throughout the nucleoplasm are sufficient to splice the large nuclear accumulation of dystrophin RNA with its 74 introns. The abundant accumulation of splicing factors associated with MyHC lies primarily adjacent to the gene, rather than on it, indicating the domain behaves more as a structure than as a diffuse accumulation of splicing factors on nascent or dispersed RNA. Further, the MyHC RNA track, as detected with a genomic probe, frequently does not occupy the whole SC-35 domain. The absence of overlap of dystrophin RNA and SC-35 foci gives the strong impression that this RNA is excluded from splicing factor domains; even when dystrophin and MyHC accumulations are extremely close they do not overlap, suggesting that structural constraints may separate them.


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)

Types and frequencies of RNA and gene associations with SC-35 domains.  (A) RNA foci of genes examined to date exhibit three  distributions relative to SC-35 domains. Type I associations are defined by an RNA  that overlaps the SC-35 domain. This type of association is evident in MyHC and  collagen RNAs. In Type II  associations demonstrated by  LMNA and E2F4, the RNA  signal is seen at the periphery  of the SC-35 domain. Nonassociated RNA signals such  as dystrophin, LMNB1, and  LBR are visibly separate  from the SC-35–rich domains. Inactive genes are  also not associated with SC-35 domains. (B) Frequency  of association with SC-35 of  genes and RNAs studied to  date. The five known associated genes/RNAs (all active,  green) contact SC-35 domains at least 70% of the  time with the two Type I  genes collagen and MyHC  approaching 100%. The four  active (green) nonassociated  genes/RNAs are seen with  SC-35 domains less than 20%  of the time, at a level comparable to inactive genes (red).  Association refers to overlap  or contact with SC-35 domain. XIST is an intron-containing gene that does not  code for a protein (Clemson  et al., 1996). #, from Xing et  al. (1993); *, from Xing et al.  (1995).
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Figure 9: Types and frequencies of RNA and gene associations with SC-35 domains. (A) RNA foci of genes examined to date exhibit three distributions relative to SC-35 domains. Type I associations are defined by an RNA that overlaps the SC-35 domain. This type of association is evident in MyHC and collagen RNAs. In Type II associations demonstrated by LMNA and E2F4, the RNA signal is seen at the periphery of the SC-35 domain. Nonassociated RNA signals such as dystrophin, LMNB1, and LBR are visibly separate from the SC-35–rich domains. Inactive genes are also not associated with SC-35 domains. (B) Frequency of association with SC-35 of genes and RNAs studied to date. The five known associated genes/RNAs (all active, green) contact SC-35 domains at least 70% of the time with the two Type I genes collagen and MyHC approaching 100%. The four active (green) nonassociated genes/RNAs are seen with SC-35 domains less than 20% of the time, at a level comparable to inactive genes (red). Association refers to overlap or contact with SC-35 domain. XIST is an intron-containing gene that does not code for a protein (Clemson et al., 1996). #, from Xing et al. (1993); *, from Xing et al. (1995).
Mentions: As suggested here for MyHC (see Fig. 9 B), other results directly demonstrate that, for genes that associate with domains, association is linked to cell type–specific expression (Moen et al., manuscript in preparation). For MyHC and dystrophin, the simplest interpretation might a priori be that the difference in SC-35 association merely reflects the amount of splicing factors bound to each of their pre-mRNAs. However, results show that it is not that simple, and that the domain associated with MyHC is not formed merely by splicing factors bound to unspliced MyHC transcripts. Although the RNA foci for MyHC and dystrophin are comparable in size and intensity when probes of similar size are used, suggesting similar numbers of transcripts, the difference in localization with SC-35 is striking. Clearly, the lower level factors throughout the nucleoplasm are sufficient to splice the large nuclear accumulation of dystrophin RNA with its 74 introns. The abundant accumulation of splicing factors associated with MyHC lies primarily adjacent to the gene, rather than on it, indicating the domain behaves more as a structure than as a diffuse accumulation of splicing factors on nascent or dispersed RNA. Further, the MyHC RNA track, as detected with a genomic probe, frequently does not occupy the whole SC-35 domain. The absence of overlap of dystrophin RNA and SC-35 foci gives the strong impression that this RNA is excluded from splicing factor domains; even when dystrophin and MyHC accumulations are extremely close they do not overlap, suggesting that structural constraints may separate them.

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