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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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Detection of MyHC  and dystrophin RNA foci in  myotube nuclei (left). Nuclei  of a myotube show variable  positions of MyHC and dystrophin RNA foci. Most nuclei have two MyHC signals  (green) and a single dystrophin signal (red). The image  illustrates the developmental stage-specific expression  of MyHC and dystrophin. A  single myoblast nucleus (left,  bottom left) outside of the  myotube has neither MyHC  nor dystrophin RNA foci.  The myotube cytoplasm is visualized by the presence of cytoplasmic MyHC mRNA (green). (Right) Two myotube nuclei demonstrate that the RNA foci detected  by a MyHC genomic probe (32 kb, green) and a dystrophin midgene genomic probe (16 kb, red) are of comparable size and intensity.  This suggests that there is roughly similar molar abundance of each RNA in these foci.
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Figure 1: Detection of MyHC and dystrophin RNA foci in myotube nuclei (left). Nuclei of a myotube show variable positions of MyHC and dystrophin RNA foci. Most nuclei have two MyHC signals (green) and a single dystrophin signal (red). The image illustrates the developmental stage-specific expression of MyHC and dystrophin. A single myoblast nucleus (left, bottom left) outside of the myotube has neither MyHC nor dystrophin RNA foci. The myotube cytoplasm is visualized by the presence of cytoplasmic MyHC mRNA (green). (Right) Two myotube nuclei demonstrate that the RNA foci detected by a MyHC genomic probe (32 kb, green) and a dystrophin midgene genomic probe (16 kb, red) are of comparable size and intensity. This suggests that there is roughly similar molar abundance of each RNA in these foci.

Mentions: As illustrated in Fig. 1 (top), MyHC and dystrophin nuclear RNAs were simultaneously visualized in postmitotic nuclei of cultured skeletal myofibers using fluorescence hybridization techniques optimized for detection of nuclear RNA. Initial experiments used genomic probes that targeted sequences of similar size, thereby facilitating comparison of the signals. Although in situ hybridization does not provide information on the rate of transcript production, it can provide reliable comparisons of the relative amounts of RNA present at a given time. Most often there were two striking accumulations of MyHC RNA and one for dystrophin, as expected for expression of both alleles of the autosomal MyHC gene and one allele of the X-linked dystrophin gene in female cells. Perhaps less anticipated was that both the dimensions and intensity of the dystrophin and MyHC nuclear RNA accumulations were comparable, as illustrated in Fig. 1 (right). Although dystrophin RNA is a less abundant cytoplasmic mRNA than MyHC (Hoffman et al., 1987; Chelly et al., 1990), and the probes used detect <1% of the dystrophin sequence versus ∼80% of MyHC, dystrophin nuclear RNA signal was similar to the MyHC RNA signal. This was seen in multiple experiments irrespective of the detection method used and even when the MyHC probe targeted a somewhat larger sequence than the dystrophin probe (32 versus 16 kb). Hence, the apparent nuclear abundance of the dystrophin RNA signal does not result from the extremely large size of the full dystrophin RNA. Rather, these results indicate a roughly comparable number of transcripts associated with each MyHC and dystrophin allele.


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)

Detection of MyHC  and dystrophin RNA foci in  myotube nuclei (left). Nuclei  of a myotube show variable  positions of MyHC and dystrophin RNA foci. Most nuclei have two MyHC signals  (green) and a single dystrophin signal (red). The image  illustrates the developmental stage-specific expression  of MyHC and dystrophin. A  single myoblast nucleus (left,  bottom left) outside of the  myotube has neither MyHC  nor dystrophin RNA foci.  The myotube cytoplasm is visualized by the presence of cytoplasmic MyHC mRNA (green). (Right) Two myotube nuclei demonstrate that the RNA foci detected  by a MyHC genomic probe (32 kb, green) and a dystrophin midgene genomic probe (16 kb, red) are of comparable size and intensity.  This suggests that there is roughly similar molar abundance of each RNA in these foci.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132926&req=5

Figure 1: Detection of MyHC and dystrophin RNA foci in myotube nuclei (left). Nuclei of a myotube show variable positions of MyHC and dystrophin RNA foci. Most nuclei have two MyHC signals (green) and a single dystrophin signal (red). The image illustrates the developmental stage-specific expression of MyHC and dystrophin. A single myoblast nucleus (left, bottom left) outside of the myotube has neither MyHC nor dystrophin RNA foci. The myotube cytoplasm is visualized by the presence of cytoplasmic MyHC mRNA (green). (Right) Two myotube nuclei demonstrate that the RNA foci detected by a MyHC genomic probe (32 kb, green) and a dystrophin midgene genomic probe (16 kb, red) are of comparable size and intensity. This suggests that there is roughly similar molar abundance of each RNA in these foci.
Mentions: As illustrated in Fig. 1 (top), MyHC and dystrophin nuclear RNAs were simultaneously visualized in postmitotic nuclei of cultured skeletal myofibers using fluorescence hybridization techniques optimized for detection of nuclear RNA. Initial experiments used genomic probes that targeted sequences of similar size, thereby facilitating comparison of the signals. Although in situ hybridization does not provide information on the rate of transcript production, it can provide reliable comparisons of the relative amounts of RNA present at a given time. Most often there were two striking accumulations of MyHC RNA and one for dystrophin, as expected for expression of both alleles of the autosomal MyHC gene and one allele of the X-linked dystrophin gene in female cells. Perhaps less anticipated was that both the dimensions and intensity of the dystrophin and MyHC nuclear RNA accumulations were comparable, as illustrated in Fig. 1 (right). Although dystrophin RNA is a less abundant cytoplasmic mRNA than MyHC (Hoffman et al., 1987; Chelly et al., 1990), and the probes used detect <1% of the dystrophin sequence versus ∼80% of MyHC, dystrophin nuclear RNA signal was similar to the MyHC RNA signal. This was seen in multiple experiments irrespective of the detection method used and even when the MyHC probe targeted a somewhat larger sequence than the dystrophin probe (32 versus 16 kb). Hence, the apparent nuclear abundance of the dystrophin RNA signal does not result from the extremely large size of the full dystrophin RNA. Rather, these results indicate a roughly comparable number of transcripts associated with each MyHC and dystrophin allele.

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