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RNA-mediated interaction of Cajal bodies and U2 snRNA genes.

Frey MR, Matera AG - J. Cell Biol. (2001)

Bottom Line: Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not.Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs.Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Program in Cell Biology, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Cajal bodies (CBs) are nuclear structures involved in RNA metabolism that accumulate high concentrations of small nuclear ribonucleoproteins (snRNPs). Notably, CBs preferentially associate with specific genomic loci in interphase human cells, including several snRNA and histone gene clusters. To uncover functional elements involved in the interaction of genes and CBs, we analyzed the expression and subcellular localization of stably transfected artificial arrays of U2 snRNA genes. Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not. Additional experiments identified factors within CBs that are important for association with the native U2 genes. Inhibition of nuclear export or targeted degradation of U2 snRNPs caused a marked decrease in the levels of U2 snRNA in CBs and strongly disrupted the interaction with U2 genes. Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs. Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

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Constructs used to create artificial tandem arrays. The top three constructs were described previously (Frey et al. 1999); the bottom three were used in this study. DSE and PSE (circles) are distal and PSEs, respectively. Deletions are denoted by parentheses. Arrowheads mark coding regions; the filled ones represent the U2 coding region, whereas the open arrowheads depict the replacement sequence. The 3′ box is an element required for proper 3′ end processing of snRNAs. CT is a microsatellite repeat. The RSV-U2 construct incorporates an RSV promoter upstream of the U2 coding region and a bovine growth hormone polyadenylation signal (BGH poly A) downstream of the CT element. Primers used for RT-PCR are indicated by arrows. The dashed line denotes the location of a cryptic intron (see text). Results of transcription (Txn) and CB association (CB Assoc.) experiments for stable arrays of the corresponding constructs are also shown.
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fig1: Constructs used to create artificial tandem arrays. The top three constructs were described previously (Frey et al. 1999); the bottom three were used in this study. DSE and PSE (circles) are distal and PSEs, respectively. Deletions are denoted by parentheses. Arrowheads mark coding regions; the filled ones represent the U2 coding region, whereas the open arrowheads depict the replacement sequence. The 3′ box is an element required for proper 3′ end processing of snRNAs. CT is a microsatellite repeat. The RSV-U2 construct incorporates an RSV promoter upstream of the U2 coding region and a bovine growth hormone polyadenylation signal (BGH poly A) downstream of the CT element. Primers used for RT-PCR are indicated by arrows. The dashed line denotes the location of a cryptic intron (see text). Results of transcription (Txn) and CB association (CB Assoc.) experiments for stable arrays of the corresponding constructs are also shown.

Mentions: Our previous work showed that deletion of promoter and enhancer elements within U2 repeats abolished association of artificial U2 arrays with CBs (Fig. 1 ; Frey et al., 1999). This led us to test a construct in which the U2 coding region was replaced by a sequence from the adenovirus 2 major late intron (Hernandez and Weiner, 1986). Interestingly, arrays of this construct were transcribed but never colocalized with CBs (Fig. 1; Frey et al., 1999). In light of the recent evidence that U2 downstream sequences are transcribed, and coupled with the fact that our original “Replacement” construct did not contain these downstream sequences, we created a new construct termed Replacement+CT (Fig. 1). Replacement+CT contains not only the adenovirus sequence driven by the U2 promoter, but includes the identical U2 downstream sequences that are present in the minigene construct, mU2+CT (Fig. 1).


RNA-mediated interaction of Cajal bodies and U2 snRNA genes.

Frey MR, Matera AG - J. Cell Biol. (2001)

Constructs used to create artificial tandem arrays. The top three constructs were described previously (Frey et al. 1999); the bottom three were used in this study. DSE and PSE (circles) are distal and PSEs, respectively. Deletions are denoted by parentheses. Arrowheads mark coding regions; the filled ones represent the U2 coding region, whereas the open arrowheads depict the replacement sequence. The 3′ box is an element required for proper 3′ end processing of snRNAs. CT is a microsatellite repeat. The RSV-U2 construct incorporates an RSV promoter upstream of the U2 coding region and a bovine growth hormone polyadenylation signal (BGH poly A) downstream of the CT element. Primers used for RT-PCR are indicated by arrows. The dashed line denotes the location of a cryptic intron (see text). Results of transcription (Txn) and CB association (CB Assoc.) experiments for stable arrays of the corresponding constructs are also shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196410&req=5

fig1: Constructs used to create artificial tandem arrays. The top three constructs were described previously (Frey et al. 1999); the bottom three were used in this study. DSE and PSE (circles) are distal and PSEs, respectively. Deletions are denoted by parentheses. Arrowheads mark coding regions; the filled ones represent the U2 coding region, whereas the open arrowheads depict the replacement sequence. The 3′ box is an element required for proper 3′ end processing of snRNAs. CT is a microsatellite repeat. The RSV-U2 construct incorporates an RSV promoter upstream of the U2 coding region and a bovine growth hormone polyadenylation signal (BGH poly A) downstream of the CT element. Primers used for RT-PCR are indicated by arrows. The dashed line denotes the location of a cryptic intron (see text). Results of transcription (Txn) and CB association (CB Assoc.) experiments for stable arrays of the corresponding constructs are also shown.
Mentions: Our previous work showed that deletion of promoter and enhancer elements within U2 repeats abolished association of artificial U2 arrays with CBs (Fig. 1 ; Frey et al., 1999). This led us to test a construct in which the U2 coding region was replaced by a sequence from the adenovirus 2 major late intron (Hernandez and Weiner, 1986). Interestingly, arrays of this construct were transcribed but never colocalized with CBs (Fig. 1; Frey et al., 1999). In light of the recent evidence that U2 downstream sequences are transcribed, and coupled with the fact that our original “Replacement” construct did not contain these downstream sequences, we created a new construct termed Replacement+CT (Fig. 1). Replacement+CT contains not only the adenovirus sequence driven by the U2 promoter, but includes the identical U2 downstream sequences that are present in the minigene construct, mU2+CT (Fig. 1).

Bottom Line: Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not.Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs.Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Program in Cell Biology, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
Cajal bodies (CBs) are nuclear structures involved in RNA metabolism that accumulate high concentrations of small nuclear ribonucleoproteins (snRNPs). Notably, CBs preferentially associate with specific genomic loci in interphase human cells, including several snRNA and histone gene clusters. To uncover functional elements involved in the interaction of genes and CBs, we analyzed the expression and subcellular localization of stably transfected artificial arrays of U2 snRNA genes. Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not. Additional experiments identified factors within CBs that are important for association with the native U2 genes. Inhibition of nuclear export or targeted degradation of U2 snRNPs caused a marked decrease in the levels of U2 snRNA in CBs and strongly disrupted the interaction with U2 genes. Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs. Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

Show MeSH