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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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Inhibition of snRNA export disrupts RNU2–CB association. (A) HeLa cells were incubated in the presence or absence of LMB for 3 h and then stained with anti-U2B″ (green) and anticoilin antibodies (red). At early time points (3–5 h) CBs remain prominent; although, diffuse accumulation of coilin within nucleoli is also observed (Carvalho et al., 1999). The presence of U2 snRNPs in CBs on the left is demonstrated by the yellow color. The lack of signal overlap is evident in the middle (note red color). On the right, FISH reveals that CBs (red) and U2 genes (green) do not colocalize. (B) CB association with U2 genes decreases with increasing incubation time in LMB. (C) HeLa cells were transfected with PHAX–GFP and costained with anticoilin. In addition to localization throughout the nucleoplasm, note accumulation of PHAX–GFP (green) within the CBs (red) of the transfected cell.
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fig6: Inhibition of snRNA export disrupts RNU2–CB association. (A) HeLa cells were incubated in the presence or absence of LMB for 3 h and then stained with anti-U2B″ (green) and anticoilin antibodies (red). At early time points (3–5 h) CBs remain prominent; although, diffuse accumulation of coilin within nucleoli is also observed (Carvalho et al., 1999). The presence of U2 snRNPs in CBs on the left is demonstrated by the yellow color. The lack of signal overlap is evident in the middle (note red color). On the right, FISH reveals that CBs (red) and U2 genes (green) do not colocalize. (B) CB association with U2 genes decreases with increasing incubation time in LMB. (C) HeLa cells were transfected with PHAX–GFP and costained with anticoilin. In addition to localization throughout the nucleoplasm, note accumulation of PHAX–GFP (green) within the CBs (red) of the transfected cell.

Mentions: Carvalho et al. (1999) showed that treatment of cells with leptomycin B (LMB), a known inhibitor of exportin1/CRM1-mediated nuclear export (Kudo et al., 1998), resulted in the progressive depletion of snRNPs from CBs. Presumably, if newly transcribed snRNAs are prevented from leaving the nucleus, then newly assembled snRNPs will be depleted first from the cytoplasm and then from CBs (Carvalho et al., 1999). We reasoned that such a disruption of the snRNP cycle would also have an effect on the association of CBs with U2 genes. HeLa cells were thus incubated in media containing LMB and assayed. As shown in Fig. 6 A (compare −LMB with +LMB), we monitored the presence of U2B″ protein in CBs as a marker for the U2 snRNP. The kinetics of disappearance of U2 snRNPs from CBs were very similar to those observed by Carvalho et al. (1999). Interestingly, the effects of U2 snRNP depletion from CBs are mirrored by a striking decrease in the RNU2–CB association frequency (Fig. 6, A and B). Although the effects of LMB on the RNU2–CB interaction can be seen in as short as 2 h of treatment, the association frequency decreases with increasing incubation time (Fig. 6 B). Thus, U2 genes no longer associate with CBs when snRNA export is blocked by LMB.


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

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

Inhibition of snRNA export disrupts RNU2–CB association. (A) HeLa cells were incubated in the presence or absence of LMB for 3 h and then stained with anti-U2B″ (green) and anticoilin antibodies (red). At early time points (3–5 h) CBs remain prominent; although, diffuse accumulation of coilin within nucleoli is also observed (Carvalho et al., 1999). The presence of U2 snRNPs in CBs on the left is demonstrated by the yellow color. The lack of signal overlap is evident in the middle (note red color). On the right, FISH reveals that CBs (red) and U2 genes (green) do not colocalize. (B) CB association with U2 genes decreases with increasing incubation time in LMB. (C) HeLa cells were transfected with PHAX–GFP and costained with anticoilin. In addition to localization throughout the nucleoplasm, note accumulation of PHAX–GFP (green) within the CBs (red) of the transfected cell.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196410&req=5

fig6: Inhibition of snRNA export disrupts RNU2–CB association. (A) HeLa cells were incubated in the presence or absence of LMB for 3 h and then stained with anti-U2B″ (green) and anticoilin antibodies (red). At early time points (3–5 h) CBs remain prominent; although, diffuse accumulation of coilin within nucleoli is also observed (Carvalho et al., 1999). The presence of U2 snRNPs in CBs on the left is demonstrated by the yellow color. The lack of signal overlap is evident in the middle (note red color). On the right, FISH reveals that CBs (red) and U2 genes (green) do not colocalize. (B) CB association with U2 genes decreases with increasing incubation time in LMB. (C) HeLa cells were transfected with PHAX–GFP and costained with anticoilin. In addition to localization throughout the nucleoplasm, note accumulation of PHAX–GFP (green) within the CBs (red) of the transfected cell.
Mentions: Carvalho et al. (1999) showed that treatment of cells with leptomycin B (LMB), a known inhibitor of exportin1/CRM1-mediated nuclear export (Kudo et al., 1998), resulted in the progressive depletion of snRNPs from CBs. Presumably, if newly transcribed snRNAs are prevented from leaving the nucleus, then newly assembled snRNPs will be depleted first from the cytoplasm and then from CBs (Carvalho et al., 1999). We reasoned that such a disruption of the snRNP cycle would also have an effect on the association of CBs with U2 genes. HeLa cells were thus incubated in media containing LMB and assayed. As shown in Fig. 6 A (compare −LMB with +LMB), we monitored the presence of U2B″ protein in CBs as a marker for the U2 snRNP. The kinetics of disappearance of U2 snRNPs from CBs were very similar to those observed by Carvalho et al. (1999). Interestingly, the effects of U2 snRNP depletion from CBs are mirrored by a striking decrease in the RNU2–CB association frequency (Fig. 6, A and B). Although the effects of LMB on the RNU2–CB interaction can be seen in as short as 2 h of treatment, the association frequency decreases with increasing incubation time (Fig. 6 B). Thus, U2 genes no longer associate with CBs when snRNA export is blocked by LMB.

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
Related in: MedlinePlus