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Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product.

Tucker KE, Berciano MT, Jacobs EY, LePage DF, Shpargel KB, Rossire JJ, Chan EK, Lafarga M, Conlon RA, Matera AG - J. Cell Biol. (2001)

Bottom Line: Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals.Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds.These so-called "residual" CBs neither condense Sm proteins nor recruit members of the SMN protein complex.

View Article: PubMed Central - PubMed

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

ABSTRACT
Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called "residual" CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

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Knockout mice display “residual” CBs. (A) Frozen tissues were sectioned and stained with antibodies against coilin and Fb. In wild-type tissues, coilin is colocalized with Fb in CBs (small arrows) and sometimes is visible in nucleolar caps (see wild-type brain panels). Coilin staining is absent from knockout tissues, but residual CBs (large arrows) are evident as extranucleolar Fb foci. For better visualization, insets display the boxed CBs or residual CBs at higher magnification. (B) Confocal imaging of sensory neurons from dorsal root ganglion of a knockout animal costained for coilin/Gemin2, Nopp140/SMN, or Nopp140/U2B′′. Note the residual CBs denoted by white arrows in the Nopp140 channel and the absence of SMN or U2B′′ within these structures. (C) Sensory ganglion neurons from wild-type and knockout animals were stained with silver to visualize nucleoli and CBs (black arrows). Note the absence of extranucleolar silver deposition in the knockout cells.
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fig3: Knockout mice display “residual” CBs. (A) Frozen tissues were sectioned and stained with antibodies against coilin and Fb. In wild-type tissues, coilin is colocalized with Fb in CBs (small arrows) and sometimes is visible in nucleolar caps (see wild-type brain panels). Coilin staining is absent from knockout tissues, but residual CBs (large arrows) are evident as extranucleolar Fb foci. For better visualization, insets display the boxed CBs or residual CBs at higher magnification. (B) Confocal imaging of sensory neurons from dorsal root ganglion of a knockout animal costained for coilin/Gemin2, Nopp140/SMN, or Nopp140/U2B′′. Note the residual CBs denoted by white arrows in the Nopp140 channel and the absence of SMN or U2B′′ within these structures. (C) Sensory ganglion neurons from wild-type and knockout animals were stained with silver to visualize nucleoli and CBs (black arrows). Note the absence of extranucleolar silver deposition in the knockout cells.

Mentions: In an effort to understand the effect of the coilin deletion on CBs, we analyzed cryosections from adult animals and mouse embryonic fibroblast (MEF) cell lines derived from day 13.5–14.5 embryos (for details see Materials and methods and Table II). Labeling of tissues and MEFs from homozygous mutant animals with anticoilin antibodies again confirmed the absence of the wild-type coilin protein in these animals (Fig. 3, A and B , and Fig. 4 A). In contrast, wild-type tissues and MEFs displayed prominent CBs when stained with this antibody (Fig. 3 A and Fig. 4 A). Importantly, sensory ganglion neurons from knockout animals lacked the robust extranucleolar silver staining common to the nuclei of wild-type neurons (Fig. 3 C).


Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product.

Tucker KE, Berciano MT, Jacobs EY, LePage DF, Shpargel KB, Rossire JJ, Chan EK, Lafarga M, Conlon RA, Matera AG - J. Cell Biol. (2001)

Knockout mice display “residual” CBs. (A) Frozen tissues were sectioned and stained with antibodies against coilin and Fb. In wild-type tissues, coilin is colocalized with Fb in CBs (small arrows) and sometimes is visible in nucleolar caps (see wild-type brain panels). Coilin staining is absent from knockout tissues, but residual CBs (large arrows) are evident as extranucleolar Fb foci. For better visualization, insets display the boxed CBs or residual CBs at higher magnification. (B) Confocal imaging of sensory neurons from dorsal root ganglion of a knockout animal costained for coilin/Gemin2, Nopp140/SMN, or Nopp140/U2B′′. Note the residual CBs denoted by white arrows in the Nopp140 channel and the absence of SMN or U2B′′ within these structures. (C) Sensory ganglion neurons from wild-type and knockout animals were stained with silver to visualize nucleoli and CBs (black arrows). Note the absence of extranucleolar silver deposition in the knockout cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150753&req=5

fig3: Knockout mice display “residual” CBs. (A) Frozen tissues were sectioned and stained with antibodies against coilin and Fb. In wild-type tissues, coilin is colocalized with Fb in CBs (small arrows) and sometimes is visible in nucleolar caps (see wild-type brain panels). Coilin staining is absent from knockout tissues, but residual CBs (large arrows) are evident as extranucleolar Fb foci. For better visualization, insets display the boxed CBs or residual CBs at higher magnification. (B) Confocal imaging of sensory neurons from dorsal root ganglion of a knockout animal costained for coilin/Gemin2, Nopp140/SMN, or Nopp140/U2B′′. Note the residual CBs denoted by white arrows in the Nopp140 channel and the absence of SMN or U2B′′ within these structures. (C) Sensory ganglion neurons from wild-type and knockout animals were stained with silver to visualize nucleoli and CBs (black arrows). Note the absence of extranucleolar silver deposition in the knockout cells.
Mentions: In an effort to understand the effect of the coilin deletion on CBs, we analyzed cryosections from adult animals and mouse embryonic fibroblast (MEF) cell lines derived from day 13.5–14.5 embryos (for details see Materials and methods and Table II). Labeling of tissues and MEFs from homozygous mutant animals with anticoilin antibodies again confirmed the absence of the wild-type coilin protein in these animals (Fig. 3, A and B , and Fig. 4 A). In contrast, wild-type tissues and MEFs displayed prominent CBs when stained with this antibody (Fig. 3 A and Fig. 4 A). Importantly, sensory ganglion neurons from knockout animals lacked the robust extranucleolar silver staining common to the nuclei of wild-type neurons (Fig. 3 C).

Bottom Line: Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals.Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds.These so-called "residual" CBs neither condense Sm proteins nor recruit members of the SMN protein complex.

View Article: PubMed Central - PubMed

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

ABSTRACT
Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called "residual" CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

Show MeSH
Related in: MedlinePlus