Limits...
Gemin3: A novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of gems.

Charroux B, Pellizzoni L, Perkinson RA, Shevchenko A, Mann M, Dreyfuss G - J. Cell Biol. (1999)

Bottom Line: The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems.Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction.The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148, USA.

ABSTRACT
The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.

Show MeSH

Related in: MedlinePlus

The Gemin3 protein colocalizes with SMN in gems. A, Laser confocal image of indirect immunofluorescence on HeLa cells using mAb 12H12 against the Gemin3 protein. Note the general cytoplasmic staining, as well as nucleoplasmic and discrete nuclear structures. B, DIC image of the same cell shown in A, arrows indicate gems. C and D, Superimposed laser confocal images of double-label immunofluorescence microscopy experiments using antibodies against coiled bodies marker, p80 coilin (C and D, green), anti-Gemin3 11G9 (C, red), and anti-SMN antibody 2B1 (D, red). E, Superimposed laser confocal images of double-label immunofluorescence microscopy experiments using mAb against Gemin3 (red) and a rabbit affinity-purified antibody against exon7 of the human SMN protein (green). Colocalization of green and red results in yellow color. Dashed lines demarcate the nucleus.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2168095&req=5

Figure 5: The Gemin3 protein colocalizes with SMN in gems. A, Laser confocal image of indirect immunofluorescence on HeLa cells using mAb 12H12 against the Gemin3 protein. Note the general cytoplasmic staining, as well as nucleoplasmic and discrete nuclear structures. B, DIC image of the same cell shown in A, arrows indicate gems. C and D, Superimposed laser confocal images of double-label immunofluorescence microscopy experiments using antibodies against coiled bodies marker, p80 coilin (C and D, green), anti-Gemin3 11G9 (C, red), and anti-SMN antibody 2B1 (D, red). E, Superimposed laser confocal images of double-label immunofluorescence microscopy experiments using mAb against Gemin3 (red) and a rabbit affinity-purified antibody against exon7 of the human SMN protein (green). Colocalization of green and red results in yellow color. Dashed lines demarcate the nucleus.

Mentions: Indirect laser confocal immunofluorescence microscopy using antibodies 11G9 and 12H12 was performed on HeLa cells to determine the subcellular localization of Gemin3. Fig. 5 A shows that Gemin3 is found throughout the cytoplasm and also displays intense staining of prominent discrete nuclear bodies that are readily discernible by differential interference contrast (DIC; Fig. 5 B). This pattern is similar to that seen for SMN and Gemin2 (Liu and Dreyfuss 1996; Liu et al. 1997), except that the nucleoplasmic staining of Gemin3 is stronger. To determine if the nuclear structures stained by 11G9 are gems or coiled bodies, we performed double-label immunofluorescence experiments using antibodies against Gemin3 and either p80 coilin as a marker of coiled bodies (Andrade et al. 1991) or SMN as a marker of gems (Liu and Dreyfuss 1996; Fig. 5). In many cell lines, gems and coiled bodies entirely overlap by antibody staining, however, in the HeLa PV strain used here, these two bodies are frequently found separate from each other (Liu and Dreyfuss 1996; Matera and Frey 1998). Therefore, we used HeLa PV cells to examine whether Gemin3 is located in gems or in coiled bodies. As can be seen in Fig. 5C and Fig. D, the nuclear structures that contain Gemin3 are clearly distinct from coiled bodies, but Gemin3 completely colocalized with SMN in gems (Fig. 5 E). The colocalization of Gemin3 with SMN strongly supports the conclusion that these two proteins exist as a complex in the cell. Gemin3 is thus the third constituent of gems described so far. Compared with visual observation, the confocal photographs in Fig. 5, slightly overestimates the nuclear staining and underestimates the cytoplasmic staining for Fig. 5C and Fig. D.


Gemin3: A novel DEAD box protein that interacts with SMN, the spinal muscular atrophy gene product, and is a component of gems.

Charroux B, Pellizzoni L, Perkinson RA, Shevchenko A, Mann M, Dreyfuss G - J. Cell Biol. (1999)

The Gemin3 protein colocalizes with SMN in gems. A, Laser confocal image of indirect immunofluorescence on HeLa cells using mAb 12H12 against the Gemin3 protein. Note the general cytoplasmic staining, as well as nucleoplasmic and discrete nuclear structures. B, DIC image of the same cell shown in A, arrows indicate gems. C and D, Superimposed laser confocal images of double-label immunofluorescence microscopy experiments using antibodies against coiled bodies marker, p80 coilin (C and D, green), anti-Gemin3 11G9 (C, red), and anti-SMN antibody 2B1 (D, red). E, Superimposed laser confocal images of double-label immunofluorescence microscopy experiments using mAb against Gemin3 (red) and a rabbit affinity-purified antibody against exon7 of the human SMN protein (green). Colocalization of green and red results in yellow color. Dashed lines demarcate the nucleus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168095&req=5

Figure 5: The Gemin3 protein colocalizes with SMN in gems. A, Laser confocal image of indirect immunofluorescence on HeLa cells using mAb 12H12 against the Gemin3 protein. Note the general cytoplasmic staining, as well as nucleoplasmic and discrete nuclear structures. B, DIC image of the same cell shown in A, arrows indicate gems. C and D, Superimposed laser confocal images of double-label immunofluorescence microscopy experiments using antibodies against coiled bodies marker, p80 coilin (C and D, green), anti-Gemin3 11G9 (C, red), and anti-SMN antibody 2B1 (D, red). E, Superimposed laser confocal images of double-label immunofluorescence microscopy experiments using mAb against Gemin3 (red) and a rabbit affinity-purified antibody against exon7 of the human SMN protein (green). Colocalization of green and red results in yellow color. Dashed lines demarcate the nucleus.
Mentions: Indirect laser confocal immunofluorescence microscopy using antibodies 11G9 and 12H12 was performed on HeLa cells to determine the subcellular localization of Gemin3. Fig. 5 A shows that Gemin3 is found throughout the cytoplasm and also displays intense staining of prominent discrete nuclear bodies that are readily discernible by differential interference contrast (DIC; Fig. 5 B). This pattern is similar to that seen for SMN and Gemin2 (Liu and Dreyfuss 1996; Liu et al. 1997), except that the nucleoplasmic staining of Gemin3 is stronger. To determine if the nuclear structures stained by 11G9 are gems or coiled bodies, we performed double-label immunofluorescence experiments using antibodies against Gemin3 and either p80 coilin as a marker of coiled bodies (Andrade et al. 1991) or SMN as a marker of gems (Liu and Dreyfuss 1996; Fig. 5). In many cell lines, gems and coiled bodies entirely overlap by antibody staining, however, in the HeLa PV strain used here, these two bodies are frequently found separate from each other (Liu and Dreyfuss 1996; Matera and Frey 1998). Therefore, we used HeLa PV cells to examine whether Gemin3 is located in gems or in coiled bodies. As can be seen in Fig. 5C and Fig. D, the nuclear structures that contain Gemin3 are clearly distinct from coiled bodies, but Gemin3 completely colocalized with SMN in gems (Fig. 5 E). The colocalization of Gemin3 with SMN strongly supports the conclusion that these two proteins exist as a complex in the cell. Gemin3 is thus the third constituent of gems described so far. Compared with visual observation, the confocal photographs in Fig. 5, slightly overestimates the nuclear staining and underestimates the cytoplasmic staining for Fig. 5C and Fig. D.

Bottom Line: The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems.Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction.The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148, USA.

ABSTRACT
The survival of motor neurons (SMN) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The SMN protein is part of a complex containing several proteins, of which one, SIP1 (SMN interacting protein 1), has been characterized so far. The SMN complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, SMN and SIP1 interact with the Sm core proteins of spliceosomal small nuclear ribonucleoproteins (snRNPs), and they play a critical role in snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing, likely by serving in the regeneration of snRNPs. Here, we report the identification of another component of the SMN complex, a novel DEAD box putative RNA helicase, named Gemin3. Gemin3 interacts directly with SMN, as well as with SmB, SmD2, and SmD3. Immunolocalization studies using mAbs to Gemin3 show that it colocalizes with SMN in gems. Gemin3 binds SMN via its unique COOH-terminal domain, and SMN mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in Gemin3 suggests that it may provide the catalytic activity that plays a critical role in the function of the SMN complex on RNPs.

Show MeSH
Related in: MedlinePlus