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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.

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mAbs 11G9 and 12H12 are specific for Gemin3. A, Myc-tagged Gemin3, hnRNP A1, and SMN proteins were produced in rabbit reticulocyte lysate in the presence of [35S]methionine. The labeled proteins were immunoprecipitated using mAb 11G9 and 12H12, and the immunoprecipitated material analyzed by SDS-PAGE and autoradiography. 10% of the in vitro translated protein is shown on the left panel. B, Immunoblotting using mAb 11G9 on purified 6His-Gemin2 and 6His-Gemin3. C, Immunoblotting using mAbs 12H12 and 11G9 on total HeLa extract. Note that, in addition to the strong p105 signal, 12H12, but not 11G9, weakly detects a protein of ∼55 kD. The position of the molecular weight markers is indicated on the left (in kD).
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Figure 4: mAbs 11G9 and 12H12 are specific for Gemin3. A, Myc-tagged Gemin3, hnRNP A1, and SMN proteins were produced in rabbit reticulocyte lysate in the presence of [35S]methionine. The labeled proteins were immunoprecipitated using mAb 11G9 and 12H12, and the immunoprecipitated material analyzed by SDS-PAGE and autoradiography. 10% of the in vitro translated protein is shown on the left panel. B, Immunoblotting using mAb 11G9 on purified 6His-Gemin2 and 6His-Gemin3. C, Immunoblotting using mAbs 12H12 and 11G9 on total HeLa extract. Note that, in addition to the strong p105 signal, 12H12, but not 11G9, weakly detects a protein of ∼55 kD. The position of the molecular weight markers is indicated on the left (in kD).

Mentions: To investigate the interaction of Gemin3 with SMN and to characterize Gemin3 further, we generated mAbs to it by immunizing mice with a purified bacterially produced recombinant 6His-Gemin3 fragment (amino acids 368–548). Two hybridomas, 11G9 and 12H12, were selected for additional studies. Several lines of evidence demonstrate that these hybridomas indeed produce mAbs that recognize Gemin3 specifically. First, both 11G9 and 12H12 specifically immunoprecipitate Gemin3 produced by in vitro transcription and translation from the Gemin3 cDNA, but do not immunoprecipitate similarly produced hnRNP A1 or SMN proteins (Fig. 4 A). Second, the mAb 11G9 efficiently recognized purified 6His-Gemin3 on Western blots, but did not recognize similarly produced and purified 6His-Gemin2 (Fig. 4 B). Finally, on an immunoblot of total HeLa lysate, both 11G9 and 12H12 recognized a single protein of ∼105 kD (Fig. 4 C). mAbs 11G9 or 12H12 did not recognized a specific protein on a Western blot with total mouse 3T3 cell lysate or Xenopus laevis XL-177 cell lysate. However, 11G9 specifically immunoprecipitated a single protein of ∼105 kD from these cell lysates (data not shown), suggesting that Gemin3, like SMN, is conserved in vertebrates.


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)

mAbs 11G9 and 12H12 are specific for Gemin3. A, Myc-tagged Gemin3, hnRNP A1, and SMN proteins were produced in rabbit reticulocyte lysate in the presence of [35S]methionine. The labeled proteins were immunoprecipitated using mAb 11G9 and 12H12, and the immunoprecipitated material analyzed by SDS-PAGE and autoradiography. 10% of the in vitro translated protein is shown on the left panel. B, Immunoblotting using mAb 11G9 on purified 6His-Gemin2 and 6His-Gemin3. C, Immunoblotting using mAbs 12H12 and 11G9 on total HeLa extract. Note that, in addition to the strong p105 signal, 12H12, but not 11G9, weakly detects a protein of ∼55 kD. The position of the molecular weight markers is indicated on the left (in kD).
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Related In: Results  -  Collection

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

Figure 4: mAbs 11G9 and 12H12 are specific for Gemin3. A, Myc-tagged Gemin3, hnRNP A1, and SMN proteins were produced in rabbit reticulocyte lysate in the presence of [35S]methionine. The labeled proteins were immunoprecipitated using mAb 11G9 and 12H12, and the immunoprecipitated material analyzed by SDS-PAGE and autoradiography. 10% of the in vitro translated protein is shown on the left panel. B, Immunoblotting using mAb 11G9 on purified 6His-Gemin2 and 6His-Gemin3. C, Immunoblotting using mAbs 12H12 and 11G9 on total HeLa extract. Note that, in addition to the strong p105 signal, 12H12, but not 11G9, weakly detects a protein of ∼55 kD. The position of the molecular weight markers is indicated on the left (in kD).
Mentions: To investigate the interaction of Gemin3 with SMN and to characterize Gemin3 further, we generated mAbs to it by immunizing mice with a purified bacterially produced recombinant 6His-Gemin3 fragment (amino acids 368–548). Two hybridomas, 11G9 and 12H12, were selected for additional studies. Several lines of evidence demonstrate that these hybridomas indeed produce mAbs that recognize Gemin3 specifically. First, both 11G9 and 12H12 specifically immunoprecipitate Gemin3 produced by in vitro transcription and translation from the Gemin3 cDNA, but do not immunoprecipitate similarly produced hnRNP A1 or SMN proteins (Fig. 4 A). Second, the mAb 11G9 efficiently recognized purified 6His-Gemin3 on Western blots, but did not recognize similarly produced and purified 6His-Gemin2 (Fig. 4 B). Finally, on an immunoblot of total HeLa lysate, both 11G9 and 12H12 recognized a single protein of ∼105 kD (Fig. 4 C). mAbs 11G9 or 12H12 did not recognized a specific protein on a Western blot with total mouse 3T3 cell lysate or Xenopus laevis XL-177 cell lysate. However, 11G9 specifically immunoprecipitated a single protein of ∼105 kD from these cell lysates (data not shown), suggesting that Gemin3, like SMN, is conserved in vertebrates.

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