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

Sequencing of Gemin3 by nano-ES MS/MS. A, Part of the spectrum of the unseparated in-gel tryptic digest of p105 band. Peptide ions, designated by T, are autolysis products of trypsin and were identified by comparison with the spectrum acquired from the control sample. Other peptide ions observed in the spectrum were in turn isolated by the first mass analyzer of a triple quadrupole instrument, fragmented in the collision cell, and their tandem mass spectra acquired. Upon searching a comprehensive protein sequence database using tandem mass spectrometric data (see below) peptide ions designated with filled triangles were identified as tryptic peptides originating from PTB-associated splicing factor (P23246). The presence of PTB-associated splicing factor in the SMN complex turned out to be negative by both coimmunoprecipitation and direct binding to several components of the SMN complex (data not shown). Peptide ions designated with asterisks were identified as peptides from immunoglobulins used for immunoaffinity purification. Tandem mass spectra acquired from the peptide ions having m/z 552.9 and 622.5 did not identify any protein in a protein sequence database. However when the search was performed against a comprehensive database of expressed sequence tags (dbEST), the peptide sequence VLISTDLTSR from EST clone W65908 was identified as matching the tandem mass spectrum. After full-length sequence had been obtained (see Materials and Methods) the tandem mass spectrum acquired from the peptide ion at m/z 622.5 was matched to the peptide LNSSDPSLIGLK present in the sequence of Gemin3. B, Tandem mass spectrum, acquired from doubly charged peptide precursor ion having m/z 552.9. Continuous series of the fragment ions containing the COOH terminus of the peptide (Y′′-ions; Roepstorff and Fohlman 1984) is produced upon collisional fragmentation of tryptic peptides. A short stretch of the peptide sequence was deduced unambiguously by considering precise mass differences between adjacent Y′′-ions (in bold) observed in a part of the spectrum above m/z of the parent ion. Note that Leu and Ile residues have the same nominal mass and, therefore, are usually not distinguished by MS. The determined piece of a peptide sequence was combined with the masses of correspondent Y′′-ions and with the mass of intact peptide into a peptide sequence tag (Mann and Wilm 1994) that was subsequently used for searching protein and EST databases by the program PeptideSearch. Once the database search produced a hit, the correspondent peptide sequence was retrieved from a database and masses of the ions from the NH2-terminal fragment series (A- and B-ions) were used to verify the match. This enabled highly confident protein identification albeit a single peptide containing only ten amino acid residues was matched to the sequence of EST clone.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2168095&req=5

Figure 2: Sequencing of Gemin3 by nano-ES MS/MS. A, Part of the spectrum of the unseparated in-gel tryptic digest of p105 band. Peptide ions, designated by T, are autolysis products of trypsin and were identified by comparison with the spectrum acquired from the control sample. Other peptide ions observed in the spectrum were in turn isolated by the first mass analyzer of a triple quadrupole instrument, fragmented in the collision cell, and their tandem mass spectra acquired. Upon searching a comprehensive protein sequence database using tandem mass spectrometric data (see below) peptide ions designated with filled triangles were identified as tryptic peptides originating from PTB-associated splicing factor (P23246). The presence of PTB-associated splicing factor in the SMN complex turned out to be negative by both coimmunoprecipitation and direct binding to several components of the SMN complex (data not shown). Peptide ions designated with asterisks were identified as peptides from immunoglobulins used for immunoaffinity purification. Tandem mass spectra acquired from the peptide ions having m/z 552.9 and 622.5 did not identify any protein in a protein sequence database. However when the search was performed against a comprehensive database of expressed sequence tags (dbEST), the peptide sequence VLISTDLTSR from EST clone W65908 was identified as matching the tandem mass spectrum. After full-length sequence had been obtained (see Materials and Methods) the tandem mass spectrum acquired from the peptide ion at m/z 622.5 was matched to the peptide LNSSDPSLIGLK present in the sequence of Gemin3. B, Tandem mass spectrum, acquired from doubly charged peptide precursor ion having m/z 552.9. Continuous series of the fragment ions containing the COOH terminus of the peptide (Y′′-ions; Roepstorff and Fohlman 1984) is produced upon collisional fragmentation of tryptic peptides. A short stretch of the peptide sequence was deduced unambiguously by considering precise mass differences between adjacent Y′′-ions (in bold) observed in a part of the spectrum above m/z of the parent ion. Note that Leu and Ile residues have the same nominal mass and, therefore, are usually not distinguished by MS. The determined piece of a peptide sequence was combined with the masses of correspondent Y′′-ions and with the mass of intact peptide into a peptide sequence tag (Mann and Wilm 1994) that was subsequently used for searching protein and EST databases by the program PeptideSearch. Once the database search produced a hit, the correspondent peptide sequence was retrieved from a database and masses of the ions from the NH2-terminal fragment series (A- and B-ions) were used to verify the match. This enabled highly confident protein identification albeit a single peptide containing only ten amino acid residues was matched to the sequence of EST clone.

Mentions: Immunoprecipitations from [35S]methionine-labeled HeLa cell lysates with anti-SMN and anti-SIP1 mAbs revealed the presence of several protein components in the SMN–SIP1 complex (Liu et al. 1997). Among the proteins that can be coimmunopurified with anti-SMN and anti-SIP1 antibodies, only some of the major low molecular mass proteins, identified as the Sm proteins, have been characterized so far (Liu et al. 1997; Fig. 1A and Fig. B). In addition to SMN, SIP1, and the Sm proteins, there is a doublet at ∼97 kD, and additional bands at 175, 95, 60, and 50 kD that coimmunopurified with the anti-SMN antibody. The two proteins of the 97-kD doublet were eluted from the gel, digested with trypsin, and the resulting peptides sequenced by nano-ES MS as described previously (Shevchenko et al. 1996; Wilm et al. 1996; Fig. 2). In this paper, we describe the molecular cloning and characterization of the high molecular weight protein of this doublet (p105). Several peptides from this band identified a human EST sequence (clone #AA303940) using the peptide sequence tag algorithm (Fig. 2). Several additional cDNA clones were obtained by hybridization screening of a human leukemia 5′-STRETCH PLUS cDNA library, using this EST clone as a probe. We isolated 12 independent partial cDNA clones with insert sizes ranging from 1–2.5 kb, all of which contained overlapping regions of the same ORF. 5′ RACE PCR was used to extend this cDNA further upstream. A cDNA clone containing the longest ORF was constructed and conceptual translation of its nucleotide sequence revealed a potential initiator methionine, preceded by an in frame stop codon. This cDNA encodes a putative protein of 824 amino acids with a calculated molecular mass of 92.2 kD and a pI of 6.5. We then determined that this cDNA encodes the p105 protein that coimmunoprecipitates with SMN (see Gemin3 and SMN Colocalize in Gems). Thus, this is a full-length cDNA clone for a novel component of the SMN complex that we termed Gemin3, for component of gems number 3 (see below). Because of the existence of several unrelated proteins called SIP1 (Mylin et al. 1994; Zhang and Wu 1998; Verschueren et al. 1999), we tentatively rename the SMN-interacting SIP1 (Liu et al. 1997) Gemin2, for component of gems number 2 (SMN is the first component of gems identified; Liu and Dreyfuss 1996). Gemin3 has high amino acid sequence similarities with the RNA helicase core region of the human eukaryotic initiation factor 4A-II (eIF4A-II). eIF4A-II is a DEAD box RNA helicase that belongs to the SFII superfamily of helicase (reviewed in De la Cruz et al. 1999). A scheme depicting the modular structure of Gemin3 and the predicted amino acid sequence of Gemin3 aligned with the sequence of eIF4A-II are presented in Fig. 3. This alignment showed the presence of seven motifs in the Gemin3 protein, which are characteristic of the RNA helicase core region. Database searches with the COOH-terminal nonconserved region did not reveal significant homology to any other protein or any recognizable motifs.


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)

Sequencing of Gemin3 by nano-ES MS/MS. A, Part of the spectrum of the unseparated in-gel tryptic digest of p105 band. Peptide ions, designated by T, are autolysis products of trypsin and were identified by comparison with the spectrum acquired from the control sample. Other peptide ions observed in the spectrum were in turn isolated by the first mass analyzer of a triple quadrupole instrument, fragmented in the collision cell, and their tandem mass spectra acquired. Upon searching a comprehensive protein sequence database using tandem mass spectrometric data (see below) peptide ions designated with filled triangles were identified as tryptic peptides originating from PTB-associated splicing factor (P23246). The presence of PTB-associated splicing factor in the SMN complex turned out to be negative by both coimmunoprecipitation and direct binding to several components of the SMN complex (data not shown). Peptide ions designated with asterisks were identified as peptides from immunoglobulins used for immunoaffinity purification. Tandem mass spectra acquired from the peptide ions having m/z 552.9 and 622.5 did not identify any protein in a protein sequence database. However when the search was performed against a comprehensive database of expressed sequence tags (dbEST), the peptide sequence VLISTDLTSR from EST clone W65908 was identified as matching the tandem mass spectrum. After full-length sequence had been obtained (see Materials and Methods) the tandem mass spectrum acquired from the peptide ion at m/z 622.5 was matched to the peptide LNSSDPSLIGLK present in the sequence of Gemin3. B, Tandem mass spectrum, acquired from doubly charged peptide precursor ion having m/z 552.9. Continuous series of the fragment ions containing the COOH terminus of the peptide (Y′′-ions; Roepstorff and Fohlman 1984) is produced upon collisional fragmentation of tryptic peptides. A short stretch of the peptide sequence was deduced unambiguously by considering precise mass differences between adjacent Y′′-ions (in bold) observed in a part of the spectrum above m/z of the parent ion. Note that Leu and Ile residues have the same nominal mass and, therefore, are usually not distinguished by MS. The determined piece of a peptide sequence was combined with the masses of correspondent Y′′-ions and with the mass of intact peptide into a peptide sequence tag (Mann and Wilm 1994) that was subsequently used for searching protein and EST databases by the program PeptideSearch. Once the database search produced a hit, the correspondent peptide sequence was retrieved from a database and masses of the ions from the NH2-terminal fragment series (A- and B-ions) were used to verify the match. This enabled highly confident protein identification albeit a single peptide containing only ten amino acid residues was matched to the sequence of EST clone.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Sequencing of Gemin3 by nano-ES MS/MS. A, Part of the spectrum of the unseparated in-gel tryptic digest of p105 band. Peptide ions, designated by T, are autolysis products of trypsin and were identified by comparison with the spectrum acquired from the control sample. Other peptide ions observed in the spectrum were in turn isolated by the first mass analyzer of a triple quadrupole instrument, fragmented in the collision cell, and their tandem mass spectra acquired. Upon searching a comprehensive protein sequence database using tandem mass spectrometric data (see below) peptide ions designated with filled triangles were identified as tryptic peptides originating from PTB-associated splicing factor (P23246). The presence of PTB-associated splicing factor in the SMN complex turned out to be negative by both coimmunoprecipitation and direct binding to several components of the SMN complex (data not shown). Peptide ions designated with asterisks were identified as peptides from immunoglobulins used for immunoaffinity purification. Tandem mass spectra acquired from the peptide ions having m/z 552.9 and 622.5 did not identify any protein in a protein sequence database. However when the search was performed against a comprehensive database of expressed sequence tags (dbEST), the peptide sequence VLISTDLTSR from EST clone W65908 was identified as matching the tandem mass spectrum. After full-length sequence had been obtained (see Materials and Methods) the tandem mass spectrum acquired from the peptide ion at m/z 622.5 was matched to the peptide LNSSDPSLIGLK present in the sequence of Gemin3. B, Tandem mass spectrum, acquired from doubly charged peptide precursor ion having m/z 552.9. Continuous series of the fragment ions containing the COOH terminus of the peptide (Y′′-ions; Roepstorff and Fohlman 1984) is produced upon collisional fragmentation of tryptic peptides. A short stretch of the peptide sequence was deduced unambiguously by considering precise mass differences between adjacent Y′′-ions (in bold) observed in a part of the spectrum above m/z of the parent ion. Note that Leu and Ile residues have the same nominal mass and, therefore, are usually not distinguished by MS. The determined piece of a peptide sequence was combined with the masses of correspondent Y′′-ions and with the mass of intact peptide into a peptide sequence tag (Mann and Wilm 1994) that was subsequently used for searching protein and EST databases by the program PeptideSearch. Once the database search produced a hit, the correspondent peptide sequence was retrieved from a database and masses of the ions from the NH2-terminal fragment series (A- and B-ions) were used to verify the match. This enabled highly confident protein identification albeit a single peptide containing only ten amino acid residues was matched to the sequence of EST clone.
Mentions: Immunoprecipitations from [35S]methionine-labeled HeLa cell lysates with anti-SMN and anti-SIP1 mAbs revealed the presence of several protein components in the SMN–SIP1 complex (Liu et al. 1997). Among the proteins that can be coimmunopurified with anti-SMN and anti-SIP1 antibodies, only some of the major low molecular mass proteins, identified as the Sm proteins, have been characterized so far (Liu et al. 1997; Fig. 1A and Fig. B). In addition to SMN, SIP1, and the Sm proteins, there is a doublet at ∼97 kD, and additional bands at 175, 95, 60, and 50 kD that coimmunopurified with the anti-SMN antibody. The two proteins of the 97-kD doublet were eluted from the gel, digested with trypsin, and the resulting peptides sequenced by nano-ES MS as described previously (Shevchenko et al. 1996; Wilm et al. 1996; Fig. 2). In this paper, we describe the molecular cloning and characterization of the high molecular weight protein of this doublet (p105). Several peptides from this band identified a human EST sequence (clone #AA303940) using the peptide sequence tag algorithm (Fig. 2). Several additional cDNA clones were obtained by hybridization screening of a human leukemia 5′-STRETCH PLUS cDNA library, using this EST clone as a probe. We isolated 12 independent partial cDNA clones with insert sizes ranging from 1–2.5 kb, all of which contained overlapping regions of the same ORF. 5′ RACE PCR was used to extend this cDNA further upstream. A cDNA clone containing the longest ORF was constructed and conceptual translation of its nucleotide sequence revealed a potential initiator methionine, preceded by an in frame stop codon. This cDNA encodes a putative protein of 824 amino acids with a calculated molecular mass of 92.2 kD and a pI of 6.5. We then determined that this cDNA encodes the p105 protein that coimmunoprecipitates with SMN (see Gemin3 and SMN Colocalize in Gems). Thus, this is a full-length cDNA clone for a novel component of the SMN complex that we termed Gemin3, for component of gems number 3 (see below). Because of the existence of several unrelated proteins called SIP1 (Mylin et al. 1994; Zhang and Wu 1998; Verschueren et al. 1999), we tentatively rename the SMN-interacting SIP1 (Liu et al. 1997) Gemin2, for component of gems number 2 (SMN is the first component of gems identified; Liu and Dreyfuss 1996). Gemin3 has high amino acid sequence similarities with the RNA helicase core region of the human eukaryotic initiation factor 4A-II (eIF4A-II). eIF4A-II is a DEAD box RNA helicase that belongs to the SFII superfamily of helicase (reviewed in De la Cruz et al. 1999). A scheme depicting the modular structure of Gemin3 and the predicted amino acid sequence of Gemin3 aligned with the sequence of eIF4A-II are presented in Fig. 3. This alignment showed the presence of seven motifs in the Gemin3 protein, which are characteristic of the RNA helicase core region. Database searches with the COOH-terminal nonconserved region did not reveal significant homology to any other protein or any recognizable motifs.

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