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

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.

Figure 1

Immunoprecipitation of the SMN complex with an mAb against SMN. A, Immunoprecipitations using anti-SMN mAb 2B1 and [³⁵S]methionine-labeled HeLa cell. The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography (24-h exposure). Antibody 2B1 immunoprecipitates SMN, Gemin2, Sm proteins B, B′, D1-3, E, F, and G, and a group of proteins indicated as p175, p105, p97, p95, p60, and p50. The SP2/O lane shows the background of immunoprecipitation. B, A longer exposure (36 h) of the bottom part of the gel to visualize the Sm proteins more clearly. The positions of the molecular weight markers are indicated on the left (in kD).

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.

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).

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.