Charroux B et al. (2000), Gemin4. A novel component of the SMN complex th...

XB-ART-11327

J Cell Biol 2000 Mar 20;1486:1177-86.

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Gemin4. A novel component of the SMN complex that is found in both gems and nucleoli.

Charroux B , Pellizzoni L , Perkinson RA , Yong J , Shevchenko A , Mann M , Dreyfuss G .

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The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly.

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Species referenced: Xenopus laevis
Genes referenced: acta2 arhgef5 arhgef7 coil dio3 eif4g2 gemin2 gemin4 herpud1 hnrnpa1 hnrnpc il1r1 myc smn1 sp2 tbx2

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	Figure 1. Immunoprecipitation of the SMN complex with mAb against SMN. Immunoprecipitations using anti-SMN mAb 2B1 and [35S]methionine-labeled HeLa cell cytoplasmic extract. The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography. Antibody 2B1 (lane 2B1) immunoprecipitates SMN, Gemin2, Gemin3, Sm proteins B, Bâ€², D1-3, E, F, and G, and a group of proteins indicated as p175, p97, p95, p60, and p50. The SP2/0 lane shows the background of immunoprecipitation (lane SP2/0). The position of the molecular mass markers is indicated on the left in kilodaltons.
	Figure 2. mAb 22C10 is specific for Gemin4. (A) Myc-tagged Gemin4, hnRNP A1, and SMN proteins were produced in rabbit reticulocyte lysate in the presence of [35S]methionine. The labeled proteins were immunoprecipitated using mAb 22C10, and the immunoprecipitated material was analyzed by SDS-PAGE and autoradiography. The left panel shows 10% of the in vitro translated proteins. (B) Immunoblotting using mAb 22C10 on total HeLa extract. (C) Immunoprecipitations using anti-Gemin4 mAb 22C10 and [35S]methionine-labeled HeLa or mouse 3T3 cell extracts. The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography. The position of the molecular mass markers is indicated on the left in kilodaltons.
	Figure 3. The Gemin4 protein colocalizes with SMN in gems and is detected in nucleoli. (A) Laser confocal image of indirect immunofluorescence on HeLa cells using mAb 22C10 against the Gemin4 protein, arrows in A indicate gems. (B and C) Superimposed laser confocal images of double label immunofluorescence microscopy experiments using antibodies against p80 coilin (B and C, red), SMN (B, green), and Gemin4 (C, green). (D) Differential interference contrast image of the same cell shown in C, arrows indicate gems and arrowheads indicate CBs. (E) Superimposed laser confocal images of double label immunofluorescence microscopy experiments using mAb against Gemin4 (green) and a rabbit affinity-purified antibody against exon 7 epitope of the human SMN protein (red). (F) Superimposed laser confocal images of double label immunofluorescence microscopy experiments using mAbs against Gemin4 (green) and against fibrillarin (red). Dashed lines demarcate the nucleus. A is a z-series composite stack of confocal sections that shows the cytoplasmic staining of Gemin4. B, C, E, and F, are confocal sections through the nucleus that underestimate the cytoplasmic staining of Gemin4 and/or SMN.
	Figure 4. Gemin4 is in a complex with SMN, Gemin2, and Gemin3. (A and B) SMN, Gemin2, Gemin3, and Gemin4 can be coimmunoprecipitated in vivo. (A) mAbs against Gemin4 coimmunoprecipitate SMN, Gemin2, and Gemin3. Immunoprecipitation from total the HeLa extract was done with mAb anti-Gemin4 (22C10) and the immunoprecipitated proteins were analyzed by Western blot using 2B1 (anti-SMN), 2S7 (anti-Gemin2), or 11G9 (anti-Gemin3) antibodies. (B) mAbs against SMN, Gemin2, and Gemin3 coimmunoprecipitate Gemin4. Immunoprecipitation from total HeLa extract was done with mAb anti-SMN (lane 2B1 IP), anti-Gemin2 (lane 2S7 IP), anti-Gemin3 (lane 11G9 IP), or SP2/O (lane SP2/O IP) as a negative control. The immunoprecipitated proteins were analyzed by Western blot using the anti-Gemin4 mAb 22C10. The position of the molecular mass markers is indicated on the left (in kD) and the positions of the light chain (l.c) and heavy chain (h.c) of the antibodies used for immunoprecipitation are indicated. (C) Gel filtration analysis of SMN complexes. Total HeLa extract was fractionated on a Superose 6 HR 10/30 column. The fractions were analyzed by SDS-PAGE, and SMN, Gemin2, Gemin3, and Gemin4 proteins were detected by Western blotting. The fraction number and the molecular mass standards are indicated.
	Figure 5. Gemin4 interacts directly with the DEAD box protein Gemin3 in vitro. (A) Gemin4 interacts with Gemin3 in vitro. In vitro translated [35S]methionine-labeled myc-tagged SMN, Gemin2, Gemin3, and Gemin4 proteins were incubated with purified GST-Gemin4 as described in Materials and Methods. Bound proteins were analyzed by SDS-PAGE and fluorography. The in vitro translation panel shows 5% of the input. The position of the molecular mass markers is indicated on the left in kilodaltons. (B) Gemin4 interacts tightly with Gemin3. In vitro translated [35S]methionine-labeled myc-Gemin4 was incubated with purified GST-Gemin3 in increasing salt concentrations as indicated (see Materials and Methods). Bound proteins were analyzed by SDS-PAGE and fluorography. The in vitro translation panel shows 10% of the input.
	Figure 6. Gemin4 is associated with snRNPs in vivo. (A) Immunoprecipitations of [35S]methionine-labeled total HeLa cell extract using mAbs specific to SMN (2B1), Gemin4 (22C10), and the snRNP Sm core proteins (Y12). The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography. The several proteins immunoprecipitated are indicated on the right. (B) Gemin4 is coimmunoprecipitated with Sm proteins. Immunoprecipitation from the total HeLa extract was performed with mAb anti-Sm (Y12) and the immunoprecipitated proteins were analyzed by Western blotting using 22C10 (anti-Gemin4). The position of the molecular mass markers is indicated on the left (in kD). The positions of the light chain (l.c) and heavy chain (h.c) of the antibody used for immunoprecipitation are indicated. The total lane shows 2% of the input. (C) Gemin4 interacts with several Sm proteins in vitro. In vitro translated [35S]methionine-labeled myc-tagged Sm proteins B, D1, D2, D3, E, F, and G were incubated with purified GST-Gemin4 as described in Materials and Methods. Bound proteins were analyzed by SDS-PAGE and fluorography. The in vitro translation panel shows 5% of the input. The position of the molecular mass markers is indicated on the left in kilodaltons. (D) Gemin4 interacts directly with SmB in vitro. Recombinant His-tagged SmB protein was incubated with purified GST or GST-Gemin4 proteins as described in Materials and Methods. Bound proteins were analyzed by SDS-PAGE and Western blotting with an anti-His tag antibodies.
	Figure 7. Gemin4 is associated with spliceosomal U snRNAs in the cytoplasm of Xenopus oocytes. (A) Immunoprecipitation of spliceosomal U snRNAs with anti-Gemin2 and anti-Gemin4 antibodies. A mixture of different in vitro synthesized 32P-labeled RNAs were injected into the cytoplasm of oocytes. 3 h later, immunoprecipitations were carried out with either anti-Gemin4 (22C10) or anti-Gemin2 (2E17) or control antibodies (SP2/O). Immunoprecipitated RNA (IP) was analyzed by gel electrophoresis. In the supernatant (SUP) panel 2.5% of each immunoprecipitation is shown. (B) Anti-Gemin4 antibodies can immunoprecipitate U1 and U5 snRNAs only from the cytoplasm. The indicated 32P-labeled RNAs were injected as a mixture into the cytoplasm of oocytes. After incubation for 18 h, the oocytes were dissected into nuclear (N) and cytoplasmic (C) fractions, and RNAs from both fractions were immunoprecipitated (IP) with either anti-Gemin4 (22C10), anti-Sm (Y12), or control antibodies (SP2/O). 2.5% of the input is shown.
	Figure 8. A schematic representation of the SMN complexes. (A) The monomeric SMNÎ”Ex7, associates with Gemin2 to form a dimeric complex. SMNÎ”Ex7 lacks the ability to self-associate as well as to interact with Gemin3 and the Sm proteins (Lorson et al. 1998; Charroux et al. 1999; Pellizzoni et al. 1999). This monomeric SMNÎ”Ex7â€“Gemin2 complex is very likely an inactive complex (see text for more details). (B) Monomeric SMN, associated with Gemin2 which binds to SMN but not itself (Liu et al. 1997; data not shown), contains a low affinity binding site for the Sm proteins (Pellizzoni et al. 1999). Gemin3 associates with Gemin4 (this work). SMN self-associates, forming at least a SMNâ€“Gemin2 dimeric complex (Pellizzoni et al. 1999). Gemin3â€“Gemin4 complex associates with SMNâ€“Gemin2 dimeric complex via an SMNâ€“Gemin3 interaction (Charroux et al. 1999). In this oligomeric conformation, a binding site with a much higher affinity for the Sm proteins is formed. The oligomeric form likely represents the active form of the SMN complex (see text for more details). (C) Gemin4 interacts with Gemin3* to form a dimeric complex that lacks SMN and Gemin2 (see text for details). Gemin3* represents the faster migrating form of Gemin3 observed in Fig. 4 B. Only one Sm snRNP protein is included in the drawing to simplify the model.

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