Purification of Native Survival of Motor Neurons Complexes and Identification of Gemin6 as a Novel Component

doi:10.1074/jbc.M110141200

Purification of Native Survival of Motor Neurons Complexes and Identification of Gemin6 as a Novel Component*

From the ^‡Howard Hughes Medical Institute and Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148 and the ^¶Protein Interaction Laboratory, Center for Experimental Bioinformatics and Department of Biochemistry & Molecular Biology, University of Southern Denmark, DK-5230 Odense M, Denmark

Abstract

The survival of motor neurons (SMN) protein, the product of the gene responsible for the motor neuron degenerative disease spinal muscular atrophy (SMA), is part of a large macromolecular complex. The SMN complex is localized in both the cytoplasm and the nucleus and contains SMN, Gemin2, Gemin3, Gemin4, Gemin5, and a few not yet identified proteins. The SMN complex plays a key role in the biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and other ribonucleoprotein particles. As a step toward the complete characterization of the components of the SMN complex, we generated stable cell lines that express FLAG-tagged SMN or Gemin2 under the control of a tetracycline-inducible promoter. Native SMN complexes of identical protein composition to those isolated by immunoprecipitation with anti-SMN antibodies were purified by affinity chromatography from extracts of both cell lines. Here we report the identification by mass spectrometry of a novel protein component of the SMN complex termed Gemin6. Co-immunoprecipitation, immunolocalization, and in vitro binding experiments demonstrate that Gemin6 is a component of the SMN complex that localizes to gems and interacts with several Sm proteins of the spliceosomal snRNPs.

Footnotes

↵* This work was supported in part by a grant from the National Institute of Health (to G. D.) and by a grant from the Danish National Research Foundation to the Center of Experimental Bioinformatics.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EBI Data Bank with accession number(s) .
↵§ These authors have contributed equally to this work.
↵‖ A Marie Curie Fellow.
↵** An Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute and Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148. Tel.: 215-898-0398; Fax: 215-573-2000; E-mail: gdreyfuss@hhmi.upenn.edu.
Published, JBC Papers in Press, December 17, 2001, DOI 10.1074/jbc.M110141200
Abbreviations:

SMA

spinal muscular atrophy

SMN

survival of motor neurons protein

snoRNP

small nucleolar ribonucleoprotein

hnRNP

heterogeneous nuclear ribonucleoprotein

snRNP

small nuclear ribonucleoprotein

EST

expressed sequence tag

CMV

cytomegalovirus

GST

glutathioneS-transferase

PBS

phosphate-buffered saline

snRNA

small nuclear RNA
- Received October 22, 2001.
- Revision received December 7, 2001.
The American Society for Biochemistry and Molecular Biology, Inc.