Identification of novel interacting protein partners of SMN using tandem affinity purification.
Mutations in the survival motor neuron (SMN) gene cause spinal muscular atrophy (SMA), a neuromuscular disease associated with muscle weakness that progresses to paralysis, respiratory distress, and ultimately death. Both neurons and muscle are severely affected in this disease. Tandem affinity purification (TAP) has emerged as a useful tool for studying protein complexes in vitro. We have used this purification system to discover novel SMN interacting partners in C2C12 muscle and PC12 neuronal cells. To do so, we fused a TAP-tag, consisting of a HIS hexamer and FLAG epitope separated by the tobacco etch virus (TEV) protease cleavage site, to either the N- or C-terminal region of SMN. Interestingly, the profile of SMN interacting proteins varies depending on the cell type and stage. We have identified a number of novel SMN interacting proteins in both C2C12 and PC12 cells, and from among these we have validated Annexin II and myosin regulatory light chain (MRLC). The discovery of these proteins will lead to a better understanding of the mechanisms underlying the pathophysiology of SMA.
Shafey
Dina
D
Ottawa Hospital Research Institute, Ottawa, ON, Canada.
Boyer
Justin G
JG
Bhanot
Kunal
K
Kothary
Rashmi
R
D000818:Animals; D017306:Annexin A2 / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D004195:Disease Models, Animal; D006639:Histidine / Q000378:metabolism; D047468:Immunoprecipitation; D051379:Mice; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000378:metabolism; D009134:Muscular Atrophy, Spinal / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D016601:RNA-Binding Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D055532:SMN Complex Proteins / Q000235:genetics / Q000378:metabolism*; D000097796:Nucleolin
D017306:Annexin A2; D006639:Histidine; D018994:Myosin Light Chains; D009842:Oligopeptides; D010455:Peptides; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; D000097796:Nucleolin; C471213:His-His-His-His-His-His; C085706:FLAG peptide