Identification of Novel Interacting Protein Partners of SMN Using Tandem Affinity Purification

Abstract

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.

The many faces of SMN: deciphering the function critical to spinal muscular atrophy pathogenesis

“…Our findings suggest that the nature of the SMN-interacting protein varies during growth, early differentiation and late differentiation [48]. Indeed, the levels of snRNP assembly activity significantly decrease during C2C12 myoblast differentiation, suggesting unique functions for SMN during growth and differentiation of muscle cells [110].…”

“…Moreover, it was found to interact with a-actinin, a crosslinking protein that stabilizes actin microfilaments [119]. Follow-up experiments in mice confirm these findings and specifically identify SMN as a Z-disc component in skeletal and cardiac muscle [19,48]. Other proteins, such as Gemins and Unrip, are also present at Z-discs, likely forming a muscle-specific SMN complex [19].…”

“…In this process, the methylosome bound to the Sm proteins also associates with the SMN complex [45][46][47]. The pICLn chaperone dissociates from the methylosome, while PRMT5 and MEP50 appear to associate with the SMN complex given that both proteins were recovered in recent SMN interaction screens [32,48,49]. In the final step of cytoplasmic snRNP assembly, hypermethylation of the snRNA by the trimethylguanosine synthetase 1 (TGS1) occurs, thus producing a 2,2,7-trimethylguanosine (TMG) cap [50].…”

“…Myosin light chains are downstream targets of the Rho GTPase signaling pathway. Interestingly, recent findings show that SMN directly interacts with myosin regulatory light chain in skeletal muscle [48]. At the moment, it is unclear whether the increased activation of the RhoA/ROCK pathway observed in SMA models affects the motor neuron, the muscle or both via the NMJ.…”

Evaluation of the safety and pharmacokinetic profile of the broad spectrum antiviral lectin Griffithsin.

“…One form, Tandem Affinity Purification has been used successfully to purify proteins for identification by Mass-Spectrometry [218][219][220][221][222][223][224]. In another type of AP, a recombinant "bait" protein is used to pull down proteins and protein complexes from biological samples, and these interacting proteins are identified by Mass Spectrometry [225,226].…”