Gemin2 Plays an Important Role in Stabilizing the Survival of Motor Neuron Complex

Fuyu Ito,Harukazu Suzuki,Masayoshi Itoh,Makoto Aoki,Kengo Usui,Yoshihide Hayashizaki,Mutsumi Kanamori-Katayama,Chikatoshi Kai,Chihiro Ogawa

doi:10.1074/jbc.m609297200

Abstract

The survival of motor neuron (SMN) protein, responsible for the neurodegenerative disease spinal muscular atrophy (SMA), oligomerizes and forms a stable complex with seven other major components, the Gemin proteins. Besides the SMN protein, Gemin2 is a core protein that is essential for the formation of the SMN complex, although the mechanism by which it drives formation is unclear. We have found a novel interaction, a Gemin2 self-association, using the mammalian two-hybrid system and the in vitro pull-down assays. Using in vitro dissociation assays, we also found that the self-interaction of the amino-terminal SMN protein, which was confirmed in this study, became stable in the presence of Gemin2. In addition, Gemin2 knockdown using small interference RNA treatment revealed a drastic decrease in SMN oligomer formation and in the assembly activity of spliceosomal small nuclear ribonucleoprotein (snRNP). Taken together, these results indicate that Gemin2 plays an important role in snRNP assembly through the stabilization of the SMN oligomer/complex via novel self-interaction. Applying the results/techniques to amino-terminal SMN missense mutants that were recently identified from SMA patients, we successfully showed that amino-terminal self-association, Gemin2 binding, the stabilization effect of Gemin2, and snRNP assembly activity were all lowered in the mutant SMN(D44V), suggesting that instability of the amino-terminal SMN self-association may cause SMA in patients carrying this allele.

Highlights

I–III, based on the severity of motor neuron degeneration and the age of onset [1,2,3]
We found that the amino-terminal self-association, Gemin2 binding, stabilizing effect of Gemin2, and small nuclear ribonucleoprotein (snRNP) assembly activity were lowered in the spinal muscular atrophy (SMA)-derived amino-terminal missense mutant survival of motor neuron (SMN)(D44V), supporting the importance of Gemin2 in stabilizing the SMN complex
To gain insight into Gemin2 and its mechanisms, we tried to purify recombinant Gemin2 using a size-fractionation column, and we found that the main fraction of Gemin2 was ϳ65 kDa, whereas minor fractions of 30 and 120 kDa were apparent (Fig. 1A)

Summary

Introduction

I–III, based on the severity of motor neuron degeneration and the age of onset [1,2,3]. We found that the amino-terminal self-association, Gemin2 binding, stabilizing effect of Gemin2, and snRNP assembly activity were lowered in the SMA-derived amino-terminal missense mutant SMN(D44V), supporting the importance of Gemin2 in stabilizing the SMN complex.

Results

Conclusion