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

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. Spinal muscular atrophy (SMA)2 is a common autosomal recessive disease that is clinically classified into three types, I-III, based on the severity of motor neuron degeneration and the age of onset (1-3). Two nearly identical copies of the survival of motor neuron genes (SMN1 and SMN2) are located on the human chromosome, 5q13, whereas other eukaryotic species have only one copy of the SMN gene. Homozygous deletions of, or mutations in the SMN1 gene are responsible for SMA (4). SMN is expressed ubiquitously and is a core component of a self-assembling multiprotein complex, the SMN complex, consisting of SMN and Gemin proteins, which plays an essential role in the assembly of the spliceosomal small nuclear ribonucleoproteins (snRNPs) and in pre-mRNA splicing (5, 6).Most of the missense mutations in SMA patients are located in exon 6 of SMN1; whereas nonsense and frameshift mutations are widely spread throughout the entire gene (7). Exon 6 of SMN1 encodes a self-association domain, and deletions or mutations of this domain result in a decrease in oligomer formation of SMN, which correlates with the severity of SMA (8). SMN oligomerization is also a prerequisite for high affinity binding of the SMN complex to spliceosomal snRNPs (9). The self-association domains of SMN were identified by surface plasmon resonance analysis; both the carboxyl-terminal exon 6-encoded region and the amino-terminal exon 2b-encoded region contributed to self-association (10). This suggests a mechanism in which the self-association formation involves eit...

SMN complexes of nucleus and cytoplasm: A proteomic study for SMA therapy

“…There is evidence that recruitment into SMN-gemin complexes stabilizes SMN, and in particular, gemin2 has been shown to play an important role in snRNP assembly through stabilization of the SMN oligomer/complex via novel self-interaction [34]. Other SMN-binding proteins have also been shown to regulate the stability of the SMN complex.…”

Role of Survival Motor Neuron Complex Components in Small Nuclear Ribonucleoprotein Assembly

“…Recently, we have discovered that Gemin2 forms a homodimer responsible for the stability of the SMN oligomer/complex required for efficient snRNP assembly (22). Surprisingly, we also found that siRNA-mediated Gemin2 knockdown causes dissociation of several components including Gemin3 and Gemin7 from the SMN complex, suggesting that Gemin2 helps to stabilize other components in the SMN complex either directly or indirectly (22).…”

“…Mammalian Two-hybrid Assay-Mammalian two-hybrid assays, including sample construction and transfection, were carried out as previously described (22). The linear doublestrand DNA constructs to express fusion proteins with the Gal4 DNA-binding domain (BIND) or the VP16 trans-activation domain (ACT) were prepared in a two-step PCR.…”

Original article Immunoexpression of gemins 2 and 4 in the rat spinal cord. Is the SMN complex a new target in investigations of sporadic amyotrophic lateral sclerosis pathogenesis?

“…A small complex composed only of SMN and gemin 2 represents its inactive form whereas the high molecular form containing SMN, gemin 2, 3 and 4 is an active form that can bind substrates [17]. Gemin 2 plays a role in stabilization of the SMN complex [16], in export of smRNAs from the nucleus, and in their assembly in the cytoplasm [12].…”