SUMMARY Spinal muscular atrophy (SMA) is a motor neuron disease caused by deficiency of the ubiquitous survival motor neuron (SMN) protein. To define the mechanisms of selective neuronal dysfunction in SMA, we investigated the role of SMN-dependent U12 splicing events in the regulation of motor circuit activity. We show that SMN deficiency perturbs splicing and decreases the expression of a subset of U12 intron-containing genes in mammalian cells and Drosophila larvae. Analysis of these SMN target genes identifies Stasimon as a novel protein required for motor circuit function. Restoration of Stasimon expression in the motor circuit corrects defects in neuromuscular junction transmission and muscle growth in Drosophila SMN mutants and aberrant motor neuron development in SMN-deficient zebrafish. These findings directly link defective splicing of critical neuronal genes induced by SMN deficiency to motor circuit dysfunction, establishing a molecular framework for the selective pathology of SMA.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival motor neuron (SMN) protein. SMN together with Gemins2-8 and unrip proteins form a macromolecular complex that functions in the assembly of small nuclear ribonucleoproteins (snRNPs) of both the major and the minor splicing pathways. It is not known whether the levels of spliceosomal snRNPs are decreased in SMA. Here we analyzed the consequence of SMN deficiency on snRNP metabolism in the spinal cord of mouse models of SMA with differing phenotypic severities. We demonstrate that the expression of a subset of Gemin proteins and snRNP assembly activity are dramatically reduced in the spinal cord of severe SMA mice. Comparative analysis of different tissues highlights a similar decrease in SMN levels and a strong impairment of snRNP assembly in tissues of severe SMA mice, although the defect appears smaller in kidney than in neural tissue. We further show that the extent of reduction in both Gemin proteins expression and snRNP assembly activity in the spinal cord of SMA mice correlates with disease severity. Remarkably, defective SMN complex function in snRNP assembly causes a significant decrease in the levels of a subset of snRNPs and preferentially affects the accumulation of U11 snRNP—a component of the minor spliceosome—in tissues of severe SMA mice. Thus, impairment of a ubiquitous function of SMN changes the snRNP profile of SMA tissues by unevenly altering the normal proportion of endogenous snRNPs. These findings are consistent with the hypothesis that SMN deficiency affects the splicing machinery and in particular the minor splicing pathway of a rare class of introns in SMA.

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by homozygous loss of the Survival Motor Neuron 1 (SMN1) gene. In the absence of SMN1, inefficient inclusion of exon 7 in transcripts from the nearly identical SMN2 gene results in ubiquitous SMN decrease but selective motor neuron degeneration. Here we investigated whether cell type-specific differences in the efficiency of exon 7 splicing contribute to the vulnerability of SMA motor neurons. We show that normal motor neurons express markedly lower levels of full-length SMN mRNA from SMN2 than do other cells in the spinal cord. This is due to inefficient exon 7 splicing that is intrinsic to motor neurons under normal conditions. We also find that SMN depletion in mammalian cells decreases exon 7 inclusion through a negative feedback loop affecting the splicing of its own mRNA. This mechanism is active in vivo and further decreases the efficiency of exon 7 inclusion specifically in motor neurons of severe-SMA mice. Consistent with expression of lower levels of full-length SMN, we find that SMN-dependent downstream molecular defects are exacerbated in SMA motor neurons. These findings suggest a mechanism to explain the selective vulnerability of motor neurons to loss of SMN1. Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degeneration of motor neurons (MNs) in the spinal cord and by skeletal muscle atrophy (8,38,44). SMA is the most common genetic cause of infant mortality and is classified into three types based on the age of onset and clinical severity (50, 56). Regardless of disease severity, all SMA patients have homozygous deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene-the SMA-determining gene-and retain at least one copy of the nearly identical SMN2 gene (33). While the SMN1 gene produces full-length transcripts, the SMN2 gene mainly produces an alternatively spliced mRNA lacking exon 7 (SMN⌬7). Since the SMN⌬7 protein is unstable and rapidly degraded (32,36), the low levels of full-length functional SMN produced by the SMN2 gene cannot compensate for the loss of SMN1, resulting in SMA. Thus, SMA is caused by decreased expression of SMN protein, and correspondingly, disease severity correlates well with the degree of SMN reduction in SMA patients (19,34). Importantly, the SMN2 gene copy number varies in the human population and acts as the principal disease modifier in SMA because the presence of more SMN2 copies generally coincides with a milder clinical outcome (41). Studies of human SMA patients and animal models of disease indicate that low SMN levels from SMN2 are sufficient for normal function of most cells but not motor neurons (8, 44). However, the reason for the selective vulnerability of motor neurons to SMN deficiency is unknown.Since the efficiency of exon 7 splicing determines the amount of functional SMN produced by SMN2, the mechanisms underlying the alternative splicing of exon 7 in SMN1 and SMN2 mRNAs have been subject to extensive studies. This is for two main rea...

The survival motor neuron (SMN) protein is the product of the spinal muscular atrophy disease gene. SMN and Gemin2-7 proteins form a large macromolecular complex that localizes in the cytoplasm as well as in the nucleoplasm and in nuclear Gems. The SMN complex interacts with several additional proteins and likely functions in multiple cellular pathways. In the cytoplasm, a subset of SMN complexes containing unrip and Sm proteins mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, by mass spectrometry analysis of SMN complexes purified from HeLa cells, we identified a novel protein that is evolutionarily conserved in metazoans, and we named it Gemin8. Co-immunoprecipitation and immunolocalization experiments demonstrated that Gemin8 is associated with the SMN complex and is localized in the cytoplasm and in the nucleus, where it is highly concentrated in Gems. Gemin8 interacts directly with the Gemin6-Gemin7 heterodimer and, together with unrip, these proteins form a heteromeric subunit of the SMN complex. Gemin8 is also associated with Sm proteins, and Gemin8-containing SMN complexes are competent to carry out snRNP assembly. Importantly, RNA interference experiments indicate that Gemin8 knock-down impairs snRNP assembly, and Gemin8 expression is down-regulated in cells with low levels of SMN. These results demonstrate that Gemin8 is a novel integral component of the SMN complex and extend the repertoire of cellular proteins involved in the pathway of snRNP biogenesis.Eukaryotic genes are transcribed as long precursors that need to undergo post-transcriptional processing to produce functional, protein-coding mRNAs. The spliceosome, a large dynamic macromolecular assembly of hundreds of proteins and a few RNAs, is responsible for the proper excision of introns and ligation of exons during pre-mRNA splicing (1). Spliceosomal small nuclear ribonucleoproteins (snRNPs) 2 are a class of abundant cellular particles and the essential components of the spliceosome. Major snRNPs are composed of an snRNA molecule (U1, U2, U4/U6, and U5) and a set of common (Sm proteins) and snRNP-specific proteins (2). The hallmark of snRNPs is the presence of a heptameric ring of Sm proteins, known as the Sm core, around a conserved sequence called the Sm site (3). Although snRNPs function in the nucleus, the biogenesis of spliceosomal snRNPs in higher eukaryotes follows a complex pathway that requires the functions of many cellular proteins and includes nuclear as well as cytoplasmic phases (2).The survival motor neuron (SMN) protein has emerged in recent years as a key player in the biogenesis of snRNPs (4, 5). Understanding the molecular function(s) of SMN also bears direct relevance to human disease because reduced levels of SMN expression, due to homozygous mutations or deletions of the SMN1 gene, cause the fatal neurodegenerative disease spinal muscular atrophy (SMA) (6). SMN localizes in the cytoplasm, in the nucleoplasm, and is highly concentrated in Gems, nuclear structures that are often associ...