Ribonucleoprotein Assembly Defects Correlate with Spinal Muscular Atrophy Severity and Preferentially Affect a Subset of Spliceosomal snRNPs

Abstract

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.

Splice-site pairing is an intrinsically high fidelity process

“…Significantly, rescue of SMN-depleted animals was achieved by the injection of purified snRNPs, suggesting a critical role for snRNPs to the SMA phenotype (27). This proposal was recently supported by the demonstration that reduced SMN results in altered levels of snRNPs, which alter the splicing profile (26,28).…”

“…The low amounts of SMN produced from SMN2 are adequate for a fetus to develop, but insufficient to maintain healthy motor neurons throughout life (24). SMN is the central component of the SMN complex, which is required for snRNP recycling, reassembly, and maintenance of high snRNP concentrations (25,26). Previous work has shown that depletion of SMN recapitulated the SMA phenotype in zebrafish.…”

“…Alternative exons, represented within the center of this distribution, represent exons with the greatest chance of being altered by minor changes in splicing efficiency. In the SMA cell culture model analyzed, a reduction in SMN and its accompanying reduction in snRNP concentrations (26,28) has the potential to trigger a change from preferential exon inclusion to exclusion, thus shifting the spectrum of exon recognition (Fig. 4C).…”

Gemin3Is an Essential Gene Required for Larval Motor Function and Pupation inDrosophila

“…In addition to larval Smn mutants, our laboratory has previously reported SMA-like phenotypes in adult flies containing a hypomorphic Smn E33 mutation (Rajendra et al, 2007). Thus, although it is clear that perturbations in the SMN complex can indeed result in neuromuscular dysfunction, the contribution that snRNP biogenesis plays in the etiology of these phenotypes remains a subject of ongoing investigation (Shpargel and Matera, 2005;Wan et al, 2005;Winkler et al, 2005;Gabanella et al, 2007). Further complicating interpretation of the various SMA models is the fact that the SMN complex appears to function in tissue-specific pathways involved in both neuronal (McWhorter et al, 2003;Zhang et al, 2006;Bowerman et al, 2007) and muscular development (Shafey et al, 2005;Rajendra et al, 2007).…”

Dysregulation of synaptogenesis genes antecedes motor neuron pathology in spinal muscular atrophy

“…Both snRNP assembly capacity and SMA severity correspond directly to the degree of SMN deficiency (24,25). Interestingly, SMN decrease results in a nonuniform change of individual snRNP's level, and thus an altered snRNP repertoire (abundance and stoichiometry), which is apparent in mouse and Drosophila SMA models (15,26,27). It is likely that this alteration contributes to the transcriptome changes observed in the spinal cord and other tissues of the SMA mouse model at P11 (15), a symptomatic stage for these mice that typically survive to P13 (6).…”