Spinal muscular atrophy (SMA) is a motoneuron disease caused by reduced levels of survival motoneuron (SMN) protein. Previous studies have assigned SMN to uridine-rich small nuclear ribonucleoprotein particle (U snRNP) assembly, splicing, transcription, and RNA localization. Here, we have used gene silencing to assess the effect of SMN protein deficiency on U snRNP metabolism in living cells and organisms. In HeLa cells, we show that reduction of SMN to levels found in SMA patients impairs U snRNP assembly. In line with this, induced silencing of SMN expression in Xenopus laevis or zebrafish arrested embryonic development. Under less severe knock-down conditions, zebrafish embryos proceeded through development yet exhibited dramatic SMA-like motor axon degeneration. The same was observed after silencing two other essential factors in the U snRNP assembly pathway, Gemin2 and pICln. Importantly, the injection of purified U snRNPs into either SMN-or Gemin2-deficient embryos of Xenopus and zebrafish prevented developmental arrest and motoneuron degeneration, respectively. These findings suggest that motoneuron degeneration in SMA patients is a direct consequence of impaired production of U snRNPs.[Keywords: Survival motor neurons (SMN); U snRNP assembly; motoneuron; spinal muscular atrophy; zebrafish] Supplemental material is available at http://www.genesdev.org.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, which result in reduced levels of functional SMN protein. Biochemical studies have linked the ubiquitously expressed SMN protein to the assembly of pre-mRNA processing U snRNPs, raising the possibility that aberrant splicing is a major defect in SMA. Accordingly, several transcripts affected upon SMN deficiency have been reported. A second function for SMN in axonal mRNA transport has also been proposed that may likewise contribute to the SMA phenotype. The underlying etiology of SMA, however, is still not fully understood. Here, we have used a combination of genomics and live Ca(2+) imaging to investigate the consequences of SMN deficiency in a zebrafish model of SMA. In a transcriptome analyses of SMN-deficient zebrafish, we identified neurexin2a (nrxn2a) as strongly down-regulated and displaying changes in alternative splicing patterns. Importantly, the knock-down of two distinct nrxn2a isoforms phenocopies SMN-deficient fish and results in a significant reduction of motor axon excitability. Interestingly, we observed altered expression and splicing of Nrxn2 also in motor neurons from the Smn(-/-);SMN2(+/+) mouse model of SMA, suggesting conservation of nrxn2 regulation by SMN in mammals. We propose that SMN deficiency affects splicing and abundance of nrxn2a. This may explain the pre-synaptic defects at neuromuscular endplates in SMA pathophysiology.

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