Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder causing infant death in half of all patients. Homozygous absence of the survival motor neuron gene (SMN1) is the primary cause of SMA, while SMA severity is mainly determined by the number of SMN2 copies. One SMN2 copy produces only about 10% of full-length protein identical to SMN1, whereas the majority of SMN2 transcripts is aberrantly spliced due to a silent mutation within an exonic splicing enhancer in exon 7. However, correct splicing can be restored by over-expression of the SR-like splicing factor Htra2-beta 1. We show that in fibroblast cultures derived from SMA patients treated with therapeutic doses (0.5-500 microM) of valproic acid (VPA), the level of full-length SMN2 mRNA/protein increased 2- to 4-fold. Importantly, this up-regulation of SMN could be most likely attributed to increased levels of Htra2-beta 1 which facilitates the correct splicing of SMN2 RNA as well as to an SMN gene transcription activation. Especially at low VPA concentrations, the restored SMN level depended on the number of SMN2 copies. Moreover, VPA was able to increase SMN protein levels through transcription activation in organotypic hippocampal brain slices from rats. Finally, VPA also increased the expression of further SR proteins, which may have important implications for other disorders affected by alternative splicing. Since VPA is a drug highly successfully used in long-term epilepsy therapy, our findings open the exciting perspective for a first causal therapy of an inherited disease by elevating the SMN2 transcription level and restoring its correct splicing.

Spinal muscular atrophy (SMA), a common motor neuron disease in humans, results from loss of functional survival motor neuron (SMN1) alleles. A nearly identical copy of the gene, SMN2, fails to provide protection from SMA because of a single translationally silent nucleotide difference in exon 7. This likely disrupts an exonic splicing enhancer and causes exon 7 skipping, leading to abundant production of a shorter isoform, SMN2⌬7. The truncated transcript encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. This report describes the identification of an in vivo regulator of SMN mRNA processing. Htra2-␤1, an SR-like splicing factor and ortholog of Drosophila melanogaster transformer-2, promoted the inclusion of SMN exon 7, which would stimulate full-length SMN2 expression. Htra2-␤1 specifically functioned through and bound an AG-rich exonic splicing enhancer in SMN exon 7. This effect is not speciesspecific as expression of Htra2-␤1 in human or mouse cells carrying an SMN2 minigene dramatically increased production of full-length SMN2. This demonstrates that SMN2 mRNA processing can be modulated in vivo. Because all SMA patients retain at least one SMN2 copy, these results show that an in vivo modulation of SMN RNA processing could serve as a therapeutic strategy to prevent SMA. P roximal spinal muscular atrophy (SMA) is a neurodegenerative disorder with progressive paralysis caused by the loss of ␣-motor neurons in the spinal cord. With an incidence of 1 in 10,000 live births and a carrier frequency of 1 in 50, SMA is the second most common autosomal recessive disorder and the most frequent genetic cause of infantile death (1). SMA patients are subdivided into types I-III according to age of onset and achieved motor abilities (2). All three forms of proximal SMA are caused by mutations within the telomeric copy of the survival motor neuron gene, SMN1 (3). Some 96.4% of 5q-linked SMA patients show homozygous absence of SMN1 caused by deletions or gene conversions, whereas 3.6% display rare subtle mutations (3, 4). Homozygous absence of SMN2 is found in 5% of control individuals; however, loss of SMN2 has no phenotypic effect (3). SMN1 produces exclusively full-length (FL) SMN mRNA. In contrast, SMN2 expresses dramatically reduced FL and abundant levels of transcript lacking exon 7, SMN2⌬7. SMN2 is retained by all patients and a correlation between the SMN2 protein level and the disease state is established (5, 6). This spliced isoform encodes a truncated, less stable protein with reduced self-oligomerization activity (3,7,8). We have shown that inclusion of exon 7 in SMN1-derived transcripts and exclusion of this exon in SMN2-derived transcripts is determined by a single nucleotide difference at position ϩ6 in SMN exon 7 (C in SMN1; T in SMN2). This nucleotide difference is nonpolymorphic in the SMN2 gene and likely disrupts an exonic splicing enhancer (ESE) (9, 10).The removal of introns and joining of exons is performed by the spliceosome, a ma...

Proximal spinal muscular atrophy (SMA) is a common motor neuron disease caused by homozygous loss of the survival motor neuron gene (SMN1). SMN2, a nearly identical copy of the gene and present in all SMA patients, fails to provide protection from SMA, due to the disruption of an exonic splicing enhancer (ESE) by a single translationally silent nucleotide exchange, which causes alternative splicing of SMN2 exon 7. Identification of splicing factors that stimulate exon 7 inclusion and thereby produce sufficient amounts of full-length transcripts from the SMN2 gene is of great importance for therapy approaches. Here, by use of in vivo splicing assays, we identified the protein hnRNP-G and its paralogue RBM as two novel splicing factors that promote the inclusion of SMN2 exon 7. Moreover, hnRNP-G and RBM non-specifically bind RNA, but directly and specifically bind Htra2-beta1, an SR-like splicing factor which we have previously shown to stimulate inclusion of exon 7 through a direct interaction with the AG-rich ESE in SMN2 exon 7 pre-mRNA. By using deletion mutants of hnRNP-G, we show that the specific protein-protein interaction of hnRNP-G with Htra2-beta1 mediates the inclusion of SMN2 exon 7 rather than the non-specific interaction of hnRNP-G with SMN pre-mRNA. Additionally, we show for the first time that recombinant trans-acting splicing factors such as hnRNP-G and Htra2-beta1 are also effective on endogenous SMN2 transcripts and increase the endogenous SMN protein level. Finally, we suggest a model of how the exon 7 mRNA processing is regulated by the splicing factors identified so far.