Htra2-β 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 ( SMN2 )

Abstract

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...

An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA

“…Htra␤1 increased approximately two-fold the levels of luciferase activity from the SMN2-luciferase construct without changing the levels of activity from the SMN1-luciferase construct, suggesting that hTra2␤1 specifically affects SMN exon 7 splicing rather than nonspecifically increasing expression levels (panel a). 21 RT-PCR from parallel transfections confirmed these results (Figure 3b). Other SR proteins previously reported to be incapable of stimulating full-length SMN expression from SMN2 also had no effect upon exon 7 splicing of SMN1 or SMN2 GFP and luciferase mini-gene constructs (data not shown).…”

“…We have reported that Tra2␤1 stimulated exon 7 inclusion within SMN2 mRNA. 21 To identify factors and their potential roles in exon 7 splicing, we treated our stable cell lines with chemical compounds that stimulate or antagonize kinases and signaling pathways (Table 1). We tested the stress/MAP kinase pathway as it has been shown recently that p38 kinase/pathway played a role in alternative splicing of adenovirus E1A pre-mRNA by effecting the distribution and phosphorylation of hnRNP A1 protein.…”

“…The SR-like protein Tra2␤1 specifically binds exon 7 RNA and promotes inclusion of this exon. 21 A subset of SR proteins may also affect exon 7 splicing (Lorson et al, unpublished data). However, it is still unclear how these trans SR factors or other non-SR proteins precisely regulate exon 7 recognition and processing.…”

Transformer 2β and miR-204 regulate apoptosis through competitive binding to 3′ UTR of BCL2 mRNA

“…Tra2β specifically binds to the splicing enhancer within exon 7 of SMN1 pre-mRNA and facilitates inclusion of exon 7 in the mature SMN1 mRNA in neuroblastoma cell lines. 11 Similarly, Tra2β as well as SRSF1 (also known as ASF/SF2) and SRSF9 (SRp30c) promote inclusion of exon 10 of tau pre-mRNA. 12,13 Tra2β also facilitates alternative splicing of the CD44 gene via binding to CD44 exons v4 and v5, which is associated with breast cancer progression.…”

Survival motor neuron (SMN) polymorphism in relation to congenital arthrogryposis in two Piedmont calves (piemontese)

“…SMA and AMCN are caused in a wide number of cases by a gene named SMN. Several exon or gene duplications and deletions or point mutations of this gene have been recorded associated to arthrogryposis [8,11,23]. SMN protein plays the key role of shuttle in distributing, regenerating and assembling the spliceosome complex [2,12].…”