Identification of a Cis-acting Element for the Regulation ofSMN Exon 7 Splicing

Abstract

Spinal muscular atrophy results from the loss of functional survival motor neuron (SMN1) alleles. Two nearly identical copies of SMN exist and differ only by a single non-polymorphic C to T transition in exon 7. This transition leads to alteration of exon 7 splicing; that is, SMN1 produces a full-length transcript, whereas SMN2 expresses a low level of full-length transcript and predominantly an isoform lacking exon 7. The truncated transcript of SMN encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. In this paper, we identified a cis-acting element (element 1), which is composed of 45 bp in intron 6 responsible for the regulation of SMN exon 7 splicing. Mutations in element 1 or treatment with antisense oligonucleotides directed toward element 1 caused an increase in exon 7 inclusion. An ϳ33-kDa protein was demonstrated to associate with a pre-mRNA sequence containing both element 1 and the C to T transition in SMN exon 7 but not with the sequence containing mutated element 1, suggesting that the binding of the ϳ33-kDa protein plays crucial roles in the skipping of SMN exon 7 containing the C to T transition. Spinal muscular atrophy (SMA)1 is a common autosomal recessive disorder with progressive paralysis caused by the degeneration of motor neurons in the spinal cord (1). The survival of the motor neurons (SMN) gene has been identified as the disease gene of SMA and is present on chromosome 5 at 5q13 (2, 3). Humans contain two nearly identical copies of the SMN gene, SMN1 and SMN2. These genes encode an identical protein, a 294-amino acid RNA-binding protein. Only homozygous deletions or mutations of SMN1 result in the SMA phenotype, and the levels of SMN expression driven by SMN2 in motor neurons inversely correlate with the severity of the disease (4 -15).SMN1 mRNA expresses a full-length transcript, whereas SMN2 produces a low level of full-length transcript and predominantly an isoform lacking exon 7 (SMN⌬7) (2, 16, 17). The SMN⌬7 is less stable (18), and it was reported that SMN⌬7 cannot oligomerize or self-associate as efficiently as the protein produced from the full-length SMN transcript (2,19,20). Therefore, a deficiency in the full-length SMN protein correlates with the disease. The critical difference between SMN1 and SMN2 is a silent nucleotide transition in SMN exon 7. SMN1 contains a C located six nucleotides inside exon 7, whereas SMN2 contains a T at this position. This transition is considered to inhibit one of the splicing regulatory elements within exon 7, which are called exonic splicing enhancers (ESE) (21). A recent report demonstrated the presence of an ESE within exon 7 and that human Tra2-␤1, a member of the serine-arginine-related proteins of splicing factors, binds to the elements and stimulates an ESE (22). However, the critical C to T transition is not contained within the element. Furthermore, the transition does not change the binding activity of Tra2-␤1 to the ESE. Thus, it is still unclear why the C to T trans...

An intronic element contributes to splicing repression in spinal muscular atrophy

“…However, these experiments used nonquantitative RT-RCR and did not consider smaller effects that could be detected by quantitative analyses. In fact, a third study that used a more quantitative RT-PCR analysis did reveal a slight increase in exon 7 inclusion when SMN2 intron 7 was replaced with its SMN1 counterpart (34). It is noteworthy, however, that we observed the strongest effect of the SMN2 ϩ100 site in the context of a shortened intron 7, which also resulted in slightly less efficient exon 7 exclusion (compared with the derivative containing full-length intron 7).…”

“…23 and 24; see also ref. 34 and Discussion). We therefore constructed an SMN2 intron 7 swap derivative, using the SMN1/2 HP plasmids, which contain full-length intron 7 (see Fig.…”

“…Studies have provided evidence that intronic elements are involved both positively and negatively in control of SMN exon 7 splicing (34,40,43). However, none of these elements involve SMN2-specific sequences, and thus they likely contribute to the inherent strength of the exon 7 5Ј and 3Ј splice sites.…”

“…For example, the element intronic splicing silencer (ISS)-N1, identified by Singh et al (40), was suggested to interfere with the function of TIA-1, a factor that helps U1 snRNP assembly at weak 5Ј splice sites (41,42). This sequence and two other less characterized elements (43,34) appear to play a more general role in facilitating the efficiency of exon 7 splicing. For example, they may constitute binding sites for other hnRNP proteins.…”

Splicing of a Critical Exon of Human Survival Motor Neuron Is Regulated by a Unique Silencer Element Located in the Last Intron

“…Proteins interacting with intronic sequences may also affect regulation of exon 7 splicing. Consistently, cis elements present in intron 6 and intron 7 have been shown to modulate exon 7 splicing (39,40). These results highlight the complexity of pre-mRNA splic-ing in which exon 7 is defined by a network of interactions involving several proteins.…”