hnRNP M facilitates exon 7 inclusion of SMN2 pre-mRNA in spinal muscular atrophy by targeting an enhancer on exon 7

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, which causes death of motor neurons in the anterior horn of the spinal cord. Genetic cause of SMA is the deletion or mutation of SMN1 gene, which encodes the SMN protein. Although SMA patients include SMN2 gene, a duplicate of SMN1 gene, predominant production of exon 7 skipped isoform from SMN2 pre-mRNA, fails to rescue SMA patients. Here we show that hnRNP M, a member of hnRNP protein family, when knocked down, promotes exon 7 skipping of both SMN2 and SMN1 pre-mRNA. By contrast, overexpression of hnRNP M promotes exon 7 inclusion of both SMN2 and SMN1 pre-mRNA. Significantly, hnRNP M promotes exon 7 inclusion in SMA patient cells. Thus, we conclude that hnRNP M promotes exon 7 inclusion of both SMN1 and SMN2 pre-mRNA. We also demonstrate that hnRNP M contacts an enhancer on exon 7, which was previously shown to provide binding site for tra2β. We present evidence that hnRNP M and tra2β contact overlapped sequence on exon 7 but with slightly different RNA sequence requirements. In addition, hnRNP M promotes U2AF65 recruitment on the flanking intron of exon 7. We conclude that hnRNP M promotes exon 7 inclusion of SMN1 and SMN2 pre-mRNA through targeting an enhancer on exon 7 through recruiting U2AF65. Our results provide a clue that hnRNP M is a potential therapeutic target for SMA.     

An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy

SMN1 and SMN2 represent the two nearly identical copies of the survival of motor neuron gene in humans. The most frequent cause of spinal muscular atrophy (SMA) is loss of SMN1 accompanied by the inability of SMN2 to compensate due to an inhibitory mutation at position 6 in exon 7 (C6U) that causes exon 7 exclusion. How this single exonic nucleotide regulates exon 7 recognition has been of major interest. Based on score matrices and in vitro assays, abrogation of an exonic splicing enhancer (ESE) associated with SF2/ASF has been considered as the cause of exon 7 exclusion. However, a recent report supports the creation of an exonic splicing silencer (ESS) associated with hnRNP A1 as the determining factor for exon 7 exclusion. Here we show that C6U strengthens an inhibitory context that covers a larger sequence than the hnRNP A1 binding site. The inhibitory context can also be strengthened by the addition of a G residue at the first position of exon 7 in SMN1, promoting exon 7 skipping despite the presence of SF2/ASF binding site. Through in vivo selection and a series of mutations we demonstrate that the strengthening of the extended inhibitory context at the 5' end of exon 7 is exercised through overlapping sequence motifs that collaborate to regulate exon usage.     

Overexpressed human survival motor neurone isoforms, SMNDeltaexon7 and SMN+exon7, both form intranuclear gems but differ in cytoplasmic distribution

Homozygous mutations of the telomeric survival motor neurone gene (SMN1) cause spinal muscular atrophy (SMA). The centromeric copy gene (SMN2) generally skips exon 7 during splicing and fails to compensate for SMN1 deficits, so SMA cells have reduced SMN protein and few nuclear gems. To investigate the role of exon 7 in SMN localisation, cDNAs for full-length SMN and SMNDeltaexon 7 were overexpressed in COS cells, neurones and SMA fibroblasts. Both constructs formed discrete intranuclear bodies colocalising with p80-coilin, but produced more cytoplasmic aggregates in cells overexpressing exon 7. Hence, the exon 7 domain enhances SMN aggregation but is not critical for gem formation.     

TIA1 prevents skipping of a critical exon associated with spinal muscular atrophy

Prevention of skipping of exon 7 during pre-mRNA splicing of Survival Motor Neuron 2 (SMN2) holds the promise for cure of spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Here, we report T-cell-restricted intracellular antigen 1 (TIA1) and TIA1-related (TIAR) proteins as intron-associated positive regulators of SMN2 exon 7 splicing. We show that TIA1/TIAR stimulate exon recognition in an entirely novel context in which intronic U-rich motifs are separated from the 5' splice site by overlapping inhibitory elements. TIA1 and TIAR are modular proteins with three N-terminal RNA recognition motifs (RRMs) and a C-terminal glutamine-rich (Q-rich) domain. Our results reveal that any one RRM in combination with a Q domain is necessary and sufficient for TIA1-associated regulation of SMN2 exon 7 splicing in vivo. We also show that increased expression of TIA1 counteracts the inhibitory effect of polypyrimidine tract binding protein, a ubiquitously expressed factor recently implicated in regulation of SMN exon 7 splicing. Our findings expand the scope of TIA1/TIAR in genome-wide regulation of alternative splicing under normal and pathological conditions.     

Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron

Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to exclusion of exon 7. Here we describe a novel inhibitory element located immediately downstream of the 5' splice site in intron 7. We call this element intronic splicing silencer N1 (ISS-N1). Deletion of ISS-N1 promoted exon 7 inclusion in mRNAs derived from the SMN2 minigene. Underlining the dominant role of ISS-N1 in exon 7 skipping, abrogation of a number of positive cis elements was tolerated when ISS-N1 was deleted. Confirming the silencer function of ISS-N1, an antisense oligonucleotide against ISS-N1 restored exon 7 inclusion in mRNAs derived from the SMN2 minigene or from endogenous SMN2. Consistently, this oligonucleotide increased the levels of SMN protein in SMA patient-derived cells that carry only the SMN2 gene. Our findings underscore for the first time the profound impact of an evolutionarily nonconserved intronic element on SMN2 exon 7 splicing. Considering that oligonucleotides annealing to intronic sequences do not interfere with exon-junction complex formation or mRNA transport and translation, ISS-N1 provides a very specific and efficient therapeutic target for antisense oligonucleotide-mediated correction of SMN2 splicing in SMA.     

High Expression Level of Tra2-β1 Is Responsible for Increased SMN2 Exon 7 Inclusion in the Testis of SMA Mice

Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn^−/− SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.     

Synthesis and characterization of pseudocantharidins, novel phosphatase modulators that promote the inclusion of exon 7 into the SMN (survival of motoneuron) pre-mRNA

Alternative pre-mRNA splicing is a central element of eukaryotic gene expression. Its deregulation can lead to disease, and methods to change splice site selection are developed as potential therapies. Spinal muscular atrophy is caused by the loss of the SMN1 (survival of motoneuron 1) gene. A therapeutic avenue for spinal muscular atrophy treatment is to promote exon 7 inclusion of the almost identical SMN2 (survival of motoneuron 2) gene. The splicing factor tra2-beta1 promotes inclusion of this exon and is antagonized by protein phosphatase (PP) 1. To identify new compounds that promote exon 7 inclusion, we synthesized analogs of cantharidin, an inhibitor of PP1, and PP2A. Three classes of compounds emerged from these studies. The first class blocks PP1 and PP2A activity, blocks constitutive splicing in vitro, and promotes exon 7 inclusion in vivo. The second class has no measurable effect on PP1 activity but activates PP2A. This class represents the first compounds described with these properties. These compounds cause a dephosphorylation of Thr-33 of tra2-beta1, which promotes exon 7 inclusion. The third class had no detectable effect on phosphatase activity and could promote exon 7 via allosteric effects. Our data show that subtle changes in similar compounds can turn a phosphatase inhibitor into an activator. These chemically related compounds influence alternative splicing by distinct mechanisms.     

Validation of trans-acting elements that promote exon 7 skipping of SMN2 in SMN2-GFP stable cell line

Spinal muscular atrophy is a genetic disease in which the SMN1 gene is deleted. The SMN2 gene exists in all of the patients. Alternative splicing of these two genes are different. More than 90% of exon 7 included form is produced from SMN1 pre-mRNA, whereas only ～20% of exon 7 included form is produced from SMN2 pre-mRNA. Only exon 7 inclusion form produces functional protein. Exon 7 skipped SMN isoform is unstable. Here we constructed a GFP reporter system that recapitulates the alternative splicing of SMN1 and SMN2 pre-mRNA. We designed a system in which GFP protein is expressed only when exon 7 of is included in alternative splicing. The stable cell that expresses SMN1-GFP produces ～4 times more GFP protein than the stable cell line that expresses SMN2-GFP; as demonstrated by microscopy, FACS analysis and immunoblotting. In addition the ratio of exon 7 inclusion and skipping of SMN1-GFP and SMN2-GFP pre-mRNA was similar to endogenous SMN1 and SMN2 pre-mRNA as shown in RT-PCR. Furthermore the knockdown with hnRNP A1 shRNA, a known protein which promotes exon 7 skipping of SMN2, induces exon 7 inclusion of exon 7 in SMN2-GFP pre-mRNA in SMN2-GFP cell line. We conclude that we have established the stable cell lines that recapitulate alternative splicing of the SMN1 and SMN2 genes. The stable cell line can be used to identify the trans-acting elements with siRNA.     

Position-dependent effects of hnRNP A1/A2 in SMN1/2 exon7 splicing

Heterogeneous nuclear ribonucleoprotein A1 and A2 (hnRNP A1/2) is a ubiquitously expressed RNA binding protein known to bind intronic or exonic splicing silencer. Binding of hnRNP A1/2 to survival of motor neuron gene (SMN1/2) exon 7 and flanking sequences strongly inhibits the inclusion of exon 7, which causes spinal muscular atrophy, a common genetic disorder. However, the role of hnRNP A1/2 on the side away from exon 7 is unclear. Here using antisense oligonucleotides, we fished an intronic splicing enhancer (ISE) near the 3′-splice site (SS) of intron 7 of SMN1/2. Mutagenesis identified the efficient motif of the ISE as “UAGUAGG”, coupled with RNA pull down and protein overexpression, we proved that hnRNP A1/2 binding to the ISE promotes the inclusion of SMN1/2 exon 7. Using MS2-tethering array and “UAGGGU” motif walking, we further uncovered that effects of hnRNP A1/2 on SMN1/2 exon 7 splicing are position-dependent: exon 7 inclusion is inhibited when hnRNP A1/2 binds proximal to the 5′SS of intron 7, promoted when its binds proximal to the 3′SS. These data provide new insights into the splicing regulatory mechanism of SMN1/2.     

Modulation of survival motor neuron pre-mRNA splicing by inhibition of alternative 3' splice site pairing

Spinal muscular atrophy is caused by the loss of functional survival motor neuron (SMN1) alleles. A translationally silent nucleotide transition in the duplicated copy of the gene (SMN2) leads to exon 7 skipping and expression of a nonfunctional gene product. It has been suggested that differential SMN2 splicing is caused by the disruption of an exonic splicing enhancer. Here we show that the single nucleotide difference reduces the intrinsic strength of the 3' splice site of exon 7 2-fold, whereas the strength of the 5' splice site of the exon 7 is not affected. Thus, a decrease in splice site strength is magnified in the context of competing exons. These data suggest that lower levels of exon 7 definition not only reduce intron 6 removal but, more importantly, increase the efficiency of the competing exon 7 skipping pathway. Antisense oligonucleotides were tested to modulate exon 7 inclusion, which contains the authentic translation stop codon. Oligonucleotides directed toward the 3' splice site of exon 8 were shown to alter SMN2 splicing in favor of exon 7 inclusion. These results suggest that antisense oligonucleotides could be used as a therapeutic strategy to counteract the progression of SMA.     

Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes

Humans have two nearly identical copies of the survival motor neuron (SMN) gene, SMN1 and SMN2. Homozygous loss of SMN1 causes spinal muscular atrophy (SMA). SMN2 is unable to prevent the disease due to skipping of exon 7. Using a systematic approach of in vivo selection, we have previously demonstrated that a weak 5′ splice site (ss) serves as the major cause of skipping of SMN2 exon 7. Here we show the inhibitory impact of RNA structure on the weak 5′ ss of exon 7. We call this structure terminal stem–loop 2 (TSL2). Confirming the inhibitory nature of TSL2, point mutations that destabilize TSL2 promote exon 7 inclusion in SMN2, whereas strengthening of TSL2 promotes exon 7 skipping even in SMN1. We also demonstrate that TSL2 negatively affects the recruitment of U1snRNP at the 5′ ss of exon 7. Using enzymatic structure probing, we confirm that the sequence at the junction of exon 7/intron 7 folds into TSL2 and show that mutations in TSL2 cause predicted structural changes in this region. Our findings reveal for the first time the critical role of RNA structure in regulation of alternative splicing of human SMN.     

Determinants of exon 7 splicing in the spinal muscular atrophy genes, SMN1 and SMN2

Spinal muscular atrophy is a neurodegenerative disorder caused by the deletion or mutation of the survival-of-motor-neuron gene, SMN1. An SMN1 paralog, SMN2, differs by a C→T transition in exon 7 that causes substantial skipping of this exon, such that SMN2 expresses only low levels of functional protein. A better understanding of SMN splicing mechanisms should facilitate the development of drugs that increase survival motor neuron (SMN) protein levels by improving SMN2 exon 7 inclusion. In addition, exonic mutations that cause defective splicing give rise to many genetic diseases, and the SMN1/2 system is a useful paradigm for understanding exon-identity determinants and alternative-splicing mechanisms. Skipping of SMN2 exon 7 was previously attributed either to the loss of an SF2/ASF–dependent exonic splicing enhancer or to the creation of an hnRNP A/B–dependent exonic splicing silencer, as a result of the C→T transition. We report the extensive testing of the enhancer-loss and silencer-gain models by mutagenesis, RNA interference, overexpression, RNA splicing, and RNA-protein interaction experiments. Our results support the enhancer-loss model but also demonstrate that hnRNP A/B proteins antagonize SF2/ASF–dependent ESE activity and promote exon 7 skipping by a mechanism that is independent of the C→T transition and is, therefore, common to both SMN1 and SMN2. Our findings explain the basis of defective SMN2 splicing, illustrate the fine balance between positive and negative determinants of exon identity and alternative splicing, and underscore the importance of antagonistic splicing factors and exonic elements in a disease context.