How RNA structure dictates the usage of a critical exon of spinal muscular atrophy gene.

Role of RNA structure in pre-mRNA splicing has been implicated for several critical exons associated with genetic disorders. However, much of the structural studies linked to pre-mRNA splicing regulation are limited to terminal stem-loop structures (hairpins) sequestering splice sites. In few instances, role of long-distance interactions is implicated as the major determinant of splicing regulation. With the recent surge of reports of circular RNA (circRNAs) generated by backsplicing, role of Alu-associated RNA structures formed by long-range interactions are taking central stage. Humans contain two nearly identical copies of Survival Motor Neuron (SMN) genes, SMN1 and SMN2. Deletion or mutation of SMN1 coupled with the inability of SMN2 to compensate for the loss of SMN1 due to exon 7 skipping causes spinal muscular atrophy (SMA), one of the leading genetic diseases of children. In this review, we describe how structural elements formed by both local and long-distance interactions are being exploited to modulate SMN2 exon 7 splicing as a potential therapy for SMA. We also discuss how Alu-associated secondary structure modulates generation of a vast repertoire of SMN circRNAs. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

High-affinity RNA targets of the Survival Motor Neuron protein reveal diverse preferences for sequence and structural motifs.

The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA-SMN interactions. Our results reveal that motifs that drive RNA-SMN interactions are diverse and suggest that tight RNA-SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA-SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions.

Nusinersen: A Review in 5q Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a rare autosomal recessive neuromuscular disorder most commonly caused by a deletion or mutation in the survival motor neuron 1 (SMN1) gene, which leads to insufficient levels of survival motor neuron (SMN) protein. In such patients, SMN protein production relies on the SMN2 gene. Nusinersen (Spinraza®) is a modified antisense oligonucleotide (ASO) approved in several countries worldwide, including the USA, Japan and those of the EU, for the treatment of 5q SMA. It binds to a specific site in the intron downstream of exon 7 on the SMN2 pre-messenger ribonucleic acid (pre-mRNA), modulating the splicing of SMN2 mRNA and thus increasing the production of SMN protein. In multinational phase III studies, nusinersen (administered intrathecally) provided significant improvements in motor function in patients with infantile- and later-onset 5q SMA compared with a sham procedure. It was also associated with significant improvements in event-free survival and overall survival in patients with infantile-onset 5q SMA, with preliminary data from an ongoing multinational phase II study suggesting a potential clinical benefit with early intervention (i.e. before symptom onset) with nusinersen. Preliminary subgroup data from a phase III extension study suggested continued improvements in motor function with longer-term therapy. Nusinersen demonstrated a favourable safety profile in clinical studies in symptomatic and presymptomatic patients, with no safety concerns due to nusinersen exposure. In conclusion, although studies in presymptomatic patients and over the long term in symptomatic patients are ongoing, current evidence indicates that nusinersen modifies 5q SMA and has a favourable safety profile and, thus, is a valuable treatment for this patient population.

Mild SMN missense alleles are only functional in the presence of SMN2 in mammals.

Spinal muscular atrophy (SMA) is caused by reduced levels of full-length SMN (FL-SMN). In SMA patients with one or two copies of the Survival Motor Neuron 2 (SMN2) gene there are a number of SMN missense mutations that result in milder-than-predicted SMA phenotypes. These mild SMN missense mutation alleles are often assumed to have partial function. However, it is important to consider the contribution of FL-SMN as these missense alleles never occur in the absence of SMN2. We propose that these patients contain a partially functional oligomeric SMN complex consisting of FL-SMN from SMN2 and mutant SMN protein produced from the missense allele. Here we show that mild SMN missense mutations SMND44V, SMNT74I or SMNQ282A alone do not rescue mice lacking wild-type FL-SMN. Thus, missense mutations are not functional in the absence of FL-SMN. In contrast, when the same mild SMN missense mutations are expressed in a mouse containing two SMN2 copies, functional SMN complexes are formed with the small amount of wild-type FL-SMN produced by SMN2 and the SMA phenotype is completely rescued. This contrasts with SMN missense alleles when studied in C. elegans, Drosophila and zebrafish. Here we demonstrate that the heteromeric SMN complex formed with FL-SMN is functional and sufficient to rescue small nuclear ribonucleoprotein assembly, motor neuron function and rescue the SMA mice. We conclude that mild SMN missense alleles are not partially functional but rather they are completely non-functional in the absence of wild-type SMN in mammals.

Mechanistic studies of a small-molecule modulator of SMN2 splicing.

RG-7916 is a first-in-class drug candidate for the treatment of spinal muscular atrophy (SMA) that functions by modulating pre-mRNA splicing of the SMN2 gene, resulting in a 2.5-fold increase in survival of motor neuron (SMN) protein level, a key protein lacking in SMA patients. RG-7916 is currently in three interventional phase 2 clinical trials for various types of SMA. In this report, we show that SMN-C2 and -C3, close analogs of RG-7916, act as selective RNA-binding ligands that modulate pre-mRNA splicing. Chemical proteomic and genomic techniques reveal that SMN-C2 directly binds to the AGGAAG motif on exon 7 of the SMN2 pre-mRNA, and promotes a conformational change in two to three unpaired nucleotides at the junction of intron 6 and exon 7 in both in vitro and in-cell models. This change creates a new functional binding surface that increases binding of the splicing modulators, far upstream element binding protein 1 (FUBP1) and its homolog, KH-type splicing regulatory protein (KHSRP), to the SMN-C2/C3-SMN2 pre-mRNA complex and enhances SMN2 splicing. These findings underscore the potential of small-molecule drugs to selectively bind RNA and modulate pre-mRNA splicing as an approach to the treatment of human disease.

A novel human-specific splice isoform alters the critical C-terminus of Survival Motor Neuron protein.

Spinal muscular atrophy (SMA), a leading genetic disease of children and infants, is caused by mutations or deletions of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of SMN1 due to skipping of exon 7. SMN2 predominantly produces SMNΔ7, an unstable protein. Here we report exon 6B, a novel exon, generated by exonization of an intronic Alu-like sequence of SMN. We validate the expression of exon 6B-containing transcripts SMN6B and SMN6BΔ7 in human tissues and cell lines. We confirm generation of SMN6B transcripts from both SMN1 and SMN2. We detect expression of SMN6B protein using antibodies raised against a unique polypeptide encoded by exon 6B. We analyze RNA-Seq data to show that hnRNP C is a potential regulator of SMN6B expression and demonstrate that SMN6B is a substrate of nonsense-mediated decay. We show interaction of SMN6B with Gemin2, a critical SMN-interacting protein. We demonstrate that SMN6B is more stable than SMNΔ7 and localizes to both the nucleus and the cytoplasm. Our finding expands the diversity of transcripts generated from human SMN genes and reveals a novel protein isoform predicted to be stably expressed during conditions of stress.

Transcript, methylation and molecular docking analyses of the effects of HDAC inhibitors, SAHA and Dacinostat, on SMN2 expression in fibroblasts of SMA patients.

Several histone deacetylase inhibitors (HDACis) are known to increase Survival Motor Neuron 2 (SMN2) expression for the therapy of spinal muscular atrophy (SMA). We aimed to compare the effects of suberoylanilide hydroxamic acid (SAHA) and Dacinostat, a novel HDACi, on SMN2 expression and to elucidate their acetylation effects on the methylation of the SMN2. Cell-based assays using type I and type II SMA fibroblasts examined changes in transcript expressions, methylation levels and protein expressions. In silico methods analyzed the intermolecular interactions between each compound and HDAC2/HDAC7. SMN2 mRNA transcript levels and SMN protein levels showed notable increases in both cell types, except for Dacinostat exposure on type II cells. However, combined compound exposures showed less pronounced increase in SMN2 transcript and SMN protein level. Acetylation effects of SAHA and Dacinostat promoted demethylation of the SMN2 promoter. The in silico analyses revealed identical binding sites for both compounds in HDACs, which could explain the limited effects of the combined exposure. With the exception on the effect of Dacinostat in Type II cells, we have shown that SAHA and Dacinostat increased SMN2 transcript and protein levels and promoted demethylation of the SMN2 gene.

SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice.

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

SMN deficiency disrupts gastrointestinal and enteric nervous system function in mice.

The 2007 Consensus Statement for Standard of Care in Spinal Muscular Atrophy (SMA) notes that patients suffer from gastroesophageal reflux, constipation and delayed gastric emptying. We used two mouse models of SMA to determine whether functional GI complications are a direct consequence of or are secondary to survival motor neuron (Smn) deficiency. Our results show that despite normal activity levels and food and water intake, Smn deficiency caused constipation, delayed gastric emptying, slow intestinal transit and reduced colonic motility without gross anatomical or histopathological abnormalities. These changes indicate alterations to the intrinsic neural control of gut functions mediated by the enteric nervous system (ENS). Indeed, Smn deficiency led to disrupted ENS signaling to the smooth muscle of the colon but did not cause enteric neuron loss. High-frequency electrical field stimulation (EFS) of distal colon segments produced up to a 10-fold greater contractile response in Smn deficient tissues. EFS responses were not corrected by the addition of a neuronal nitric oxide synthase inhibitor indicating that the increased contractility was due to hyperexcitability and not disinhibition of the circuitry. The GI symptoms observed in mice are similar to those reported in SMA patients. Together these data suggest that ENS cells are susceptible to Smn deficiency and may underlie the patient GI symptoms.

The SMN structure reveals its crucial role in snRNP assembly.

The spliceosome plays a fundamental role in RNA metabolism by facilitating pre-RNA splicing. To understand how this essential complex is formed, we have used protein crystallography to determine the first complete structures of the key assembler protein, SMN, and the truncated isoform, SMNΔ7, which is found in patients with the disease spinal muscular atrophy (SMA). Comparison of the structures of SMN and SMNΔ7 shows many similar features, including the presence of two Tudor domains, but significant differences are observed in the C-terminal domain, including 12 additional amino acid residues encoded by exon 7 in SMN compared with SMNΔ7. Mapping of missense point mutations found in some SMA patients reveals clustering around three spatial locations, with the largest cluster found in the C-terminal domain. We propose a structural model of SMN binding with the Gemin2 protein and a heptameric Sm ring, revealing a critical assembly role of the residues 260-294, with the differences at the C-terminus of SMNΔ7 compared with SMN likely leading to loss of small nuclear ribonucleoprotein (snRNP) assembly. The SMN complex is proposed to form a dimer driven by formation of a glycine zipper involving α helix formed by amino acid residues 263-294. These results explain how structural changes of SMN give rise to loss of SMN-mediated snRNP assembly and support the hypothesis that this loss results in atrophy of neurons in SMA.

Solution structure of the core SMN-Gemin2 complex.

In humans, assembly of spliceosomal snRNPs (small nuclear ribonucleoproteins) begins in the cytoplasm where the multi-protein SMN (survival of motor neuron) complex mediates the formation of a seven-membered ring of Sm proteins on to a conserved site of the snRNA (small nuclear RNA). The SMN complex contains the SMN protein Gemin2 and several additional Gemins that participate in snRNP biosynthesis. SMN was first identified as the product of a gene found to be deleted or mutated in patients with the neurodegenerative disease SMA (spinal muscular atrophy), the leading genetic cause of infant mortality. In the present study, we report the solution structure of Gemin2 bound to the Gemin2-binding domain of SMN determined by NMR spectroscopy. This complex reveals the structure of Gemin2, how Gemin2 binds to SMN and the roles of conserved SMN residues near the binding interface. Surprisingly, several conserved SMN residues, including the sites of two SMA patient mutations, are not required for binding to Gemin2. Instead, they form a conserved SMN/Gemin2 surface that may be functionally important for snRNP assembly. The SMN-Gemin2 structure explains how Gemin2 is stabilized by SMN and establishes a framework for structure-function studies to investigate snRNP biogenesis as well as biological processes involving Gemin2 that do not involve snRNP assembly.

A positive modifier of spinal muscular atrophy in the SMN2 gene.

Spinal muscular atrophy (SMA) is a common autosomal-recessive motor neuron disease caused by the homozygous loss of the SMN1 gene. A nearly identical gene, SMN2, has been shown to decrease the severity of SMA in a dose-dependent manner. However SMN2 is not the sole phenotypic modifier, because there are discrepant SMA cases in which the SMN2 copy number does not explain the clinical phenotype. This report describes three unrelated SMA patients who possessed SMN2 copy numbers that did not correlate with the observed mild clinical phenotypes. A single base substitution in SMN2, c.859G>C,, was identified in exon 7 in the patients' DNA. We now show that the change creates a new exonic splicing enhancer element and increases the amount of full-length transcripts, thus resulting in the less severe phenotypes. This demonstrates that the c.859G>C substitution is a positive modifier of the SMA phenotype and that not all SMN2 genes are equivalent. We have shown not only that the SMA phenotype is modified by the number of SMN2 genes but that SMN2 sequence variations can also affect the disease severity.