Antisense Oligonucleotide Induction of the hnRNPA1b Isoform Affects Pre-mRNA Splicing of SMN2 in SMA Type I Fibroblasts

Spinal muscular atrophy (SMA) is a severe, debilitating neuromuscular condition characterised by loss of motor neurons and progressive muscle wasting. SMA is caused by a loss of expression of SMN1 that encodes the survival motor neuron (SMN) protein necessary for the survival of motor neurons. Restoration of SMN expression through increased inclusion of SMN2 exon 7 is known to ameliorate symptoms in SMA patients. As a consequence, regulation of pre-mRNA splicing of SMN2 could provide a potential molecular therapy for SMA. In this study, we explored if splice switching antisense oligonucleotides could redirect the splicing repressor hnRNPA1 to the hnRNPA1b isoform and restore SMN expression in fibroblasts from a type I SMA patient. Antisense oligonucleotides (AOs) were designed to promote exon 7b retention in the mature mRNA and induce the hnRNPA1b isoform. RT-PCR and western blot analysis were used to assess and monitor the efficiency of different AO combinations. A combination of AOs targeting multiple silencing motifs in hnRNPA1 pre-mRNA led to robust hnRNPA1b induction, which, in turn, significantly increased expression of full-length SMN (FL-SMN) protein. A combination of PMOs targeting the same motifs also strongly induced hnRNPA1b isoform, but surprisingly SMN2 exon 5 skipping was detected, and the PMO cocktail did not lead to a significant increase in expression of FL-SMN protein. We further performed RNA sequencing to assess the genome-wide effects of hnRNPA1b induction. Some 3244 genes were differentially expressed between the hnRNPA1b-induced and untreated SMA fibroblasts, which are functionally enriched in cell cycle and chromosome segregation processes. RT-PCR analysis demonstrated that expression of the master regulator of these enrichment pathways, MYBL2 and FOXM1B, were reduced in response to PMO treatment. These findings suggested that induction of hnRNPA1b can promote SMN protein expression, but not at sufficient levels to be clinically relevant.     

Genomic Variability in the Survival Motor Neuron Genes (SMN1 and SMN2): Implications for Spinal Muscular Atrophy Phenotype and Therapeutics Development

Spinal muscular atrophy (SMA) is a leading genetic cause of infant death worldwide that is characterized by loss of spinal motor neurons leading to muscle weakness and atrophy. SMA results from the loss of survival motor neuron 1 (SMN1) gene but retention of its paralog SMN2. The copy numbers of SMN1 and SMN2 are variable within the human population with SMN2 copy number inversely correlating with SMA severity. Current therapeutic options for SMA focus on increasing SMN2 expression and alternative splicing so as to increase the amount of SMN protein. Recent work has demonstrated that not all SMN2, or SMN1, genes are equivalent and there is a high degree of genomic heterogeneity with respect to the SMN genes. Because SMA is now an actionable disease with SMN2 being the primary target, it is imperative to have a comprehensive understanding of this genomic heterogeneity with respect to hybrid SMN1–SMN2 genes generated by gene conversion events as well as partial deletions of the SMN genes. This review will describe this genetic heterogeneity in SMA and its impact on disease phenotype as well as therapeutic efficacy.     

CRISPR-Mediated Enzyme Fragment Complementation Assay for Quantification of the Stability of Splice Isoforms

Small-molecule splicing modulators exemplified by an FDA-approved drug, risdiplam, are a new pharmacological modality for regulating the expression and stability of splice isoforms. We report a CRISPR-mediated enzyme fragment complementation (EFC) assay to quantify the splice isoform stability. The EFC assay harnessed a 42 amino acid split of a β-galactosidase (designate α-tag), which could be fused at the termini of the target genes using CRISPR/cas9. The α-tagged splice isoform would be quantified by measuring the enzymatic activity upon complementation with the rest of β-galactosidase. This EFC assay retained all the sequences of introns and exons of the target gene in the native genomic environment that recapitulates the cell biology of the diseases of interest. For a proof-of-concept, we developed a CRISPR-mediated EFC assay targeting the exon 7 of the survival of motor neuron 2 (SMN2) gene. The EFC assay is compatible with 384-well plates and robustly quantified the splicing modulation activity of small molecules. In this study, we also discovered that a coumarin derivative, compound 4 , potently modulated SMN2 exon 7 splicing at as low as 1.1 nM.     

An Antisense Oligonucleotide against a Splicing Enhancer Sequence within Exon 1 of the MSTN Gene Inhibits Pre-mRNA Maturation to Act as a Novel Myostatin Inhibitor

Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal muscle growth. ASOs that induce skipping of out-of-frame exon 2 of the MSTN gene have been studied for their use in increasing muscle mass. However, no ASOs are currently available for clinical use. We hypothesized that ASOs against the splicing enhancer sequence within exon 1 of the MSTN gene would inhibit maturation of pre-mRNA, thereby suppressing gene function. To explore this hypothesis, ASOs against sequences of exon 1 of the MSTN gene were screened for their ability to reduce mature MSTN mRNA levels. One screened ASO, named KMM001, decreased MSTN mRNA levels in a dose-dependent manner and reciprocally increased MSTN pre-mRNA levels. Accordingly, KMM001 decreased myostatin protein levels. KMM001 inhibited SMAD-mediated myostatin signaling in rhabdomyosarcoma cells. Remarkably, it did not decrease GDF11 mRNA levels, indicating myostatin-specific inhibition. As expected, KMM001 enhanced the proliferation of human myoblasts. We conclude that KMM001 is a novel myostatin inhibitor that inhibits pre-mRNA maturation. KMM001 has great promise for clinical applications and should be examined for its ability to treat various muscle-wasting conditions.     

Transformer2 proteins protect breast cancer cells from accumulating replication stress by ensuring productive splicing of checkpoint kinase 1

Increased expression levels of the RNA splicing regulator Transformer2β (abbreviated Tra2β) have been reported in several types of cancer. Recent work has revealed an intimate cross-regulation between Tra2β and the highly similar Tra2α protein in human breast cancer cells, though these two proteins are encoded by separate genes created by a gene duplication that occurred over 500 million years ago. This cross-regulation involves splicing control of a special class of exons, called poison exons. Down-regulation of Tra2β reduces splicing inclusion of a poison exon in the mRNA encoding Tra2α, thereby up-regulating Tra2α protein expression. This buffers any splicing changes that might be caused by individual depletion of Tra2β alone. Discovery of this cross-regulation pathway, and its by-pass by joint depletion of both human Tra2 proteins, revealed Tra2 proteins are essential for breast cancer cell viability, and led to the identification of important targets for splicing control. These exons include a critical exon within the checkpoint kinase 1 (CHK1) gene that plays a crucial function in the protection of cancer cells from replication stress. Breast cancer cells depleted for Tra2 proteins have reduced CHK1 protein levels and accumulate DNA damage. These data suggest Tra2 proteins and/or their splicing targets as possible cancer drug targets.

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Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.     

Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype–phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype–phenotype correlations and improve prognostic outcomes.     

The Importance of Digging into the Genetics of SMN Genes in the Therapeutic Scenario of Spinal Muscular Atrophy

After 26 years of discovery of the determinant survival motor neuron 1 and the modifier survival motor neuron 2 genes (SMN1 and SMN2, respectively), three SMN-dependent specific therapies are already approved by FDA and EMA and, as a consequence, worldwide SMA patients are currently under clinical investigation and treatment. Bi-allelic pathogenic variants (mostly deletions) in SMN1 should be detected in SMA patients to confirm the disease. Determination of SMN2 copy number has been historically employed to correlate with the phenotype, predict disease evolution, stratify patients for clinical trials and to define those eligible for treatment. In view that discordant genotype-phenotype correlations are present in SMA, besides technical issues with detection of SMN2 copy number, we have hypothesized that copy number determination is only the tip of the iceberg and that more deepen studies of variants, sequencing and structures of the SMN2 genes are necessary for a better understanding of the disease as well as to investigate possible influences in treatment responses. Here, we highlight the importance of a comprehensive approach of SMN1 and SMN2 genetics with the perspective to apply for better prediction of SMA in positive neonatal screening cases and early diagnosis to start treatments.     

What Genetics Has Told Us and How It Can Inform Future Experiments for Spinal Muscular Atrophy,a Perspective

Proximal spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by motor neuron loss and subsequent atrophy of skeletal muscle. SMA is caused by deficiency of the essential survival motor neuron (SMN) protein, canonically responsible for the assembly of the spliceosomal small nuclear ribonucleoproteins (snRNPs). Therapeutics aimed at increasing SMN protein levels are efficacious in treating SMA. However, it remains unknown how deficiency of SMN results in motor neuron loss, resulting in many reported cellular functions of SMN and pathways affected in SMA. Herein is a perspective detailing what genetics and biochemistry have told us about SMA and SMN, from identifying the SMA determinant region of the genome, to the development of therapeutics. Furthermore, we will discuss how genetics and biochemistry have been used to understand SMN function and how we can determine which of these are critical to SMA moving forward.     

Molecular Characterization,mRNA Expression and Alternative Splicing of Ryanodine Receptor Gene in the Brown Citrus Aphid,Toxoptera citricida (Kirkaldy)

Ryanodine receptors (RyRs) play a critical role in regulating the release of intracellular calcium, which enables them to be effectively targeted by the two novel classes of insecticides, phthalic acid diamides and anthranilic diamides. However, less information is available about this target site in insects, although the sequence and structure information of target molecules are essential for designing new control agents of high selectivity and efficiency, as well as low non-target toxicity. Here, we provided sufficient information about the coding sequence and molecular structures of RyR in T. citricida (TciRyR), an economically important pest. The full-length TciRyR cDNA was characterized with an open reading frame of 15,306 nucleotides, encoding 5101 amino acid residues. TciRyR was predicted to embrace all the hallmarks of ryanodine receptor, typically as the conserved C-terminal domain with consensus calcium-biding EF-hands (calcium-binding motif) and six transmembrane domains, as well as a large N-terminal domain. qPCR analysis revealed that the highest mRNA expression levels of TciRyR were observed in the adults, especially in the heads. Alternative splicing in TciRyR was evidenced by an alternatively spliced exon, resulting from intron retention, which was different from the case of RyR in Myzus persicae characterized with no alternative splicing events. Diagnostic PCR analysis indicated that the splicing of this exon was not only regulated in a body-specific manner but also in a stage-dependent manner. Taken together, these results provide useful information for new insecticide design and further insights into the molecular basis of insecticide action.     

Mechanistic Insights of Aberrant Splicing with Splicing Factor Mutations Found in Myelodysplastic Syndromes

Pre-mRNA splicing is an essential process for gene expression in higher eukaryotes, which requires a high order of accuracy. Mutations in splicing factors or regulatory elements in pre-mRNAs often result in many human diseases. Myelodysplastic syndrome (MDS) is a heterogeneous group of chronic myeloid neoplasms characterized by many symptoms and a high risk of progression to acute myeloid leukemia. Recent findings indicate that mutations in splicing factors represent a novel class of driver mutations in human cancers and affect about 50% of Myelodysplastic syndrome (MDS) patients. Somatic mutations in MDS patients are frequently found in genes SF3B1, SRSF2, U2AF1, and ZRSR2. Interestingly, they are involved in the recognition of 3′ splice sites and exons. It has been reported that mutations in these splicing regulators result in aberrant splicing of many genes. In this review article, we first describe molecular mechanism of pre-mRNA splicing as an introduction and mainly focus on those four splicing factors to describe their mutations and their associated aberrant splicing patterns.     

Effects of RNAi-Mediated Knockdown of Histone Methyltransferases on the Sex-Specific mRNA Expression of Imp in the Silkworm Bombyx mori

Sexual differentiation in Bombyx mori is controlled by sex-specific splicing of Bmdsx, which results in the omission of exons 3 and 4 in a male-specific manner. In B. mori, insulin-like growth factor II mRNA-binding protein (Imp) is a male-specific factor involved in male-specific splicing of Bmdsx. Male-specific Imp mRNA results from the male-specific inclusion of exon 8. To verify the link between histone methylation and alternative RNA processing in Imp, we examined the effects of RNAi-mediated knockdown of several histone methyltransferases on the sex-specific mRNA expression of Imp. As a result, male-specific expression of Imp mRNA was completely abolished when expression of the H3K79 methyltransferase DOT1L was repressed to <10% of that in control males. Chromatin immunoprecipitation-quantitative PCR analysis revealed a higher distribution of H3K79me2 in normal males than in normal females across Imp. RNA polymerase II (RNAP II) processivity assays indicated that RNAi knockdown of DOT1L in males caused a twofold decrease in RNAP II processivity compared to that in control males, with almost equivalent levels to those observed in normal females. Inhibition of RNAP II-mediated elongation in male cells repressed the male-specific splicing of Imp. Our data suggest the possibility that H3K79me2 accumulation along Imp is associated with the male-specific alternative processing of Imp mRNA that results from increased RNAP II processivity.     

A Regulatory Axis between Epithelial Splicing Regulatory Proteins and Estrogen Receptor α Modulates the Alternative Transcriptome of Luminal Breast Cancer

Epithelial splicing regulatory proteins 1 and 2 (ESRP1/2) control the splicing pattern during epithelial to mesenchymal transition (EMT) in a physiological context and in cancer, including breast cancer (BC). Here, we report that ESRP1, but not ESRP2, is overexpressed in luminal BCs of patients with poor prognosis and correlates with estrogen receptor α (ERα) levels. Analysis of ERα genome-binding profiles in cell lines and primary breast tumors showed its binding in the proximity of ESRP1 and ESRP2 genes, whose expression is strongly decreased by ERα silencing in hormone-deprived conditions. The combined knock-down of ESRP1/2 in MCF-7 cells followed by RNA-Seq, revealed the dysregulation of 754 genes, with a widespread alteration of alternative splicing events (ASEs) of genes involved in cell signaling, metabolism, cell growth, and EMT. Functional network analysis of ASEs correlated with ESRP1/2 expression in ERα+ BCs showed RAC1 as the hub node in the protein–protein interactions altered by ESRP1/2 silencing. The comparison of ERα- and ESRP-modulated ASEs revealed 63 commonly regulated events, including 27 detected in primary BCs and endocrine-resistant cell lines. Our data support a functional implication of the ERα-ESRP1/2 axis in the onset and progression of BC by controlling the splicing patterns of related genes.     

BRCA1 Exon 11, a CERES (Composite Regulatory Element of Splicing) Element Involved in Splice Regulation

Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a “silent” change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES).