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.     

Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-β and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.     

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.     

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).     

A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.     

The Influence of Microencapsulation with a Modified Chitosan (Water Soluble) on β-Galactosidase Activity

The aim of this work was to investigate the possibility of producing microparticles containing β-galactosidase, using a modified chitosan (water soluble) as encapsulating agent. β-galactosidase microparticles were prepared by a spray-drying method and were characterized in terms of particle size, surface morphology, zeta potential, and stability over a storage period of six months. Microparticles were also analyzed by FTIR spectroscopy and thermogravimetry techniques. Structural analysis of the surface of the particles was performed by scanning electron microscopy (SEM). SEM results show that the obtained microparticles have an average diameter smaller than 3.5 µm and a regular shape. The β-galactosidase activity decreases when microencapsulated. The parameters Km and Vmax of the Michaelis-Menten equation were calculated for the different experimental conditions. The optimal pH ranges from 6.4 to 7.2, approximately, depending on the enzyme concentration in the microcapsule. After six months of storage, the enzyme activity presents a small decrease, although no significant differences in the appearance, color, and particle size distribution were identified.     

Comparison of β-galactosidase immobilization by entrapment in and adsorption on poly(2-hydroxyethylmethacrylate) membranes

β-Galactosidase was immobilized in/on poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two different methods: adsorption on Cibacron F3GA derivatized pHEMA membranes (pHEMA-CB), and entrapment in the bulk of the pHEMA membranes. The maximum β-galactosidase adsorption on pHEMA-CB membranes was obtained as 95·6μgcm^-2 in 2·0mgcm^-3 enzyme solution. The adsorption phenomena appeared to follow a typical Langmuir isotherm. In the entrapment, an increase in β-galactosidase loading resulted in a consistent increase in membrane activity from 3·3×10^-2 to 17·8×10^-2Ucm^-2 pHEMA membranes. The K_m values for both immobilized β-galactosidase (adsorbed 0·32mM and entrapped 0·81mM ) were higher than that of the free enzyme (0·26mM ). The optimum reaction temperature of the adsorbed enzyme was 5°C higher than that of both the free and the entrapped enzyme. The optimum reaction pH was 7·5 for free and both immobilized preparations. After 15 successive uses the retained activity of the adsorbed and the entrapped enzymes was 80% and 95%, respectively. The storage stability of the enzyme was found to increase upon immobilization. ©1997 SCI     

A Splice Switch in SIGIRR Causes a Defect of IL-37-Dependent Anti-Inflammatory Activity in Cystic Fibrosis Airway Epithelial Cells

Cystic fibrosis (CF) is a hereditary disease typically characterized by infection-associated chronic lung inflammation. The persistent activation of toll-like receptor (TLR) signals is considered one of the mechanisms for the CF hyperinflammatory phenotype; however, how negative regulatory signals of TLRs associate with CF inflammation is still elusive. Here, we showed that the cell surface expression of a single immunoglobulin interleukin-1 receptor (IL-1R)-related molecule (SIGIRR), a membrane protein essential for suppressing TLRs- and IL-1R-dependent signals, was remarkably decreased in CF airway epithelial cells compared to non-CF cells. Notably, CF airway epithelial cells specifically and highly expressed a unique, alternative splice isoform of the SIGIRR that lacks exon 8 (Δ8-SIGIRR), which results in the production of a C-terminal truncated form of the SIGIRR. Δ8-SIGIRR was expressed intracellularly, and its over-expression abolished the cell surface expression and function of the full-length SIGIRR (WT-SIGIRR), indicating its dominant-negative effect leading to the deficiency of anti-inflammatory activity in CF cells. Consistently, IL-37, a ligand for the SIGIRR, failed to suppress viral dsRNA analogue poly(I:C)-dependent JNK activation and IL-8 production, confirming the reduction in the functional WT-SIGIRR expression in the CF cells. Together, our studies reveal that SIGIRR-dependent anti-inflammatory activity is defective in CF airway epithelial cells due to the unique splicing switch of the SIGIRR gene and provides the first evidence of IL-37-SIGIRR signaling as a target of CF airway inflammation.     

Immobilization of β-galactosidase onto magnetic poly(GMA–MMA) beads for hydrolysis of lactose in bed reactor

In the present study, novel magnetic beads were prepared from glycidylmethacrylate and methylmethacrylate via suspension polymerization in the presence of a cross-linker (i.e. ethylenedimethylmethacrylate). The magnetic poly(GMA–MMA) beads were characterized with scanning electron microscope, FT-IR and ESR spectrophotometers. The reactive character of the epoxy groups allowed the attachment of the amino groups. The aminated magnetic beads were used for the covalent immobilization of β-galactosidase via glutaric dialdehyde activation. The maximum amount of immobilized β-galactosidase on the magnetic poly(GMA–MMA) beads was 9.87 mg/g support. The values of Michaelis constants K_m for immobilized β-galactosidase was significant larger, indicating decreased affinity by the enzyme for its substrate, whereas V_max values were smaller for the immobilized β-galactosidase. However, the β-galactosidase immobilized on the magnetic poly(GMA–MMA) beads resulted in an increase in enzyme stability with time. Optimum operational temperature for immobilized enzyme was 5 °C higher than that of the free enzyme and was significantly broader. Finally, a bed reactor with β-galactosidase immobilized was used for hydrolysis of lactose. The enzyme reactor operated continuously at 35 °C for 60 h and the immobilized enzyme lost about 12% of its initial activity after this period.     

Antisense Oligonucleotide-Based Rescue of Aberrant Splicing Defects Caused by 15 Pathogenic Variants in ABCA4

The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.     

HuR and TIA1/TIAL1 Are Involved in Regulation of Alternative Splicing of SIRT1 Pre-mRNA

SIRT1 is a pleiotropic protein that plays critical and multifunctional roles in metabolism, senescence, longevity, stress-responses, and cancer, and has become an important therapeutic target across a range of diseases. Recent research demonstrated that SIRT1 pre-mRNA undergoes alternative splicing to produce different isoforms, such as SIRT1 full-length and SIRT1-ΔExon8 variants. Previous studies revealed these SIRT1 mRNA splice variants convey different characteristics and functions to the protein, which may in turn explain the multifunctional roles of SIRT1. However, the mechanisms underlying the regulation of SIRT1 alternative splicing remain to be elucidated. Our objective is to search for new pathways that regulate of SIRT1 alternative splicing. Here we describe experiments showing that HuR and TIA1/TIAL1, two kinds of RNA-binding proteins, were involved in the regulation of alternative splicing of SIRT1 pre-mRNA under normal and stress circumstances: HuR increased SIRT1-ΔExon8 by promoting SIRT1 exon 8 exclusion, whereas TIA1/TIAL1 inhibition of the exon 8 exclusion led to a decrease in SIRT1-ΔExon8 mRNA levels. This study provides novel insight into how the alternative splicing of SIRT1 pre-mRNA is regulated, which has fundamental implications for understanding the critical and multifunctional roles of SIRT1.     

Exploring Pharmacological Functions of Alternatively Spliced Variants of the Mu Opioid Receptor Gene,Oprm1, via Gene-Targeted Animal Models

The mu opioid receptor has a distinct place in the opioid receptor family, since it mediates the actions of most opioids used clinically (e.g., morphine and fentanyl), as well as drugs of abuse (e.g., heroin). The single-copy mu opioid receptor gene, OPRM1, goes through extensive alternative pre-mRNA splicing to generate numerous splice variants that are conserved from rodents to humans. These OPRM1 splice variants can be classified into three structurally distinct types: (1) full-length 7 transmembrane (TM) carboxyl (C)-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Distinct pharmacological functions of these splice variants have been demonstrated by both in vitro and in vivo studies, particularly by using several unique gene-targeted mouse models. These studies provide new insights into our understanding of the complex actions of mu opioids with regard to OPRM1 alternative splicing. This review provides an overview of the studies that used these gene-targeted mouse models for exploring the functional importance of Oprm1 splice variants.     

Evaluation of β‐Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure

β‐Amino acids have a backbone that is expanded by one carbon atom relative to α‐amino acids, and β residues have been investigated as subunits in protein‐like molecules that adopt discrete and predictable conformations. Two classes of β residue have been widely explored in the context of generating α‐helix‐like conformations: β³‐amino acids, which are homologous to α‐amino acids and bear a side chain on the backbone carbon adjacent to nitrogen, and residues constrained by a five‐membered ring, such the one derived from trans‐2‐aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their β³ homologues within an α‐helix‐forming sequence generally causes a decrease in conformational stability. Use of a ring‐constrained β residue, however, can offset the destabilizing effect of α→β substitution. Here we extend the study of α→β substitutions, involving both β³ and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a two‐, three‐, or four‐residue β‐hairpin loop, and we evaluate α→β replacements at each loop position for each variant. By referral to the ?,ψ angles of the native structure, one can choose a stereochemically appropriate ACPC residue. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three β‐containing Pin1 WW domain variants show that a native‐like tertiary structure is maintained in each case.