Splicing efficiency of minor introns in a mouse model of SMA predominantly depends on their branchpoint sequence and can involve the contribution of major spliceosome components.

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease caused by reduced amounts of the ubiquitously expressed Survival of Motor Neuron (SMN) protein. In agreement with its crucial role in the biogenesis of spliceosomal snRNPs, SMN-deficiency is correlated to numerous splicing alterations in patient cells and various tissues of SMA mouse models. Among the snRNPs whose assembly is impacted by SMN-deficiency, those involved in the minor spliceosome are particularly affected. Importantly, splicing of several, but not all U12-dependent introns has been shown to be affected in different SMA models. Here, we have investigated the molecular determinants of this differential splicing in spinal cords from SMA mice. We show that the branchpoint sequence (BPS) is a key element controlling splicing efficiency of minor introns. Unexpectedly, splicing of several minor introns with suboptimal BPS is not affected in SMA mice. Using in vitro splicing experiments and oligonucleotides targeting minor or major snRNAs, we show for the first time that splicing of these introns involves both the minor and major machineries. Our results strongly suggest that splicing of a subset of minor introns is not affected in SMA mice because components of the major spliceosome compensate for the loss of minor splicing activity.

Evaluation of suspected autosomal Alport Syndrome synonymous variants

【Background】 Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in autosomal Alport syndrome cases, synonymous mutations in these genes have rarely been identified. 【Methods】 We conducted in silico splicing analysis using the Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of 4 synonymous variants detected in 4 patients with suspected autosomal dominant Alport syndrome (COL4A3 (c.693G>A (p.Val231=) and COL4A4 (c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=))). 【Results】 Both in vivo and in vitro splicing assays showed exon skipping in 2 out of the 4 synonymous variants identified (c.735G>A and c.870G>A in COL4A4). Prediction analysis of wild-type and mutated COL4A4 sequences using the HSF and Alamut suggested that these 2 variants may lead to the loss of binding sites for several splicing factors, e.g., in acceptor sites and exonic splicing enhancers. The other 2 variants did not induce aberrant splicing. 【Conclusions】 This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing as shown in 2 of our cases. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in autosomal dominant Alport syndrome cases.

Growth Hormone Receptor (Ghr) 6ω Pseudoexon Activation: A Novel Cause Of Severe Growth Hormone Insensitivity (Ghi)

Context Severe forms of Growth Hormone Insensitivity (GHI) are characterized by extreme short stature, dysmorphism and metabolic anomalies. Objective Identification of the genetic cause of growth failure in 3 ‘classical’ GHI subjects. Design A novel intronic GHR variant was identified, and in vitro splicing assays confirmed aberrant splicing. A 6Ω pseudoexon GHR vector and patient fibroblast analysis assessed the consequences of the novel pseudoexon inclusion and the impact on GHR function. Results We identified a novel homozygous intronic GHR variant (g.5:42700940T>G, c.618 + 836T> G), 44bp downstream of the previously recognized intronic 6Ψ GHR pseudoexon mutation in the index patient. Two siblings also harbored the novel intronic 6Ω pseudoexon GHR variant in compound heterozygosity with the known GHR c.181C>T (R43X) mutation. In vitro splicing analysis confirmed inclusion of a 151bp mutant 6Ω pseudoexon not identified in wild-type constructs. Inclusion of the 6Ω pseudoexon causes a frameshift resulting in a non-functional truncated GHR lacking the transmembrane and intracellular domains. The truncated 6Ω pseudoexon protein demonstrated extracellular accumulation and diminished activation of STAT5B signaling following growth hormone stimulation. Conclusion Novel GHR 6Ω pseudoexon inclusion results in loss of GHR function consistent with a severe GHI phenotype. This represents a novel mechanism of Laron syndrome and is the first deep intronic variant identified causing severe postnatal growth failure. The 2 kindreds originate from the same town in Campania, Southern Italy, implying common ancestry. Our findings highlight the importance of studying variation in deep intronic regions as a cause of monogenic disorders.

In Vitro Splicing Assay Proves the Pathogenicity of Intronic Variants in MRAP

Introduction: Familial glucocorticoid deficiency (FGD) is characterised by isolated glucocorticoid deficiency in a patient who retains normal mineralocorticoid production. FGD causing mutations in the MC2R accessory protein, MRAP, are often splice-site or nonsense mutations resulting in a truncated protein. Many of these mutations occur at the canonical donor splice-site of intron 3, where it has been shown previously that c.106 + 2_3dupTA, for example, results in skipping of the first coding exon with unknown consequences at the protein level. Patients and methods: DNA was isolated from three consanguineous individuals diagnosed with early onset FGD (0 - 13 months) with high ACTH and/or low cortisol levels and underwent whole exome sequencing. The proband in family 1 (P1) presented at 13 months and had a hyperpigmented sibling who died in neonatal period due to adrenal failure. Patient 2 (P2), who also had a family history of adrenal insufficiency, was noted to be hyperpigmented at birth with markedly raised ACTH, patient 3 (P3) was noted to have diffuse hyperpigmentation in the early neonatal period and on formal testing at 16m was found to have low serum cortisol. Variants were confirmed using Sanger sequencing and predicted splice-site mutations were investigated using an in vitro splicing assay. Results: Homozygous mutations in MRAP were identified in all three cases which were heterozygous in their parents. Previously described mutations, c.106 + 1delG (chr21:33671388delG; rs1476574441; CD050155) in P1 and c.106 + 2dupT (Chr21: 33671390_91insT; rs761576317; CI118288) in P2 at the canonical donor splice-site of intron 3, were identified, with the former predicted to destroy the splice site and the latter to weaken it. These mutations in vitro resulted in the complete skipping of exon 3, which contains the translational start site, and presumably result in no protein product. A novel homozygous mutation in intron 4, c.206 + 5G>T; (chr21:33679055G>T rs1064796398) was identified in P3, but was not predicted to alter splicing. In vitro, this mutation negates the canonical donor splice site and creates two different alternative sites, both resulting in frameshifts and predicted early termination of the protein (p.Val44fs*50, p.Pro72fs*90). Conclusion: All mutations reported here are predicted to produce no protein, either because the start site is excluded (for c.106 + 1delG and c.106 + 2dupT) or because the transcripts are likely to undergo nonsense mediated decay (for c.206 + 5G>T), resulting in the early onset FGD seen in the patients. Splice prediction protocols, although effective for variants within 2bp of exon/intron boundaries may not predict the true outcome of a base change whereas the splice assay conclusively revealed the effect of all three variants allowing us to assign pathogenicity to them.

Design and synthesis of herboxidiene derivatives that potently inhibit in vitro splicing

Enantioselective syntheses of C-6 modified derivatives of herboxidiene and their biological evaluation in splicing inhibitory assay.

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene

Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs.