Antisense oligonucleotide-induced alternative splicing of the APOB mRNA generates a novel isoform of APOB

BACKGROUND: Apolipoprotein B (APOB) is an integral part of the LDL, VLDL, IDL, Lp(a) and chylomicron lipoprotein particles. The APOB pre-mRNA consists of 29 constitutively-spliced exons. APOB exists as two natural isoforms: the full-length APOB100 isoform, assembled into LDL, VLDL, IDL and Lp(a) and secreted by the liver in humans; and the C-terminally truncated APOB48, assembled into chylomicrons and secreted by the intestine in humans. Down-regulation of APOB100 is a potential therapy to lower circulating LDL and cholesterol levels. RESULTS: We investigated the ability of 2'O-methyl RNA antisense oligonucleotides (ASOs) to induce the skipping of exon 27 in endogenous APOB mRNA in HepG2 cells. These ASOs are directed towards the 5' and 3' splice-sites of exon 27, the branch-point sequence (BPS) of intron 26–27 and several predicted exonic splicing enhancers within exon 27. ASOs targeting either the 5' or 3' splice-site, in combination with the BPS, are the most effective. The splicing of other alternatively spliced genes are not influenced by these ASOs, suggesting that the effects seen are not due to non-specific changes in alternative splicing. The skip 27 mRNA is translated into a truncated isoform, APOB87(SKIP27). CONCLUSION: The induction of APOB87(SKIP27 )expression in vivo should lead to decreased LDL and cholesterol levels, by analogy to patients with hypobetalipoproteinemia. As intestinal APOB mRNA editing and APOB48 expression rely on sequences within exon 26, exon 27 skipping should not affect APOB48 expression unlike other methods of down-regulating APOB100 expression which also down-regulate APOB48

Antisense Oligonucleotide Therapeutics for Cystic Fibrosis: Recent Developments and Perspectives

Antisense oligonucleotide (ASO) technology has become an attractive therapeutic modality for various diseases, including Mendelian disorders. ASOs can modulate the expression of a target gene by promoting mRNA degradation or changing pre-mRNA splicing, nonsense-mediated mRNA decay, or translation. Advances in medicinal chemistry and a deeper understanding of post-transcriptional mechanisms have led to the approval of several ASO drugs for diseases that had long lacked therapeutic options. For instance, an ASO drug called nusinersen became the first approved drug for spinal muscular atrophy, improving survival and the overall disease course. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Although Trikafta and other CFTR-modulation therapies benefit most CF patients, there is a significant unmet therapeutic need for a subset of CF patients. In this review, we introduce ASO therapies and their mechanisms of action, describe the opportunities and challenges for ASO therapeutics for CF, and discuss the current state and prospects of ASO therapies for CF

Distribution of SR protein exonic splicing enhancer motifs in human protein-coding genes

Exonic splicing enhancers (ESEs) are pre-mRNA cis-acting elements required for splice-site recognition. We previously developed a web-based program called ESEfinder that scores any sequence for the presence of ESE motifs recognized by the human SR proteins SF2/ASF, SRp40, SRp55 and SC35 (). Using ESEfinder, we have undertaken a large-scale analysis of ESE motif distribution in human protein-coding genes. Significantly higher frequencies of ESE motifs were observed in constitutive internal protein-coding exons, compared with both their flanking intronic regions and with pseudo exons. Statistical analysis of ESE motif frequency distributions revealed a complex relationship between splice-site strength and increased or decreased frequencies of particular SR protein motifs. Comparison of constitutively and alternatively spliced exons demonstrated slightly weaker splice-site scores, as well as significantly fewer ESE motifs, in the alternatively spliced group. Our results underline the importance of ESE-mediated SR protein function in the process of exon definition, in the context of both constitutive splicing and regulated alternative splicing

Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo

Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3′ processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523–527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo

A novel histone exchange factor, protein phosphatase 2Cγ, mediates the exchange and dephosphorylation of H2A–H2B

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A–H2B, we identified protein phosphatase (PP) 2C γ subtype (PP2Cγ/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A–H2B. The disruption of PP2Cγ in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cγ-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes

Exon-skipping antisense oligonucleotides for cystic fibrosis therapy

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity but is expressed at a very low level due to nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable, and read-through drugs are inefficient partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site-targeting ASOs induced the expression of CFTR mRNA without the premature-termination-codon-containing exon 23 (CFTR-Δex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-Δex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation

Higher-order epistasis and phenotypic prediction

Contemporary high-throughput mutagenesis experiments are providing an increasingly detailed view of the complex patterns of genetic interaction that occur between multiple mutations within a single protein or regulatory element. By simultaneously measuring the effects of thousands of combinations of mutations, these experiments have revealed that the genotype-phenotype relationship typically reflects not only genetic interactions between pairs of sites but also higher-order interactions among larger numbers of sites. However, modeling and understanding these higher-order interactions remains challenging. Here we present a method for reconstructing sequence-to-function mappings from partially observed data that can accommodate all orders of genetic interaction. The main idea is to make predictions for unobserved genotypes that match the type and extent of epistasis found in the observed data. This information on the type and extent of epistasis can be extracted by considering how phenotypic correlations change as a function of mutational distance, which is equivalent to estimating the fraction of phenotypic variance due to each order of genetic interaction (additive, pairwise, three-way, etc.). Using these estimated variance components, we then define an empirical Bayes prior that in expectation matches the observed pattern of epistasis and reconstruct the genotype-phenotype mapping by conducting Gaussian process regression under this prior. To demonstrate the power of this approach, we present an application to the antibody-binding domain GB1 and also provide a detailed exploration of a dataset consisting of high-throughput measurements for the splicing efficiency of human pre-mRNA [Formula: see text] splice sites, for which we also validate our model predictions via additional low-throughput experiments

Therapeutic manipulation of IKBKAP mis-splicing with a small molecule to cure familial dysautonomia.

Approximately half of genetic disease-associated mutations cause aberrant splicing. However, a widely applicable therapeutic strategy to splicing diseases is yet to be developed. Here, we analyze the mechanism whereby IKBKAP-familial dysautonomia (FD) exon 20 inclusion is specifically promoted by a small molecule splice modulator, RECTAS, even though IKBKAP-FD exon 20 has a suboptimal 5' splice site due to the IVS20 + 6 T > C mutation. Knockdown experiments reveal that exon 20 inclusion is suppressed in the absence of serine/arginine-rich splicing factor 6 (SRSF6) binding to an intronic splicing enhancer in intron 20. We show that RECTAS directly interacts with CDC-like kinases (CLKs) and enhances SRSF6 phosphorylation. Consistently, exon 20 splicing is bidirectionally manipulated by targeting cellular CLK activity with RECTAS versus CLK inhibitors. The therapeutic potential of RECTAS is validated in multiple FD disease models. Our study indicates that small synthetic molecules affecting phosphorylation state of SRSFs is available as a new therapeutic modality for mechanism-oriented precision medicine of splicing diseases

Systematic characterization of short intronic splicing-regulatory elements in SMN2 pre-mRNA.

Intronic splicing enhancers and silencers (ISEs and ISSs) are two groups of splicing-regulatory elements (SREs) that play critical roles in determining splice-site selection, particularly for alternatively spliced introns or exons. SREs are often short motifs; their mutation or dysregulation of their cognate proteins frequently causes aberrant splicing and results in disease. To date, however, knowledge about SRE sequences and how they regulate splicing remains limited. Here, using an SMN2 minigene, we generated a complete pentamer-sequence library that comprises all possible combinations of 5 nucleotides in intron 7, at a fixed site downstream of the 5' splice site. We systematically analyzed the effects of all 1023 mutant pentamers on exon 7 splicing, in comparison to the wild-type minigene, in HEK293 cells. Our data show that the majority of pentamers significantly affect exon 7 splicing: 584 of them are stimulatory and 230 are inhibitory. To identify actual SREs, we utilized a motif set enrichment analysis (MSEA), from which we identified groups of stimulatory and inhibitory SRE motifs. We experimentally validated several strong SREs in SMN1/2 and other minigene settings. Our results provide a valuable resource for understanding how short RNA sequences regulate splicing. Many novel SREs can be explored further to elucidate their mechanism of action