Mouse Exon Research Articles

Rational design of a genomically humanized mouse model for dominantly inherited hearing loss, DFNA9

DFNA9 is a dominantly inherited form of adult-onset progressive hearing impairment caused by mutations in the COCH gene. COCH encodes cochlin, a crucial extracellular matrix protein. We established a genomically humanized mouse model for the Dutch/Belgian c.151C>T founder mutation in COCH. Considering upcoming sequence-specific genetic therapies, we exchanged the genomic murine Coch exons 3–6 for the corresponding human sequence. Introducing human-specific genetic information into mouse exons can be risky. To mitigate unforeseen consequences on cochlin function resulting from the introduction of the human COCH protein-coding sequence, we converted all human-specific amino acids to mouse equivalents. We furthermore optimized the recognition of the human COCH exons by the murine splicing machinery during pre-mRNA splicing. Subsequent observations in mouse embryonic stem cells revealed correct splicing of the hybrid Coch transcript. The inner ear of the established humanized Coch mice displays correctly-spliced wild-type and mutant humanized Coch alleles. For a comprehensive study of auditory function, mice were crossbred with C57BL/6 Cdh23753A>G mice to remove the Cdh23ahl allele from the genetic background of the mice. At 9 months, all humanized Coch genotypes showed hearing thresholds comparable to wild-type C57BL/6 Cdh23753A>G mice. This indicates that both the introduction of human wildtype COCH, and correction of Cdh23ahl in the humanized Coch lines was successful. Overall, our approach proved beneficial in eliminating potential adverse events of genomic humanization of mouse genes, and provides us with a model in which sequence-specific therapies directed against the human mutant COCH alle can be investigated. With the hearing and balance defects anticipated to occur late in the second year of life, a long-term follow-up study is ongoing to fully characterize the humanized Coch mouse model.

Good news for the mdx mouse community: Improved dystrophin restoration after skipping mouse dystrophin exon 23

Good news for the mdx mouse community: Improved dystrophin restoration after skipping mouse dystrophin exon 23

A humanized knockin mouse model of Duchenne muscular dystrophy and its correction by CRISPR-Cas9 therapeutic gene editing

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the X-linked dystrophin (DMD) gene. Exon deletions flanking exon 51, which disrupt the dystrophin open reading frame (ORF), represent one of the most common types of human DMD mutations. Previously, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) gene editing to restore the reading frame of exon 51 in mice and dogs with exon 50 deletions. Due to genomic sequence variations between species, the single guide RNAs (sgRNAs) used for DMD gene editing are often not conserved, impeding direct clinical translation of CRISPR-Cas therapeutic gene-editing strategies. To circumvent this potential obstacle, we generated a humanized DMD mouse model by replacing mouse exon 51 with human exon 51, followed by deletion of mouse exon 50, which disrupted the dystrophin ORF. Systemic CRISPR-Cas9 gene editing using an sgRNA that targets human exon 51 efficiently restored dystrophin expression and ameliorated pathologic hallmarks of DMD, including histopathology and grip strength in this mouse model. This unique DMD mouse model with the human genomic sequence allows in vivo assessment of clinically relevant gene editing strategies as well as other therapeutic approaches and represents a significant step toward therapeutic translation of CRISPR-Cas9 gene editing for correction of DMD.

Establishment and Phenotypic Analysis of the Novel Gaucher Disease Mouse Model With the Partially Humanized Gba1 Gene and F213I Mutation.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the GBA1 gene, which produces the glucocerebrosidase (GCase) protein. There are more than 500 mutations reported in GBA1, among which L444P (p.Leu444Pro) and F213I (p.Phe213Ile) are the most common in the Chinese population, while the function of F213I mutation remains elusive. This study aims to establish the GD mouse model of partially humanized Gba1 gene with F213I mutation. In vitro GCase activity assays showed that the product of partially humanized Gba1 gene, in which the mouse exons 5-7 were replace by the corresponding human exons, displayed similar activity with the wild-type mouse Gba1, while the F213I mutation in the humanized Gba1 led to significant decrease in enzyme activity. ES cell targeting was used to establish the mice expressing the partially humanized Gba1-F213I. Gba1 F213I/+ mice did not show obviously abnormal phenotypes, but homozygous Gba1 F213I/F213I mice died within 24 h after birth, whose epidermal stratum corneum were abnormal from the wild-type. The GCase activity in Gba1 F213I/F213I mice greatly decreased. In conclusion, our results showed that the partially humanized GD mouse model with the F213I mutation was developed and homozygous F213I mutation is lethal for newborn mice.

Dysregulation of Skeletal Muscle Mitochondrial Turnover and Dynamics Occurs During Late Symptomatic Stages of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder caused by a mutation in the survival motor neuron 1 (SMN1) gene and it is the leading genetic cause of infant mortality. Recently approved genetic therapies designed to augment full length SMN protein in the central nervous system fail to ameliorate abnormal skeletal muscle features, which strongly suggests an important role for SMN protein in skeletal muscle homeostasis. This study aimed to characterize key modifiers of skeletal muscle mitochondrial turnover and dynamics during disease progression in a pre‐clinical murine model of SMA in vivo. Additionally, we investigated the effects of a single dose of exercise on these mitochondrial biology‐regulating pathways in the skeletal muscle of SMA mice. Muscle samples were collected from wild‐type (WT) and Smn2B/‐ (SMA) mice at postnatal day 9 (P9), P13, and P21 to examine skeletal muscle‐specific disease progression. Mitochondrial content did not differ between genotypes at all timepoints as evident by similar levels of mitochondrial oxidative phosphorylation proteins, succinate dehydrogenase staining, and citrate synthase content. However, mRNA content of key genes responsible for regulating mitochondrial quality such as nuclear respiratory factor 2, mitochondrial transcription factor A, and p53 were significantly higher in SMA mice versus WT at P21. Additionally, we observed elevated mitochondrial fission activity as indicated by a 2‐fold increase (p < 0.05) in dynamin related protein 1 (DRP1) activation. Concomitantly, the expression of several mitophagy proteins including BLC2 interacting protein 3, parkin, and PTEN‐induced kinase 1 were significantly increased by 2.9‐, 2.7‐, and 2‐fold, respectively, compared to WT animals at P21. Interestingly, we observed blunted (p < 0.05) inclusion of optic atrophy 1 exon 4b, a key exon in mitochondrial biogenesis, between WT and SMA mice at P21. Acute exercise significantly increased the inhibition of DRP1‐mediated mitochondrial fission activity in the muscles of SMA mice relative to sedentary SMA mice. However, a single exercise dose failed to alter the expression of mitophagy proteins up to three hours after running. Our data demonstrate that skeletal muscle mitochondrial health is compromised in SMA mice due to elevated fission and mitophagy processes. Furthermore, SMA mice display aberrant alternative splicing of Opa1 transcripts that may in turn hinder mitochondrial biogenesis in later disease stages. We also highlight that an acute bout of treadmill running elicits pro‐fusion signaling to potentially improve the mitochondrial reticulum. Collectively, this study is the first to reveal mitochondrial dysfunction in SMA skeletal muscle and outlines signalling associated with mitochondrial plasticity following a single dose of exercise.

TTR exon-humanized mouse optimal for verifying new therapies for FAP

Familial amyloidotic polyneuropathy (FAP) is caused by a mutation in the transthyretin (TTR) gene. In addition, deposition of wild-type TTR can cause senile systemic amyloidosis (SSA). To date, we have produced several transgenic mouse models for FAP and SSA by introducing TTR genes with different promoters or mutations. However, mouse TTR can associate with human TTR to produce hybrid tetramers in transgenic mice. Thus, these transgenic mice cannot be used to test the efficacy of a new therapy. In this study, we attempted to construct an optimized mouse model to verify a new therapy. The TTR gene consists of 4 exons and 3 introns. We prepared two gRNAs, one for the exon 1 and the other for exon 4, and a single donor vector carrying the whole TTR gene in which mouse exons were replaced with human exons. Using these vectors, we produced a TTR exon-humanized mouse with human exons and mouse introns using genome editing technology. These TTR exon-humanized mice showed normal TTR expression patterns in terms of serum TTR level and spatial specificity. These TTR exon-humanized mice will be useful for devising new treatment methods for FAP, including gene therapy.

TRITHORAX-dependent arginine methylation of HSP68 mediates circadian repression by PERIOD in the monarch butterfly

Transcriptional repression drives feedback loops that are central to the generation of circadian (∼24-h) rhythms. In mammals, circadian repression of circadian locomotor output cycles kaput, and brain and muscle ARNT-like 1 (CLOCK:BMAL1)-mediated transcription is provided by a complex formed by PERIOD (PER) and CRYPTOCHROME (CRY) proteins. PER initiates transcriptional repression by binding CLK:BMAL1, which ultimately results in their removal from DNA. Although PER's ability to repress transcription is widely recognized, how PER binding triggers repression by removing CLK:BMAL1 from DNA is not known. Here, we use the monarch butterfly as a model system to address this problem because it harbors a simplified version of the CLK:BMAL1-activated circadian clock present in mammals. We report that an intact CLOCK mouse exon 19 homologous region (CLKe19r) and the histone methyltransferase TRITHORAX (TRX) are both necessary for monarch CLK:BMAL1-mediated transcriptional activation, CLK-PER interaction, and PER repression. Our results show that TRX catalytic activity is essential for CLK-PER interaction and PER repression via the methylation of a single arginine methylation site (R45) on heat shock protein 68 (HSP68). Our study reveals TRX and HSP68 as essential links between circadian activation and PER-mediated repression and suggests a potential conserved clock function for HSPs in eukaryotes.

Intronic enhancer region governs transcript-specific Bdnf expression in rodent neurons.

Brain-derived neurotrophic factor (BDNF) controls the survival, growth, and function of neurons both during the development and in the adult nervous system. Bdnf is transcribed from several distinct promoters generating transcripts with alternative 5' exons. Bdnf transcripts initiated at the first cluster of exons have been associated with the regulation of body weight and various aspects of social behavior, but the mechanisms driving the expression of these transcripts have remained poorly understood. Here, we identify an evolutionarily conserved intronic enhancer region inside the Bdnf gene that regulates both basal and stimulus-dependent expression of the Bdnf transcripts starting from the first cluster of 5' exons in mouse and rat neurons. We further uncover a functional E-box element in the enhancer region, linking the expression of Bdnf and various pro-neural basic helix-loop-helix transcription factors. Collectively, our results shed new light on the cell-type- and stimulus-specific regulation of the important neurotrophic factor BDNF.

A novel mutation in the mouse Pcsk1 gene showing obesity and diabetes

The proprotein convertase subtilisin/Kexin type 1 (PCSK1/PC1) protein processes inactive pro-hormone precursors into biologically active hormones in a number of neuroendocrine and endocrine cell types. Patients with recessive mutations in PCSK1 exhibit a complex spectrum of traits including obesity, diarrhoea and endocrine disorders. We describe here a new mouse model with a point mutation in the Pcsk1 gene that exhibits obesity, hyperphagia, transient diarrhoea and hyperproinsulinaemia, phenotypes consistent with human patient traits. The mutation results in a pV96L amino acid substitution and changes the first nucleotide of mouse exon 3 leading to skipping of that exon and in homozygotes very little full-length transcript. Overexpression of the exon 3 deleted protein or the 96L protein results in ER retention in Neuro2a cells. This is the second Pcsk1 mouse model to display obesity phenotypes, contrasting knockout mouse alleles. This model will be useful in investigating the basis of endocrine disease resulting from prohormone processing defects.

Phenotype onset in Huntington's disease knock-in mice is correlated with the incomplete splicing of the mutant huntingtin gene.

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by an expanded CAG repeat within the huntingtin (HTT) gene. The Q140 and HdhQ150 knock‐in HD mouse models were generated such that HdhQ150 mice have an expanded CAG repeat inserted into the mouse Htt gene, whereas in the Q140s, mouse exon 1 Htt was replaced with a mutated version of human exon 1. By standardizing mouse strain background, breeding to homozygosity and employing sensitive behavioral tests, we demonstrate that the onset of behavioral phenotypes occurs earlier in the Q140 than the HdhQ150 knock‐in mouse models and that huntingtin (HTT) aggregation appears earlier in the striata of Q140 mice. We have previously found that the incomplete splicing of mutant HTT from exon 1 to exon 2 results in the production of a small polyadenylated transcript that encodes the highly pathogenic mutant HTT exon 1 protein. In this report, we have identified a functional consequence of the sequence differences between these two models at the RNA level, in that the level of incomplete splicing, and of the mutant exon 1 HTT protein, are greater in the brains of Q140 mice. While differences in the human and mouse exon 1 HTT proteins (e.g., proline rich sequences) could also contribute to the phenotypic differences, our data indicate that the incomplete splicing of HTT and approaches to lower the levels of the exon 1 HTT transcript should be pursued as therapeutic targets.

Abstract 4976: Ex vivo functional characterization and in vivo efficacy validation of human CD40 agonistic antibodies in the human CD40 knock-in model (CD40 HuGEMM)

Abstract CD40 receptor is a member of TNF-receptor superfamily, broadly expressed on the surface of antigen-presenting cells (APC) including B cells, dendritic cells (DC), macrophages, monocytes, and non-APC like platelets, endothelial cells and even some tumor cells. CD40 is essential for APC activation and signalling via ligation of CD40 ligand located on helper T cells and CD40 on APC to mediate licensing (activating) of APC. Ligation of CD40 on DCs, for example, can induce increased surface expression of costimulatory and MHC molecules, production of pro-inflammatory cytokines, and enhanced T cell activation. CD40 ligation on resting B cells can increase its antigen-presenting function and proliferation. In contrast to its critical role in the induction of effective innate and adaptive immune responses, CD40 signalling on certain malignant cells, particularly B cell lymphomas, triggers tumor apoptosis. The dual function of CD40 signalling provide unique opportunities for the use of agonistic CD40 antibodies in cancer immunotherapy. Indeed, several CD40 monoclonal antibodies are now in different stages of clinical development, some shown promising preliminary results in combination with other cancer immunotherapies. However, we are lacking preclinical models to evaluate efficacy of therapeutic CD40 agonistic antibodies, which usually don’t cross-bind to mouse targets. To fill this gap, we developed a human CD40 knock-in model (CD40 HuGEMM), of which mouse exon 2-5 are replaced by human counterparts. These KI mice express a chimeric CD40 with human extracellular domain & mouse trans-membrane and intracellular sequences. We have characterized the human CD40 expression of CD40 HuGEMM on a broad spectrum of mouse cells, such as lymphocytes, macrophages, DCs, and endothelial cells. In addition, we confirmed that human CD40 antibodies bind to the chimeric m/hCD40 recombinant protein, and lead to increased expression of MHCII on the B cells. Finally, the in vivo efficacy study indicates that human CD40 antibody treatment lead to robust anti-tumor response and contribute to better survival of the mice. Taken together, our CD40 HuGEMM provides a powerful preclinical model to assess the efficacy of the human-specific CD40 agonistic antibodies. It may also serve as a model for proof of concept studies of CD40 agonistic antibodies with other therapies. Citation Format: Daniel Xianfei He, Lei Zheng, Ruilin Sun, Annie Xiaoyu An, Jian Fei, Henry Qixiang Li, Davy Xuesong Ouyang. Ex vivo functional characterization and in vivo efficacy validation of human CD40 agonistic antibodies in the human CD40 knock-in model (CD40 HuGEMM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4976.

CRISPR/Cas9-mediated genome editing induces gene knockdown by altering the pre-mRNA splicing in mice

BackgroundClustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has been wildly used to generate gene knockout models through inducing indels causing frame-shift. However, there are few studies concerning the post-transcript effects caused by CRISPR-mediated genome editing.ResultsIn the present study, we showed that gene knockdown model also could be generated using CRISPR-mediated gene editing by disrupting the boundary of exon and intron in mice (C57BL/6 J). CRISPR induced indel at the boundary of exon and intron (5′ splice site) caused alternative splicing and produced multiple different mRNAs, most of these mRNAs introduced premature termination codon causing down expression of the gene.ConclusionsThese results showed that alternative splicing mutants were able to generate through CRISPR-mediated genome editing by deleting the boundary of exon and intron causing disruption of 5′ splice site. Although alternative splicing was an unexpected outcome, this finding could be developed as a technology to generate gene knockdown models or to investigate pre-mRNA splicing.

A wide variety of Mitf transcript variants are expressed in the Xenopus laevis periodic albino mutant.

The periodic albino mutant of Xenopus laevis has been used to study the development of pigment cells because both the retinal pigment epithelium (RPE) and melanophores are affected. In this mutant, "white pigment cells" containing both melanophore-specific and iridophore-specific pigment organelles appear. The present experiments were designed to investigate the structural organization and expression of microphthalmia-associated transcription factor (Mitf) in the mutant since Mitf is known to regulate the development of melanocytes and RPE. The exon structures of X.laevis mitf genes (mitf.L and mitf.S) were defined using newly obtained Mitf transcripts and X.laevis genomic data. Compared to mouse mitf, exons 3 and 6a were absent in X.laevis mitf. The four exons between exons 4 and 6b in X.laevis mitf were named 5α, 5β, 5γ, and 5δ. Exons 5α and 5δ were specific to X.laevis mitf, whereas the continuous exons 5β/γ were identical to exon 5 of mouse mitf. A wide variety of A-Mitf and M-Mitf transcript variants lacking one or more exons were found in X.laevis; however, different types of Mitf transcripts were expressed in the mutant. In addition, white pigment cells and melanophores expressed both the mitf and dopachrome tautomerase (dct) genes.

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases.

Abstract A203: Generation of human TIM3 knock-in mice for preclinical efficacy assessment of therapeutic antibodies

Abstract Immune checkpoint inhibitors, i.e., PD1 and CTLA4 antibody therapeutics, have led to long-term survival in many late-stage solid tumor patients. Despite this revolutionary clinical impact, the overall response rate is still low. One major roadblock is that compensatory immune inhibitory pathways were turned on to protect tumor cells from being attacked by T cells. T cell immunoglobulin- and mucin domain-containing molecule 3 (Tim-3) is a type I transmembrane protein expressed in T cells, monocytes, macrophages, and dendritic cells. PD-1 and Tim-3 coexpression is often associated with an exhausted phenotype of tumor-infiltrating lymphocyte (TIL) in various tumor types. TIM-3 expression has been reported as a compensatory mechanism in the PD1 nonresponsive patients. Simultaneously targeting PD1 and TIM3 is now being actively tested in clinical trials. However, we still do not have an animal model that can efficiently evaluate the efficacy of therapeutic TIM3 antibodies or combinations. Here we report generation of a human TIM3 knock-in C57BL/6 mouse model, in which we replaced mouse exon 2-5 of HAVCR2 gene with human counterparts. The engineered knock-in mice express a chimeric TIM3 where its extracellular and transmembrane domains are human sequence, while the signal peptide and intracellular domain are kept intact. We confirmed that homozygous knock-in mice only express this chimeric TIM3, recognizable by human TIM3 antibody in NK cells, macrophages, and stimulated T cells. In an efficacy study, we inoculated the TIM3 knock-in mice with MC38 syngeneic tumor cells. Treating the mice with 5 mg/kg of human TIM3 antibody resulted in modest tumor growth inhibition (TGI), while in combination with anti-mouse PD1 antibody, the TGI improved to 62% following 2 weeks of treatment. We are now breeding the human PD1 and human TIM3 double knock-in mice to test the combinatory therapeutic antibodies. We have also developed human PD-L1 expressing MC38 cells, so that when inoculated in human TIM3 knock-in mice, PD-L1 and TIM3 therapeutic combination can be evaluated. Citation Format: Lei Zheng, Xuesong Huang, Wenyi Ouyang, Gang Chen, Annie Xiaoyu An, Xin Dong, Jay Liu, Jean Pierre Wery, Qian Shi, Davy Xuesong Ouyang. Generation of human TIM3 knock-in mice for preclinical efficacy assessment of therapeutic antibodies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A203.

CiRS-7 exonic sequence is embedded in a long non-coding RNA locus.

ciRS-7 is an intensely studied, highly expressed and conserved circRNA. Essentially nothing is known about its biogenesis, including the location of its promoter. A prevailing assumption has been that ciRS-7 is an exceptional circRNA because it is transcribed from a locus lacking any mature linear RNA transcripts of the same sense. To study the biogenesis of ciRS-7, we developed an algorithm to define its promoter and predicted that the human ciRS-7 promoter coincides with that of the long non-coding RNA, LINC00632. We validated this prediction using multiple orthogonal experimental assays. We also used computational approaches and experimental validation to establish that ciRS-7 exonic sequence is embedded in linear transcripts that are flanked by cryptic exons in both human and mouse. Together, this experimental and computational evidence generates a new model for regulation of this locus: (a) ciRS-7 is like other circRNAs, as it is spliced into linear transcripts; (b) expression of ciRS-7 is primarily determined by the chromatin state of LINC00632 promoters; (c) transcription and splicing factors sufficient for ciRS-7 biogenesis are expressed in cells that lack detectable ciRS-7 expression. These findings have significant implications for the study of the regulation and function of ciRS-7, and the analytic framework we developed to jointly analyze RNA-seq and ChIP-seq data reveal the potential for genome-wide discovery of important biological regulation missed in current reference annotations.

The Use of Antisense Oligonucleotides for the Treatment of Duchenne Muscular Dystrophy.

Antisense oligonucleotides (AONs) hold great promise for therapeutic splice-switching correction in many genetic diseases and in particular for Duchenne muscular dystrophy (DMD), where AONs can be used to reframe the dystrophin transcript and give rise to a partially deleted but yet functional dystrophin protein. Many different chemistries of AONs can be used for splice switching modulation, and some of them have been evaluated in clinical trials for DMD. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake, and sufficient therapeutic efficacy is difficult to achieve. Therefore, there is still a critical need to develop efficient AONs able to restore the expression of dystrophin in all relevant tissues and international efforts are currently on going to develop new compounds or alternative chemistries with higher therapeutic potential. Here, we describe the methods to evaluate the potency of antisense oligonucleotides, and in particular of tricyclo-DNA (tcDNA)-AONs, a novel class of AONs which displays unique pharmacological properties and unprecedented uptake in many tissues after systemic administration. We focus on the most widely used mouse model for DMD, the mdx mouse and detail methods to analyze the skipping of the mouse exon 23 both in vitro in H2K mdx cells and in vivo in the mdx mouse model.

Muscle developmental defects in heterogeneous nuclear Ribonucleoprotein A1 knockout mice.

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is crucial for regulating alternative splicing. Its integrated function within an organism has not, however, been identified. We generated hnRNP A1 knockout mice to study the role of hnRNP A1 in vivo. The knockout mice, hnRNP A1−/−, showed embryonic lethality because of muscle developmental defects. The blood pressure and heart rate of the heterozygous mice were higher than those of the wild-type mice, indicating heart function defects. We performed mouse exon arrays to study the muscle development mechanism. The processes regulated by hnRNP A1 included cell adhesion and muscle contraction. The expression levels of muscle development-related genes in hnRNP A1+/− mice were significantly different from those in wild-type mice, as detected using qRT-PCR. We further confirmed the alternative splicing patterns of muscle development-related genes including mef2c, lrrfip1, usp28 and abcc9. Alternative mRNA isoforms of these genes were increased in hnRNP A1+/− mice compared with wild-type mice. Furthermore, we revealed that the functionally similar hnRNP A2/B1 did not compensate for the expression of hnRNP A1 in organisms. In summary, our study demonstrated that hnRNP A1 plays a critical and irreplaceable role in embryonic muscle development by regulating the expression and alternative splicing of muscle-related genes.

Identification of the Mtus1 Splice Variant as a Novel Inhibitory Factor Against Cardiac Hypertrophy.

BackgroundIn cardiac hypertrophy and failure, there is a widespread alteration in mRNA splicing, but the role of splice variants in cardiac hypertrophy has not yet been fully elucidated. In this study, we used an exon array to identify novel splice variants associated with cardiac hypertrophy.Methods and ResultsWe performed genome‐wide exon array analysis and developed a splicing profile in murine hearts with hypertrophy induced by transverse aortic constriction for 8 weeks. Following global analysis of splice variants using the Mouse Exon 1.0 ST Array, we identified 46 spliced genes and narrowed our focus to 1 gene, mitochondrial tumor suppressor 1 (Mtus1), whose splice variants were registered in the NCBI RefSeq database. Notably, one of the splice variants Mtus1A was specifically upregulated, although the total expression of the Mtus1 gene remained unchanged. We showed that Mtus1A was localized in the mitochondria, and its expression level increased with the degree of cardiac hypertrophy. In cultured cardiomyocytes, Mtus1A overexpression reduced phenylephrine‐induced reactive oxygen species production and consequent ERK phosphorylation, resulting in a decrease in both cell size and protein synthesis. In vivo, cardiac‐specific Mtus1A transgenic mice showed left ventricle wall thinning and a reduced hypertrophic response to pressure overload and phenylephrine treatment.ConclusionsWe found that Mtus1 is specifically spliced in hypertrophic hearts and that the Mtus1A variant has an inhibitory effect on cardiac hypertrophy. Mtus1A is, therefore, a possible diagnostic and therapeutic target for cardiac hypertrophy and failure.

Deciphering targeting rules of splicing modulator compounds: case of TG003.

BackgroundRecent advances in the development of small chemical compounds that can modulate RNA splicing brought excitement to the field of splicing-targeting therapy. Splicing-targeting therapy tries to ameliorate the disease by altering the exon combination of transcripts to reduce the undesired effect of genetic mutations. However, the knowledge and tools to understand factors contributing to splicing modulator compound sensitivity have been lacking. Our goal was to establish a method to characterize sequence features found in compound sensitive exons.ResultsHere we developed a comparative transcriptomic approach to explore features that make an exon sensitive to a chemical compound. In this study, we chose TG003, a potential drug for Duchenne muscular dystrophy, and performed RNA-sequencing on samples from human and mouse skeletal muscle cells, with and without TG003 treatments. We compared TG003 responsiveness between homologous exon pairs and identified 21 pairs in which human exons were skip-enhanced but not mouse exons. We compared the sequence features; splice site scores, number of splicing factor binding sites, and properties of branch sequence and polypyrimidine tracts, and found that polypyrimidine tracts were stronger (longer stretches and richer content of consecutive polypyrimidine) in the mouse TG003 insensitive exons. We also compared the features between TG003 skip-enhanced and insensitive exons within the species, and discovered that human TG003 skip-enhanced exons were shorter and had less splicing factor binding sites than the group of human TG003 insensitive exons. Mouse insensitive exons homologous to human TG003 skip-enhanced exons shared these properties. Our results suggested that these features are prerequisites for TG003 skip-enhanced exons and weak polypyrimidine tracts are defining features, which were supported by a decision tree analysis on all cassette exons in human.ConclusionsIn this study we established a comparative transcriptomic approach, which shed lights on how small chemical compounds modulate RNA splicing. The results described here was the first attempt to decipher the targeting rules of a splicing modulator compound. We expect that this approach would contribute to the precise understanding of the mechanism of TG003-induced splicing modulation, expand target diseases of splicing modulators in general, as well as the development of new splicing modulators.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0044-6) contains supplementary material, which is available to authorized users.