The role of Musashi-1 in CEP290 c.2991+1655A>G cryptic exon splicing in Leber Congenital Amaurosis

Human photoreceptors maximise alternative exon splicing to generate a unique set of gene isoforms. Conversely, the inclusion of a cryptic exon caused by the c.2991+1655A>G deep intronic change in CEP290 occurs in the human retina leading to Leber Congenital Amaurosis (LCA10). The RNA-binding protein Musashi-1 (MSI1) is a key component of alternative splicing in the developing mouse retina. Here we investigated the role of MSI1 in human photoreceptor-specific splicing and its potential role in CEP290 aberrant splicing disease. Alternative splicing was studied using human induced pluripotent stem cell derived 3D retinal organoid and RPE RNA-seq datasets and several photoreceptor gene isoforms were identified. Their temporal expression was resolved in control 3D retinal organoids in comparison to development and differentiation markers. Morpholino knockdown of MSI1 in control retinal organoids reduced the expression of several photoreceptor differentiation markers and the inclusion of photoreceptor-specific exons. Nonetheless, MSI1 knockdown in homozygous CEP290 c.2991+1655A>G LCA10 retinal organoids did not affect the inclusion of the LCA10-associated cryptic exon. These results show that while MSI1 is important for photoreceptor alternative splicing and homeostasis, it is not a major driver of the recognition of the CEP290 cryptic splice site and the manifestation of LCA10.

RNA-binding proteins PCBP1 and PCBP2 are critical determinants of murine erythropoiesis

We have previously demonstrated that the two paralogous RNA binding protein, PCBP1 and PCBP2, are individually essential for mouse development: Pcbp1 -null embryos are peri-implantation lethal while Pcbp2 -null embryos lose viability at mid-gestation. Mid-gestation Pcbp2 −/− embryos revealed a complex phenotype that included loss of certain hematopoietic determinants. Whether PCBP2 directly contributes to erythropoietic differentiation and whether PCBP1 has a role in this process remained undetermined. Here we selectively inactivate the genes encoding these two RNA-binding proteins during differentiation of the erythroid lineage in the developing mouse embryo. Individual inactivation of either locus fails to impact viability or blood formation. However, combined inactivation of the two loci results in mid-gestational repression of erythroid/hematopoietic gene expression, loss of blood formation, and fetal demise. Orthogonal ex-vivo analyses of primary erythroid progenitors selectively depleted of these two RNA binding proteins revealed that they mediate a combination of overlapping and isoform-specific impacts on hematopoietic lineage transcriptome, impacting both mRNA representation and exon splicing. These data lead us to conclude that PCBP1 and PCBP2 mediate functions critical to differentiation of the erythroid lineage.

Alternative exon splicing and differential expression in pancreatic islets reveals candidate genes and pathways implicated in early diabetes development

Type 2 diabetes (T2D) has a strong genetic component. Most of the gene variants driving the pathogenesis of T2D seem to target pancreatic β-cell function. To identify novel gene variants acting at early stage of the disease, we analyzed whole transcriptome data to identify differential expression (DE) and alternative exon splicing (AS) transcripts in pancreatic islets collected from two metabolically diverse mouse strains at 6 weeks of age after three weeks of high-fat-diet intervention. Our analysis revealed 1218 DE and 436 AS genes in islets from NZO/Hl vs C3HeB/FeJ. Whereas some of the revealed genes present well-established markers for β-cell failure, such as Cd36 or Aldh1a3, we identified numerous DE/AS genes that have not been described in context with β-cell function before. The gene Lgals2, previously associated with human T2D development, was DE as well as AS and localizes in a quantitative trait locus (QTL) for blood glucose on Chr.15 that we reported recently in our N2(NZOxC3H) population. In addition, pathway enrichment analysis of DE and AS genes showed an overlap of only half of the revealed pathways, indicating that DE and AS in large parts influence different pathways in T2D development. PPARG and adipogenesis pathways, two well-established metabolic pathways, were overrepresented for both DE and AS genes, probably as an adaptive mechanism to cope for increased cellular stress. Our results provide guidance for the identification of novel T2D candidate genes and demonstrate the presence of numerous AS transcripts possibly involved in islet function and maintenance of glucose homeostasis.

TDP-43 represses cryptic exon inclusion in FTD/ALS gene UNC13A

A hallmark pathological feature of neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing. Single nucleotide polymorphisms (SNPs) in UNC13A are among the strongest genome-wide association study (GWAS) hits associated with FTD/ALS in humans, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines, and iPSC-derived motor neurons resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. Remarkably, the top variants associated with FTD/ALS risk in humans are located in the cryptic exon harboring intron itself and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD/ALS (UNC13A genetic variants) and loss of TDP-43 function.

SRSF9 Regulates Cassette Exon Splicing of Caspase-2 by Interacting with Its Downstream Exon

Alternative splicing (AS) is an important posttranscriptional regulatory process. Damaged or unnecessary cells need to be removed though apoptosis to maintain physiological processes. Caspase-2 pre-mRNA produces pro-apoptotic long mRNA and anti-apoptotic short mRNA isoforms through AS. How AS of Caspase-2 is regulated remains unclear. In the present study, we identified a novel regulatory protein SRSF9 for AS of Caspase-2 cassette exon 9. Knock-down (KD) of SRSF9 increased inclusion of cassette exon and on the other hand, overexpression of SRSF9 decreased inclusion of this exon. Deletion mutagenesis demonstrated that exon 9, parts of intron 9, exon 8 and exon 10 were not required for the role of SRSF9 in Caspase-2 AS. However, deletion and substitution mutation analysis revealed that AGGAG sequence located at exon 10 provided functional target for SRSF9. In addition, RNA-pulldown mediated immunoblotting analysis showed that SRSF9 interacted with this sequence. Gene ontology analysis of RNA-seq from SRSF9 KD cells demonstrates that SRSF9 could regulate AS of a subset of apoptosis related genes. Collectively, our results reveal a basis for regulation of Caspase-2 AS.

Characterization and Comparative Transcriptomic Analysis of Skeletal Muscle in Pekin Duck at Different Growth Stages Using RNA-seq

Skeletal muscle, accounting for approximately 50% of body weight, is the largest and most important tissue. In this study, the gene expression profiles and pathways in skeletal muscle of Pekin duck were investigated and compared at embryonic day 17, 21, and 27 and postnatally at 6 months of age. An average of 49,555,936 reads in each sample was obtained from the transcriptome libraries. Over 70.0% of alternative splicing (AS) in each sample was mainly alternative 5’ first exon (transcription start site)-the first exon splicing (TSS) and alternative 3’ last exon (transcription terminal site)-the last exon splicing (TTS), indicating that TSS and TTS were the most common AS event in Pekin ducks, and these AS events were closely related to the regulation of muscle development at different growth stages. The results provided a valuable genomic resource for selective breeding and functional studies of genes. A total of 299 novel genes with ≥2 exons were obtained. There were 294 to 2806 differentially expressed genes (DEGs) in each pairwise comparison of Pekin duck. Notably, 90 DEGs in breast muscle and 9 DEGs in leg muscle were co-expressed at all developmental points. DEGs were validated by qPCR analysis, which confirmed the tendency of the expression. DEGs related to muscle development were involved in biological processes such as “endodermal cell differentiation”, “muscle cell cellular homeostasis”, “skeletal muscle tissue growth” and “skeletal muscle cell differentiation”, and were involved in pathways such as oxidative phosphorylation, ECM-receptor (extracellular matrix receptor) interaction, focal adhesion, carbon metabolism, and biosynthesis of amino acids. Some DEGs, including MYL4, IGF2BP1, CSRP3, SPP1 and KLHL31, as well as LAMB2, LAMA2, ITGB1 and OPN, played crucial roles in muscle growth and development. This study provides valuable information about the expression profile of mRNAs and pathways from duck skeletal muscle at different growth stages, and further functional study of these mRNAs and pathways could provide new ideas for studying the molecular networks of growth and development in duck skeletal muscle.

Transcriptome Sequencing of Patients with Hypertrophic Cardiomyopathy Reveals Novel Splice-altering Variants in MYBPC3

Background - Transcriptome sequencing can improve genetic diagnosis of Mendelian diseases but requires access to tissue expressing disease-relevant transcripts. We explored genetic testing of hypertrophic cardiomyopathy (HCM) using transcriptome sequencing of patient-specific human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). We also explored whether antisense oligonucleotides (AOs) could inhibit aberrant mRNA splicing in hiPSC-CMs. Methods - We derived hiPSC-CMs from patients with HCM due to MYBPC3 splice-gain variants, or an unresolved genetic cause. We used transcriptome sequencing of hiPSC-CM RNA to identify pathogenic splicing and used AOs to inhibit this splicing. Results - Transcriptome sequencing of hiPSC-CMs confirmed aberrant splicing in two people with previously identified MYBPC3 splice-gain variants (NM_000256.3:c.1090+453C>T and NM_000256.3:c.1224-52G>A). In a patient with an unresolved genetic cause of HCM following genome sequencing, transcriptome sequencing of hiPSC-CMs revealed diverse cryptic exon splicing due to an MYBPC3 NM_000256.3:c.1928-569G>T variant, and this was confirmed in cardiac tissue from an affected sibling. AO treatment demonstrated almost complete inhibition of cryptic exon splicing in one patient-specific hiPSC-CM line. Conclusions - Transcriptome sequencing of patient specific hiPSC-CMs solved a previously undiagnosed genetic cause of HCM and may be a useful adjunct approach to genetic testing. AO inhibition of cryptic exon splicing is a potential future personalised therapeutic option.

Skeletal Muscle Transcriptome Analysis of Hanzhong Ma Duck at Different Growth Stages Using RNA-Seq

As one of the most important poultry worldwide, ducks (Anas platyrhynchos) are raised mainly for meat and egg products, and muscle development in ducks is important for meat production. Therefore, an investigation of gene expression in duck skeletal muscle would significantly contribute to our understanding of muscle development. In this study, twenty-four cDNA libraries were constructed from breast and leg muscles of Hanzhong Ma ducks at day 17, 21, 27 of the embryo and postnatal at 6-month-old. High-throughput sequencing and bioinformatics were used to determine the abundances and characteristics of transcripts. A total of 632,172,628 (average 52,681,052) and 637,213,938 (average 53,101,162) reads were obtained from the sequencing data of breast and leg muscles, respectively. Over 71.63% and 77.36% of the reads could be mapped to the Anas platyrhynchos genome. In the skeletal muscle of Hanzhong duck, intron variant (INTRON), synonymous variant (SYNONYMOUS_CODING), and prime 3′ UTR variant (UTR_3_PRIME) were the main single nucleotide polymorphisms (SNP) annotation information, and “INTRON”, “UTR_3_PRIME”, and downstream-gene variant (DOWNSTREAM) were the main insertion-deletion (InDel) annotation information. The predicted number of alternative splicing (AS) in all samples were mainly alternative 5′ first exon (transcription start site)-the first exon splicing (TSS) and alternative 3′ last exon (transcription terminal site)-the last exon splicing (TTS). Besides, there were 292 to 2801 annotated differentially expressed genes (DEGs) in breast muscle and 304 to 1950 annotated DEGs in leg muscle from different databases. It is worth noting that 75 DEGs in breast muscle and 49 DEGs in leg muscle were co-expressed at all developmental points of comparison, respectively. The RNA-Seq data were confirmed to be reliable by qPCR. The identified DEGs, such as CREBL2, RHEB, GDF6, SHISA2, MYLK2, ACTN3, RYR3, and STMN1, were specially highlighted, indicating their strong associations with muscle development in the Hanzhong Ma duck. KEGG pathway analysis suggested that regulation of actin cytoskeleton, oxidative phosphorylation, and focal adhesion were involved in the development of skeletal muscle. The findings from this study can contribute to future investigations of the growth and development mechanism in duck skeletal muscle.

Sense-oriented AluYRa1 elements provide a lineage-specific transcription environment for polyadenylation

Transposable elements cause alternative splicing (AS) in different ways, contributing to transcript diversification. Alternative polyadenylation (APA), one of the AS events, is related to the generation of mRNA isoforms in 70% of human genes. In this study, we tried to investigate AluYRa1s located at the terminal region of cynomolgus monkey genes, utilizing both computational analysis and molecular experimentation. We found that ten genes had AluYRa1 at their 3′ end, and nine of these AluYRa1s were sense-oriented. Furthermore, in seven genes, AluYRa1s were expected to have a similar consensus sequence for polyadenylation cleavage. Additional computational analysis using the annotation files from the UCSC database showed that AluYRa1 was more involved in polyadenylation than in open reading frame exon splicing. To examine the extent of AluYRa1 involvement in polyadenylation, RNA-seq data from 30 normal cynomolgus monkeys were analyzed using TAPAS, a recently devised software that detects all the promising polyadenylation sites including APA sites. We observed that approximately 74% of possible polyadenylation sites in the analyzed genes were provided by sense-oriented AluYRa1. In conclusion, AluYRa1 is an Old-World monkey-specific TE, and its sense-oriented insertion at the 3′UTR region tends to provide a favorable environment for polyadenylation, diversifying gene transcripts.

Prediction of Alzheimer’s disease-specific phospholipase c gamma-1 SNV by deep learning-based approach for high-throughput screening

Exon splicing triggered by unpredicted genetic mutation can cause translational variations in neurodegenerative disorders. In this study, we discover Alzheimer’s disease (AD)-specific single-nucleotide variants (SNVs) and abnormal exon splicing of phospholipase c gamma-1 (PLCγ1) gene, using genome-wide association study (GWAS) and a deep learning-based exon splicing prediction tool. GWAS revealed that the identified single-nucleotide variations were mainly distributed in the H3K27ac-enriched region of PLCγ1 gene body during brain development in an AD mouse model. A deep learning analysis, trained with human genome sequences, predicted 14 splicing sites in human PLCγ1 gene, and one of these completely matched with an SNV in exon 27 of PLCγ1 gene in an AD mouse model. In particular, the SNV in exon 27 of PLCγ1 gene is associated with abnormal splicing during messenger RNA maturation. Taken together, our findings suggest that this approach, which combines in silico and deep learning-based analyses, has potential for identifying the clinical utility of critical SNVs in AD prediction.