Splicing Patterns Research Articles

The pattern of alternative splicing and DNA methylation alteration and their interaction in linseed (Linum usitatissimum L.) response to repeated drought stresses

BackgroundDrought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions.ResultsWe analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3’ splice site (Alt3’SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes.ConclusionOur study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.

Transcriptomic Analysis Reveals mRNA and Alternative Splicing Events in Ovine Skeletal Muscle Satellite Cells during Proliferation and Differentiation

Skeletal muscle satellite cells (SMSCs), which are highly multifunctional muscle-derived stem cells, play an essential role in myogenesis and regeneration. Here, the transcriptional profile of SMSCs during proliferation and differentiation were constructed using the RNA-Seq method. A total of 1954 differentially expressed genes (DEGs) and 1092 differentially alternative splicing genes (DAGs) were identified including 1288 upregulated genes as well as 666 downregulated genes. GO and KEGG analyses showed that the DEGs and DAGs were enriched in the MAPK (mitogen-activated protein kinase) signaling pathway, the PI3K-Akt (phosphatidylinositol-tris-phosphate kinase 3/protein kinase B) signaling pathway, the Wnt signaling pathway, and the Ras signaling pathway. In total, 1479 alternative splice events (AS) were also identified during SMSC proliferation and differentiation. Among them, a unique AS event was the major per-mRNA splicing type, and SE was the predominant splicing pattern. Furthermore, transcription factors with AS were scanned during SMSC differentiation such as myocyte enhancer factor-2C (MEF2C) and the nuclear receptor subfamily 4 group A member 2 (NR4A2). Our results imply that MEF2C and NR4A2 can interact, and we speculate that NR4A2 and MEF2C might regulate the myogenesis of ovine SMSCs through interaction. Together, our study provides useful information on the transcriptional regulation of SMSCs during proliferation and differentiation at the transcriptional level, and provides a valuable resource for understanding the molecular mechanism of myogenesis and muscle development.

Evaluation of the clinical significance of global mRNA alternative splicing in patients with acute myeloid leukemia.

Aberrant alternative splicing (AS) is involved in leukemogenesis. This study explored the clinical impact of alterations in global AS patterns in 341 patients with acute myeloid leukemia (AML) newly diagnosed at the National Taiwan University Hospital and validated it using The Cancer Genome Atlas (TCGA) cohort. While studying normal cord blood CD34+ /CD38- cells, we found that AML cells exhibited significantly different global splicing patterns. AML with mutated TP53 had a particularly high degree of genome-wide aberrations in the splicing patterns. Aberrance in the global splicing pattern was an independent unfavorable prognostic factor affecting the overall survival of patients with AML receiving standard intensive chemotherapy. The integration of global splicing patterns into the 2022 European LeukemiaNet risk classification could stratify AML patients into four groups with distinct prognoses in both our experimental and TCGA cohorts. We further identified four genes with AS alterations that harbored prognostic significance in both of these cohorts. Moreover, these survival-associated AS events are involved in several important cellular processes that might be associated with poor response to intensive chemotherapy. In summary, our study demonstrated the clinical and biological implications of differential global splicing patterns in AML patients. Further studies with larger prospective cohorts are required to confirm these findings.

Genome-wide analysis of bromodomain gene family in Arabidopsis and rice.

The bromodomain-containing proteins (BRD-proteins) belongs to family of 'epigenetic mark readers', integral to epigenetic regulation. The BRD-members contain a conserved 'bromodomain' (BRD/BRD-fold: interacts with acetylated-lysine in histones), and several additional domains, making them structurally/functionally diverse. Like animals, plants also contain multiple Brd-homologs, however the extent of their diversity and impact of molecular events (genomic duplications, alternative splicing, AS) therein, is relatively less explored. The present genome-wide analysis of Brd-gene families of Arabidopsis thaliana and Oryza sativa showed extensive diversity in structure of genes/proteins, regulatory elements, expression pattern, domains/motifs, and the bromodomain (w.r.t. length, sequence, location) among the Brd-members. Orthology analysis identified thirteen ortholog groups (OGs), three paralog groups (PGs) and four singleton members (STs). While more than 40% Brd-genes were affected by genomic duplication events in both plants, AS-events affected 60% A. thaliana and 41% O. sativa genes. These molecular events affected various regions (promoters, untranslated regions, exons) of different Brd-members with potential impact on expression and/or structure-function characteristics. RNA-Seq data analysis indicated differences in tissue-specificity and stress response of Brd-members. Analysis by RT-qPCR revealed differential abundance and salt stress response of duplicate A. thaliana and O. sativa Brd-genes. Further analysis of AtBrd gene, AtBrdPG1b showed salinity-induced modulation of splicing pattern. Bromodomain (BRD)-region based phylogenetic analysis placed the A. thaliana and O. sativa homologs into clusters/sub-clusters, mostly consistent with ortholog/paralog groups. The bromodomain-region displayed several conserved signatures in key BRD-fold elements (α-helices, loops), along with variations (1-20 sites) and indels among the BRD-duplicates. Homology modeling and superposition identified structural variations in BRD-folds of divergent and duplicate BRD-members, which might affect their interaction with the chromatin histones, and associated functions. The study also showed contribution of various duplication events in Brd-gene family expansion among diverse plants, including several monocot and dicot plant species.

Computational analysis of crosstalk between transcriptional regulators and RNA-binding proteins suggests mutual regulation of polycomb proteins and SRSF1 influencing adult hippocampal neurogenesis

BackgroundAdult hippocampal neurogenesis (AHN) is a clinically significant neural phenomenon. Understanding its molecular regulation would be important. In this regard, most studies have focused on transcriptional regulators (TRs), epigenetic modifiers, or non-coding RNAs. RNA-binding proteins (RBPs) have emerged as dominant molecular regulators. It would be significant to understand the potential cross-talk between RBPs and TRs, which could influence AHN.MethodsThe present study employed computational analyses to identify RBPs and TRs regulating AHN, followed by the analysis of their interaction networks and detection of hub proteins. Next, the potential mutual regulation of hub TRs and RBPs was analyzed. Additionally, hippocampal genes differentially expressed upon exercise were analyzed for potential regulation by the identified TRs and RBPs.Results105 TRs and 26 RBPs were found to influence AHN, which could also form interactive networks. Polycomb complex proteins were among the TR network hubs, while HNRNP and SRSF family members were among the hub RBPs. Further, the polycomb complex proteins and SRSF1 could have a mutual regulatory relationship, suggesting a cross-talk between epigenetic/transcriptional and post-transcriptional regulatory pathways. A number of exercise-induced hippocampal genes were also found to be potential targets of the identified TRs and RBPs.ConclusionSRSF1 may influence post-transcriptional stability, localization, and alternative splicing patterns of polycomb complex transcripts, and the polycomb proteins may in turn epigenetically influence the SRSF1. Further experimental validation of these regulatory loops/networks could provide novel insights into the molecular regulation of AHN, and unravel new targets for disease-treatment.

Cohesin regulates alternative splicing.

Cohesin, a trimeric complex that establishes sister chromatid cohesion, has additional roles in chromatin organization and transcription. We report that among those roles is the regulation of alternative splicing through direct interactions and in situ colocalization with splicing factors. Degradation of cohesin results in marked changes in splicing, independent of its effects on transcription. Introduction of a single cohesin point mutation in embryonic stem cells alters splicing patterns, demonstrating causality. In primary human acute myeloid leukemia, mutations in cohesin are highly correlated with distinct patterns of alternative splicing. Cohesin also directly interacts with BRD4, another splicing regulator, to generate a pattern of splicing that is distinct from either factor alone, documenting their functional interaction. These findings identify a role for cohesin in regulating alternative splicing in both normal and leukemic cells and provide insights into the role of cohesin mutations in human disease.

SWATH-MS-based proteogenomic analysis reveals the involvement of alternative splicing in poplar upon lead stress.

Alternative splicing (AS) regulates gene expression and increases proteomic diversity for the fine tuning of stress responses in plants, but the exact mechanism through which AS functions in plant stress responses is not thoroughly understood. Here, we investigated how AS functions in poplar (Populus trichocarpa), a popular plant for bioremediation, in response to lead (Pb) stress. Using a proteogenomic analysis, we determine that Pb stress induced alterations in AS patterns that are characterized by an increased use of nonconventional splice sites and a higher abundance of Pb-responsive splicing factors (SFs) associated with Pb-responsive transcription factors. A strong Pb(II)-inducible chaperone protein, PtHSP70, that undergoes AS was further characterized. Overexpression of its two spliced isoforms, PtHSP70-AS1 and PtHSP70-AS2, in poplar and Arabidopsis significantly enhances the tolerance to Pb. Further characterization shows that both isoforms can directly bind to Pb(II), and PtHSP70-AS2 exhibits 10-fold higher binding capacities and a greater increase in expression under Pb stress, thereby reducing cellular toxicity through Pb(II) extrusion and conferring Pb tolerance. AS of PtHSP70 is found to be regulated by PtU1-70K, a Pb(II)-inducible core SF involved in 5'-splice site recognition. Because the same splicing pattern is also found in HSP70 orthologs in other plant species, AS of HSP70 may be a common regulatory mechanism to cope with Pb(II) toxicity. Overall, we have revealed a novel post-transcriptional machinery that mediates heavy metal tolerance in diverse plant species. Our findings offer new molecular targets and bioengineering strategies for phytoremediation and provide new insight for future directions in AS research.

Farnesyl diphosphate synthase promotes cell proliferation by regulating gene expression and alternative splicing profiles in HeLa cells.

Farnesyl diphosphate synthase (FDPS), an essential enzyme involved in the mevalonate pathway, is implicated in various diseases, including multiple types of cancer. As an RNA-binding protein (RBP), FDPS is also involved in transcriptional and post-transcriptional regulation. However, to the best of our knowledge, transcriptome-wide targets of FDPS still remain unknown. In the present study, FDPS expression patterns in pan-cancer were analyzed. In addition, it was investigated how FDPS overexpression (FDPS-OE) regulates the transcriptome in HeLa cells. FDPS-OE increased the proliferation rate in HeLa cells by MTT assay. Using transcriptome-wide high throughput sequencing and bioinformatics analysis, it was found that FDPS upregulated the expression levels of genes enriched in cell proliferation and extracellular matrix organization, including the laminin subunit γ2, interferon-induced proteins with tetratricopeptide repeats 2 and matrix metallopeptidase 19 genes. According to alternative splicing (AS) analysis, FDPS modulated the splicing patterns of the bone morphogenic protein 1, semaphorin 4D, annexin A2 and sirtuin 2 genes, which are enriched in the cell cycle and DNA repair, and are related to cell proliferation. To corroborate the FDPS-regulated transcriptome findings, FDPS was overexpressed in human osteosarcoma cells. Differentially expressed genes and regulated AS genes in the cells were both validated by reverse transcription-quantitative PCR. The results suggested that, as an emerging RBP, FDPS may serve an important role in transcriptome profiles by altering gene expression and regulating AS. FDPS also affected the cell proliferation rate. These findings broaden the understanding of the molecular functions of FDPS, and the potential of FDPS as a target in therapy should be investigated.

Comparative transcriptome responses of leaf and root tissues to salt stress in wheat strains with different salinity tolerances.

Background: Salinity stress is a major adverse environmental factor that can limit crop yield and restrict normal land use. The selection of salt-tolerant strains and elucidation of the underlying mechanisms by plant breeding scientists are urgently needed to increase agricultural production in arid and semi-arid regions. Results: In this study, we selected the salt-tolerant wheat (Triticum aestivum) strain ST9644 as a model to study differences in expression patterns between salt-tolerant and salt-sensitive strains. High-throughput RNA sequencing resulted in more than 359.10Gb of clean data from 54 samples, with an average of 6.65Gb per sample. Compared to the IWGSC reference annotation, we identified 50,096 new genes, 32,923 of which have functional annotations. Comparisons of abundances between salt-tolerant and salt-sensitive strains revealed 3,755, 5,504, and 4,344 genes that were differentially expressed at 0, 6, and 24h, respectively, in root tissue under salt stress. KEGG pathway analysis of these genes showed that they were enriched for phenylpropanoid biosynthesis (ko00940), cysteine and methionine metabolism (ko00270), and glutathione metabolism (ko00480). We also applied weighted gene co-expression network analysis (WGCNA) analysis to determine the time course of root tissue response to salt stress and found that the acute response lasts >6h and ends before 12h. We also identified key alternative splicing factors showing different splicing patterns in salt-sensitive and salt-tolerant strains; however, only few of them were differentially expressed in the two groups. Conclusion: Our results offer a better understanding of wheat salt tolerance and improve wheat breeding.

Pitfalls of using sequence databases for heterologous expression studies - a technical review.

Synthesis of DNA fragments based on gene sequences that are available in public resources has become an efficient and affordable method that has gradually replaced traditional cloning efforts such as PCR cloning from cDNA. However, database entries based on genome sequencing results are prone to errors which can lead to false sequence information and, ultimately, errors in functional characterisation of proteins such as ion channels and transporters in heterologous expression systems. We have identified five common problems that repeatedly appear in public resources: (1) Not every gene has yet been annotated; (2) not all gene annotations are necessarily correct; (3) transcripts may contain automated corrections; (4) there are mismatches between gene, mRNA and protein sequences; and (5) splicing patterns often lack experimental validation. This technical review highlights and provides a strategy to bypass these issues in order to avoid critical mistakes that could impact future studies of any gene/protein of interest in heterologous expression systems.

One pipeline to predict them all? On the prediction of alternative splicing from RNA-Seq data

RNA-Seq has become the standard approach to quantify and compare gene expression and alternative splicing in different conditions. In many cases the limiting factor is not the sequencing itself but the bioinformatic analysis. A variety of software tools exist that predict alternative splicing patterns from RNA-Seq data, but surprisingly, a systematic comparison of the predictions obtained from different pipelines has not been performed. Here we compare results from frequently used bioinformatic tools using a high-quality RNA-Seq dataset. We show that there is little overlap in the splicing changes predicted by different tools and that GO-term analysis of the splicing changes predicted by the individual targets yields very different results. Validation of bioinformatic predictions by RT-PCR suggest a high number of false positives in the splicing changes predicated by each pipeline, which probably dominates GO-term analysis. The validation rate is strongly increased for targets predicted by several tools, offering a strategy to reduce false positives. Based on these results we offer some guidelines that may contribute to make alternative splicing predictions more reliable and may thus increase the impact of conclusions drawn from RNA-Seq studies. Furthermore, we created rmappet, a nextflow pipeline that performs alternative splicing analysis using rMATS and Whippet with subsequent overlapping of the results, enabling robust splicing analysis with only one command (https://github.com/didrikolofsson/rmappet/).

Subgenome-dominant expression and alternative splicing in response to Sclerotinia infection in polyploid Brassica napus and progenitors.

Polyploidy has played an extensive role in the evolution of flowering plants. Allopolyploids, with subgenomes containing duplicated gene pairs called homeologs, can show rapid transcriptome changes including novel alternative splicing (AS) patterns. The extent to which abiotic stress modulates AS of homeologs is a nascent topic in polyploidy research. We subjected both resynthesized and natural lines of polyploid Brassica napus, along with the progenitors Brassica rapa and Brassica oleracea, to infection with the fungal pathogen Sclerotinia sclerotiorum. RNA-sequencing analyses revealed widespread divergence between polyploid subgenomes in both gene expression and AS patterns. Resynthesized B. napus displayed significantly more A and C subgenome biased homeologs under pathogen infection than during uninfected growth. Differential AS (DAS) in response to infection was highest in natural B. napus (12 709 DAS events) and lower in resynthesized B. napus (8863 DAS events). Natural B. napus had more upregulated events and fewer downregulated events. There was a global expression bias towards the B. oleracea-derived (C) subgenome in both resynthesized and natural B. napus, enhanced by widespread non-parental downregulation of the B. rapa-derived (A) homeolog. In the resynthesized B. napus, this resulted in a disproportionate C subgenome contribution to the pathogen defense response, characterized by biases in both transcript expression levels and the proportion of induced genes. Our results elucidate the complex ways in which Sclerotinia infection affects expression and AS of homeologous genes in resynthesized and natural B. napus.

Alzheimer's Disease-Associated Alternative Splicing of CD33 Is Regulated by the HNRNPA Family Proteins.

Genetic variations of CD33 have been implicated as a susceptibility factor of Alzheimer's disease (AD). A polymorphism on exon 2 of CD33, rs12459419, affects the alternative splicing of this exon. The minor allele is associated with a reduced risk of AD and promotes the skipping of exon 2 to produce a shorter CD33 isoform lacking the extracellular ligand-binding domain, leading to decreased suppressive signaling on microglial activity. Therefore, factors that regulate the splicing of exon 2 may alter the disease-associated properties of CD33. Herein, we sought to identify the regulatory proteins of CD33 splicing. Using a panel of RNA-binding proteins and a human CD33 minigene, we found that exon 2 skipping of CD33 was promoted by HNRNPA1. Although the knockdown of HNRNPA1 alone did not reduce exon 2 skipping, simultaneous knockdown of HNRNPA1 together with that of HNRNPA2B1 and HNRNPA3 promoted exon 2 inclusion, suggesting functional redundancy among HNRNPA proteins. Similar redundant regulation by HNRNPA proteins was observed in endogenous CD33 of THP-1 and human microglia-like cells. Although mouse Cd33 showed a unique splicing pattern of exon 2, we confirmed that HNRNPA1 promoted the skipping of this exon. Collectively, our results revealed novel regulatory relationships between CD33 and HNRNPA proteins.

An iterative gene-editing strategy broadens eIF4E1 genetic diversity in Solanum lycopersicum and generates resistance to multiple potyvirus isolates.

Resistance to potyviruses in plants has been largely provided by the selection of natural variant alleles of eukaryotic translation initiation factors (eIF) 4E in many crops. However, the sources of such variability for breeding can be limited for certain crop species, while new virus isolates continue to emerge. Different methods of mutagenesis have been applied to inactivate the eIF4E genes to generate virus resistance, but with limited success due to the physiological importance of translation factors and their redundancy. Here, we employed genome editing approaches at the base level to induce non-synonymous mutations in the eIF4E1 gene and create genetic diversity in cherry tomato (Solanum lycopersicum var. cerasiforme). We sequentially edited the genomic sequences coding for two regions of eIF4E1 protein, located around the cap-binding pocket and known to be important for susceptibility to potyviruses. We show that the editing of only one of the two regions, by gene knock-in and base editing, respectively, is not sufficient to provide resistance. However, combining amino acid mutations in both regions resulted in resistance to multiple potyviruses without affecting the functionality in translation initiation. Meanwhile, we report that extensive base editing in exonic region can alter RNA splicing pattern, resulting in gene knockout. Altogether our work demonstrates that precision editing allows to design plant factors based on the knowledge on evolutionarily selected alleles and enlarge the gene pool to potentially provide advantageous phenotypes such as pathogen resistance.

Decoding allelic diversity, transcript variants and transcriptional complexity of CENH3 gene in Brassica oleracea var. botrytis.

Histone proteins play a critical role in the primary organization of nucleosomes, which is the fundamental unit of chromatin. Among the fivetypes of the histones, histone H3 has multiple variants, and the number differs among thespecies. Amongst histone H3 variants, centromeric histone H3 (CENH3) is crucial for centromere identification and proper chromosomal segregation during cell division. In the present study, we have identified 17 putative histone H3 genes of Brassica oleracea. Furthermore, we have done a detailed characterization of the CENH3 gene of B. oleracea. We showed that a single CENH3 gene exhibits allelic diversity with at least two alleles and alternative splicing pattern. Also, we have identified a CENH3 gene-specific co-dominant cleaved amplified polymorphic sequence marker SNP34(A/C) to distinguish CENH3 alleles and follow their expression in leaf and flower tissues. The gene structure analysis of theCENH3 gene revealed the conserved 5'-CAGCAG-3' sequence at the intron 3-exon 4 junction in B. oleracea, which serves as an alternative splicing site with one-codon (alanine) addition/deletion. However, this one-codon alternative splicing feature is not conserved in the CENH3 genes of wild allied Brassica species. Our finding suggests that transcriptional complexity and alternative splicing might play a key role in the transcriptional regulation and function of the CENH3 gene in B. oleracea. Altogether, data generated from the present study can serve as a primary information resource and can be used to engineer CENH3 gene towards developing haploid inducer lines in B. oleracea.

The Integrative Studies on the Functional A-to-I RNA Editing Events in Human Cancers

Adenosine-to-inosine (A-to-I) RNA editing, constituting nearly 90% of all RNA editing events in humans, has been reported to contribute to the tumorigenesis in diverse cancers. However, the comprehensive map for functional A-to-I RNA editing events in cancers is still insufficient. To fill this gap, we systematically and intensively analyzed multiple tumorigenic mechanisms of A-to-I RNA editing events in samples across 33 cancer types from The Cancer Genome Atlas. For individual candidate among ∼ 1,500,000 quantified RNA editing events, we performed diverse types of downstream functional annotations. Finally, we identified 24,236 potentially functional A-to-I RNA editing events, including the cases in APOL1, IGFBP3, GRIA2, BLCAP, and miR-589-3p. These events might play crucial roles in the scenarios of tumorigenesis, due to their tumor-related editing frequencies or probable effects on altered expression profiles, protein functions, splicing patterns, and microRNA regulations of tumor genes. Our functional A-to-I RNA editing events (https://ccsm.uth.edu/CAeditome/) will help better understand the cancer pathology from the A-to-I RNA editing aspect.

Identification of tumor antigens and immune landscapes for bladder urothelial carcinoma mRNA vaccine.

Bladder urothelial carcinoma (BLCA) is associated with high mortality and recurrence. Although mRNA-based vaccines are promising treatment strategies for combating multiple solid cancers, their efficacy against BLCA remains unclear. We aimed to identify potential effective antigens of BLCA for the development of mRNA-based vaccines and screen for immune clusters to select appropriate candidates for vaccination. Gene expression microarray data and clinical information were retrieved from The Cancer Genome Atlas and GSE32894, respectively. The mRNA splicing patterns were obtained from the SpliceSeq portal. The cBioPortal for Cancer Genomics was used to visualize genetic alteration profiles. Furthermore, nonsense-mediated mRNA decay (NMD) analysis, correlation analysis, consensus clustering analysis, immune cell infiltration analysis, and weighted co-expression network analysis were conducted. Six upregulated and mutated tumor antigens related to NMD, and infiltration of APCs were identified in patients with BLCA, including HP1BP3, OSBPL9, SSH3, ZCCHC8, FANCI, and EIF4A2. The patients were subdivided into two immune clusters (IC1 and IC2) with distinct clinical, cellular and molecular features. Patients in IC1 represented immunologically 'hot' phenotypes, whereas those in IC2 represented immunologically 'cold' phenotypes. Moreover, the survival rate was better in IC2 than in IC1, and the immune landscape of BLCA indicated significant inter-patient heterogeneity. Finally, CALD1, TGFB3, and ANXA6 were identified as key genes of BLCA through WGCNA analysis, and their mRNA expression levels were measured using qRT-PCR. HP1BP3, OSBPL9, SSH3, ZCCHC8, FANCI, and EIF4A2 were identified as potential antigens for developing mRNA-based vaccines against BLCA, and patients in IC2 might benefit more from vaccination.

Exon definitive regions for MPC1 microexon splicing and its usage for splicing modulation

Alternative splicing of microexons (3-30 base pairs [bp]) is involved in important biological processes in brain development and human cancers. However, understanding a splicing process of non-3x bp microexons is scarce. We showed that 4bp microexon of mitochondrial pyruvate carrier1 (MPC1) is constitutively included in mRNA. Based on our studies with minigene and exon island constructs, we found the strong exon definition region in the proximal introns bordering MPC1 microexon. Ultimately, we defined a nucleotide fragment from the 3'ss 67bp of MPC1 microexon to the 5'ss consensus sequence, as a core exon island, which can concatenate its microexon and neighboring exons by splicing. Furthermore, we showed that insertion of the core exon island into a target exon or intron induced skip the target exon or enhance the splicing of an adjacent exon, respectively. Collectively, we suggest that the exon island derived from MPC1 microexon modifies genuine splicing patterns depending on its position, thereby providing insights on strategies for splicing-mediated gene correction.

NR3C1 overexpression regulates the expression and alternative splicing of inflammation-associated genes involved in PTSD

NR3C1-encoding glucocorticoid receptors have dual roles as RNA-binding protein and transcription factor. Recent studies revealed that NR3C1 might play an important role in the pathogenesis of PTSD (Post-traumatic stress disorder). However, its molecular mechanism remained unclear. In the present study, a neuronal cell model was constructed by transfecting a NR3C1-overexpressing plasmid pIRES-hrGFP-1a-NR3C1 or empty vector into HT22 cells. The changes in global transcription levels and alternative splicing events in HT22 cells after NR3C1 overexpression were analyzed by RNA sequencing. Compared with the empty vector control, the expression of inflammatory factors were differentially regulated by NR3C1, including genes involved in chemokine signal pathway, PI3K-Akt signal pathway, cytokine receptor interaction, neural ligand-receptor interaction and so on. In addition, NR3C1 regulated the alternative splicing of many genes involved in immune response, axon formation, stress response and inflammation. This study was the first to perform a transcriptome analysis of differential gene expression and alternative splicing in a NR3C1-overexpressing HT22 cell model. Our results suggested that NR3C1 could manipulate the expression of inflammatory transcription factors and their alternative splicing patterns, subsequently affecting the expression of downstream targets, may be leading to the onset of PTSD. This study will provide new insights in the NR3C1-mediated gene regulation in relation to PTSD.

Minigene Splicing Assays and Long-Read Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital Aniridia.

PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis.