Alternative splicing (AS) is a substantial contributor to the high complexity of transcriptomes in multicellular eukaryotes. Fast chemical reprogramming (FCR) system is an innovative approach that facilitates the rapid transition of somatic cells into induced pluripotent stem cells (iPSCs).

In this study, we used the FCR system to delve into the dynamics of AS during cell fate transition. The trajectory of FCR, as characterized by gene expression profiles, consistently aligned with that observed in AS patterns, revealing a complex interplay between AS and gene expression regulation. Additionally, we discovered that the exon exclusion events were more prevalent than the exon inclusion events, indicating a predominant mode of splicing regulation during FCR. Compared to transcription factor-induced reprogramming (TFR), FCR showed a distinct AS pattern, underscoring the unique regulatory mechanisms governing AS in each reprogramming system. Further investigation uncovered polypyrimidine tract-binding protein 3 (Ptbp3) as an important splicing factor, possibly participating in epigenetic regulation in late stage of FCR by affecting AS of epigenetic regulators. Moreover, we found an abundance of intron retention events caused by decrease in spliceosome activity, potentially contributing to the downregulation of key diapause-related genes in the middle and late stages of FCR.

This research provided a comprehensive characterization of AS during FCR, highlighting the pivotal roles of AS in regulating cell fate transitions. Our findings advanced the understanding of the molecular mechanisms governing cell fate decisions and offered new insights into the potential of FCR for regenerative medicine and therapeutic applications.

Hepatitis B virus (HBV) is the primary etiological agent of chronic hepatitis B (CHB) infection, posing a serious threat to human health. The pregenomic RNA (pgRNA) of HBV is the template for HBV reverse transcription, and the epsilon stem-loop (ε) is required for nucleocapsid assembly. The host factor serine/arginine (SR)-rich splicing factor 7 (SRSF7) is a splicing regulator and RNA-binding protein that was involved in regulating viral RNA splicing and export from the nucleus during the viral life cycle, but its biological function and regulatory mechanisms in HBV remain unclear. In this study, SRSF7 was found to promote HBV replication and upregulate HBV RNA levels through knockdown or overexpression of SRSF7 in different cell lines using the HBV replication model. Surprisingly, we found that SRSF7 enhanced HBV RNA stability at the post-transcriptional level, rather than regulating its splicing. We further demonstrated that SRSF7 could bind to pgRNA; deletion of the bulge and loop structures of the ε element significantly reduced its binding capacity. In addition, we confirmed that SRSF7 supports HBV replication in CHB patients. Our study suggests that the host factor SRSF7 promotes HBV replication, which provides new perspectives for further elucidation of HBV-host interactions and the development of host-targeted anti-HBV drugs.

Alternative splicing (AS) is a fundamental mechanism for enhancing transcriptome diversity and regulating gene expression, crucial for various cellular processes and the development of complex traits. This review examines the role of AS in metabolic disorders, including obesity, weight loss, dyslipidemias, and metabolic syndrome. We explore the molecular mechanisms underlying AS regulation, focusing on the interplay between cis-acting elements and trans-acting factors, and the influence of RNA-binding proteins (RBPs). Advances in high-throughput sequencing and bioinformatics have unveiled the extensive landscape of AS events across different tissues and conditions, highlighting the importance of tissue-specific splicing in metabolic regulation. We discuss the impact of genetic variants on AS, with a particular emphasis on splicing quantitative trait loci (sQTLs) and their association with cardiometabolic traits. The review also covers the regulation of spliceosome components by phosphorylation, the role of m6A modification in AS, and the interaction between transcription and splicing. Additionally, we address the clinical relevance of AS, illustrating how splicing misregulation contributes to metabolic diseases and the potential for therapeutic interventions targeting splicing mechanisms. This comprehensive overview underscores the significance of AS in metabolic health and disease, advocating for further research to harness its therapeutic potential.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has become the leading cause of chronic liver disease worldwide, with fibrosis recognized as the main prognostic factor and therapeutic target. While early-stage fibrosis is reversible, advanced fibrosis poses a significant clinical challenge due to limited treatment options, highlighting the need for innovative management strategies. Recent studies have shown that alternative pre-mRNA splicing, a critical mechanism regulating gene expression and protein diversity, plays a fundamental role in the pathogenesis of MAFLD and associated fibrosis. Understanding the complex relationship between alternative splicing and fibrosis progression in MAFLD could pave the way for novel therapeutic approaches and improve clinical outcomes. In this review, we describe the intricate mechanisms of alternative splicing in fibrosis associated with MAFLD. Specifically, we explored the pivotal of splicing factors, and RNA-binding proteins, highlighting their critical interactions with metabolic and epigenetic regulators. Furthermore, we provide an overview of the latest advancements in splicing-based therapeutic strategies and biomarker development. Particular emphasis is placed on the potential application of antisense oligonucleotides for rectifying splicing anomalies, thereby laying the foundation for precision medicine approaches in the treatment of MAFLD-associated fibrosis.

Understanding the molecular mechanisms of the malarial parasites in hosts is crucial for developing effective treatments. Epitranscriptomic research on pathogens has unveiled the significance of RNA methylation in gene regulation and pathogenesis. This is the first report investigating methylation signatures and alternative splicing events using Nanopore Direct RNA Sequencing to single-base resolution in Plasmodium falciparum and Plasmodium vivax clinical isolates with hepatic dysfunction complications.

Direct RNA Sequencing using Nanopore from clinical isolates of P. falciparum and P. vivax showing hepatic dysfunction manifestation was performed. Subsequently, transcriptome reconstruction using FLAIR and transcript classification using SQANTI3, followed by methylation detection using CHEUI and m6Anet to identify N6-methyladenosine (m6A) and 5-methylcytosine (m5C) methylation signatures, was done. The alternative splicing events from both the datasets were documented.

The reference genome of Plasmodium reports > 5000 genes out of which ~ 50% was identified as expressed in the two sequenced isolates, including novel isoforms and intergenic transcripts, highlighting extensive transcriptome diversity. The distinct RNA methylation profiles of m6A and m5C from the expressed transcripts were observed in sense, Natural Antisense Transcripts (NATs) and intergenic categories hinting at species-specific regulatory mechanisms. Dual modification events were observed in a significant number of transcripts in both the parasites. Modified transcripts originating from apicoplast and mitochondrial genomes have also been detected. These modifications are unevenly present in the annotated regions of the mRNA, potentially influencing mRNA export and translation. Several splicing events were observed, with alternative 3' and 5' end splicing predominating in the datasets suggesting differences in translational kinetics and possible protein characteristics in these disease conditions.

The data shows the presence of modified sense, NATs and alternatively spliced transcripts. These phenomena together suggest the presence of multiple regulatory layers which decides the post-translational proteome of the parasites in particular disease conditions. Studies like these will help to decipher the post-translational environments of malaria parasites in vivo and elucidate their inherent proteome plasticity, thus allowing the conceptualization of novel strategies for interventions.

Ninein is a multifunctional protein involved in microtubule (MT) organization and dynein/dynactin complex recruitment and activation. Several isoforms of ninein have been identified in various tissues, however, their relative contribution(s) are not clear. Here, we identify two ninein isoforms in mouse macrophages with distinct C-termini and disproportionate expression levels; a canonical ninein (nineinCAN) isoform and ninein isoform 2 (nineinISO2). Analysis of ninein pre-mRNA exon-intron boundaries revealed that nineinISO2 transcript is likely generated by two alternative splicing site selection events predicted to result in a distinct 3D structure compared to nineinCAN. We used selective and total protein knockdown experiments to assess the intracellular and functional roles of ninein in macrophages. Live cell imaging analyses of macrophages implicated both isoforms in regulating cell proliferation. MT regrowth following nocodazole depolymerization showed that both isoforms contributed to MT nucleation and structural integrity of the centrosome, as cells lacking nineinCAN or nineinISO2 contained multiple ectopic γ-tubulin foci. However, nineinCAN, but not nineinISO2, was important for the separation of duplicated centrosomes during cell division. Despite a requirement of both ninein isoforms to recruit dynein/dynactin to the centrosome, only nineinCAN was required for Golgi positioning and morphology, dynein-dependent events. We additionally found that nineinISO2 was the primary isoform required for F-actin recruitment during the internalization of IgG-opsonized particles. Our study indicates that alternative splicing promotes both redundant and differential activities for ninein in MT organization, organelle positioning, and macrophage function.

The epigenetic regulation of gene expression through the covalent modification of histones is crucial for developing germline cells. To study the regulatory role of alternative splicing (AS) of euchromatic histone lysine methyltransferase 2 (EHMT2/G9A) in spermatogenesis in Mongolian horses, this study first examines the localization of the EHMT2 gene in testicular support cells and then predicts the higher-order structures of sequences with and without AS. Two types of lentiviral vectors for overexpression were subsequently constructed for the EHMT2 gene, one with AS and one without, to infect support cells. The proliferation and activity of infected cells were measured using CCK8, and the differential expression of spermatogenesis-related genes in the two types of support cells was analyzed via qRT-PCR. We analyzed the expression of EHMT2 by immunofluorescence staining. EHMT2 was expressed in the nuclei of Sertoli cells. The expression of spermatogenesis-related genes was measured in the two types of cells. The results reveal that the expression levels of the FSH, Stra8, CCNB2, CDC27, NRG1, PPP2R5C, CCNB2, and YWHAZ genes in the AS group were greater than those in the control group. These results indicate that AS events in EHMT2 affect gene expression and thus affect spermatogenesis.

RBM39 is an essential component of the spliceosome, playing a critical role in maintaining mRNA integrity. Its depletion significantly exacerbates RNA splicing defects and demonstrates potent anticancer activity. To identify key effectors following RBM39 depletion, we employed a multiomics approach to directly compare two structurally distinct compounds, CB039 and Indisulam. Through proteomic analysis, RNA sequencing, and DepMap dependency assessment, CEP192 emerged as a crucial gene, exhibiting dependency in 96% of the 1,100 analyzed cancer cell lines. In eight cancer cell lines, treatment with both CB039 and Indisulam consistently induced CEP192 exon 42 skipping and reduced CEP192 protein levels. Mechanistically, either CB039 treatment or RNA interference-mediated CEP192 knockdown led to a significant increase in spindle disorganization, as well as chromosome condensation and failed segregation. In conclusion, our characterization of the downstream effects of RBM39 depletion provides novel insights into the therapeutic potential of RBM39 degraders.

Alternative splicing commonly generates unproductive mRNA transcripts that harbor premature termination codons 1 , leading to their degradation by nonsense-mediated decay (NMD). These events reduce overall protein expression levels of affected genes, potentially contributing to gene regulation and disease mechanisms. Here, we present LeafCutter2, which enables identification and quantification of unproductive splicing from short-read RNA-seq data. LeafCutter2 requires minimal gene annotations (start and stop codons) to annotate NMD-inducing splicing events, and identifies differential unproductive splicing between groups, providing insights into its contributions to differential gene expression. Moreover, LeafCutter2 enables mapping of unproductive splicing quantitative trait loci (u-sQTLs), which often colocalize with expression QTLs and GWAS loci. Applying LeafCutter2 to RNA-seq data across human and 6 non-human species, we uncovered a broad landscape of unproductive splicing, which varies widely across tissues. Strikingly, we observed a conserved developmental-stage-specific increase in unproductive splicing during testis maturation across all species. In Alzheimer's disease (AD), we analyzed RNA-seq data from the AD Functional Genomics consortium FunGen-xQTL project and identified unproductive splicing events in 18 AD risk genes, including TSPAN14 , PICALM , and CASS4 , likely mediating genetic effects on disease risk. We performed an integrative analysis using gene expression QTLs, protein expression QTLs, and AD GWAS data, showing that unproductive splicing provides unique regulatory insights beyond traditional approaches. Thus, LeafCutter2 represents a powerful tool for understanding the functional impact of alternative splicing on gene expression and disease mechanisms.

Circular RNA (circRNA) was first discovered in viruses in 1974; they are primarily formed through back splicing, where a downstream splice donor is joined to an upstream splice acceptor, resulting in a closed circRNA transcript. Under normal conditions, most circRNAs are stably expressed; however, in pathological conditions, circRNAs can play critical roles in the disease process of multiple myeloma (MM) through mechanisms such as competing endogenous RNAs (ceRNAs), regulation of transcription and splicing, affecting protein expression and localization, and even direct encoding of peptides. In recent years, there has been increasing interest in the role of circRNAs in MM and their regulatory functions during the disease process. Numerous studies have revealed that circRNAs are involved in the pathogenesis and prognosis of MM, aiding in the identification of reliable prognostic markers and potential therapeutic targets. Therefore, this review summarizes the structural characteristics of circRNAs, and their regulatory roles in MM, and introduces the latest advancements in understanding the novel functions of circRNAs in MM.

Preeclampsia is a pregnancy-specific hypertensive disorder and is associated with an increased postpartum risk of cardiovascular morbidity for both women and their offspring. Previous studies have indicated that cord blood endothelial colony-forming cells (ECFCs) are dysfunctional in preeclampsia. The specific mechanisms are not yet fully understood, but dysregulation of alternative splicing has been proposed as one of the pathogenic pathways. To identify specific targets of alternative splicing in fetal ECFCs, we performed transcriptome-wide differential splicing analyses between cord blood ECFCs from preeclamptic (n = 16) and normal pregnancies (n = 13). Selected splicing events were validated using fragment length analysis and Sanger sequencing. In silico transcriptome-wide differential splicing analysis identified a significantly increased abundance of the CADM1 isoform ENST00000542447 in the preeclamptic cohort (P = 0.002), which was confirmed by wet-lab validation. The deleted exon 8 harbors glycosylation sites known to mediate cell-cell adhesion. To investigate the functional impact of alternative splice variants, we induced an in vitro splice switch using antisense morpholino treatment and then monitored cellular effects using migration and angiogenesis assays in ECFCs from six normal pregnancies. The CADM1 exon 8 skipping converted the normal ECFCs to a preeclampsia-like state characterized by a decreased migration ability (PANOVA = 0.005) and decreased tubule length (PANOVA = 0.02). We propose aberrant splicing of CADM1 and the resulting changes in the adherence properties of ECFCs as a potential contributor to cardiovascular sequelae in the offspring of preeclamptic pregnancies.NEW & NOTEWORTHY We investigated differential splicing between normal and preeclamptic pregnancies in endothelial colony-forming cells (ECFCs) from cord blood. Transcriptome-wide analysis identified exon 8 skipping of CADM1 mRNA to be upregulated in ECFCs from women with preeclampsia. In vitro splice switching studies indicated that induction of this isoform decreases the cell migration and tubule formation abilities of fetal ECFCs. Our findings link a specific splice isoform of CADM1 to preeclampsia, with potential implications for vascular health in the offspring.

Triclosan (TCS) is an effective broad-spectrum antibacterial agent. TCS possesses a stable structure, can easily accumulate in the environment, and may have numerous negative impacts on human health. One organ particularly susceptible to TCS damage is the liver; however, the molecular mechanisms underlying TCS-induced liver damage remain unclear. A long-term TCS exposure model was established in C57BL/6 mice through maternal administration from gestation to postnatal 8-week-old. The offspring were randomly assigned to three groups (0, 50, and 100 mg/kg TCS) with six animals per group, ensuring an equal gender distribution (3 males and 3 females). The results showed that TCS-exposed mice exhibited serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase enzyme activities increased by 1.5-2 times when compared with vehicle-treated mice, along with features of liver fibrosis. In the LX-2 cell line, used as an in vitro model, TCS promoted proliferation and migration and induced the activation of hepatic stellate cells (HSCs). The level of pyruvate kinase M2 (PKM2) dimer increased by 200 % in LX-2 cells treated with TCS. PKM2 dimer overexpression stimulated HSC activation, whereas treatment with TEPP-46 (a PKM2 dimer inhibitor) significantly decreased the activation process. The expression of heterogeneous ribonucleoprotein particle A1 (hnRNPA1) was upregulated in the TCS treatment group and promoted the PKM2 expression. Moreover, disruption of the hnRNPA1/PKM2 axis reduced HSC proliferation and migration activated by TCS. Overall, our findings highlighted that TCS could cause liver fibrosis by stimulating the proliferation and migration of HSCs activated via the hnRNPA1/PKM2 axis, providing promising treatment options for TCS-related liver damage.

Heat stress significantly impacts global rice production, highlighting the critical need to understand the genetic basis of heat resistance in rice. U2AF (U2 snRNP auxiliary factor) is an essential splicing complex with critical roles in recognizing the 3'-splice site of precursor messenger RNAs (pre-mRNAs). The U2AF small subunit (U2AF35) can bind to the 3'-AG intron border and promote U2 snRNP binding to the branch-point sequences of introns through interaction with the U2AF large subunit (U2AF65). However, the functions of U2AF35 in plants are poorly understood. In this study, we discovered that the OsU2AF35a gene was vigorously induced by heat stress and could positively regulate rice thermotolerance during both the seedling and reproductive growth stages. OsU2AF35a interacts with OsU2AF65a within the nucleus, and both of them can form condensates through liquid-liquid phase separation (LLPS) following heat stress. The intrinsically disordered regions (IDR) are accountable for their LLPS. OsU2AF35a condensation is indispensable for thermotolerance. RNA-seq analysis disclosed that, subsequent to heat treatment, the expression levels of several genes associated with water deficiency and oxidative stress in osu2af35a-1 were markedly lower than those in ZH11. In accordance with this, OsU2AF35a is capable of positively regulating the oxidative stress resistance of rice. The pre-mRNAs of a considerable number of genes in the osu2af35a-1 mutant exhibited defective splicing, among which was the OsHSA32 gene. Knocking out OsHSA32 significantly reduced the thermotolerance of rice, while overexpressing OsHSA32 could partially rescue the heat sensitivity of osu2af35a-1. Together, our findings uncovered the essential role of OsU2AF35a in rice heat stress response through protein separation and regulating alternative pre-mRNA splicing.

Alternative splicing in genes associated with neuromuscular junction (NMJ) often compromises neuromuscular signal transmission and provokes pathological consequences. Muscle-specific receptor tyrosine kinase (MuSK) is an essential molecule in the NMJ. MUSK exon 10 encodes an important part of the frizzled-like cysteine-rich domain, which is necessary for Wnt-mediated acetylcholine receptors clustering at NMJ. MUSK exon 10 is alternatively spliced in humans but not in mice. We reported that humans acquired a unique exonic splicing silencer in exon 10 compared to mice, which promotes exon skipping coordinated by hnRNP C, YB-1, and hnRNP L. Here, we have dissected the underlying mechanisms of exon inclusion. We precisely characterized the exonic splicing enhancer (ESE) elements and determined the functional motifs. We demonstrated that SRSF6 and SRSF1 coordinately enhance exon inclusion through multiple functional motifs in the ESE. Remarkably, SRSF6 exerts a stronger effect than SRSF1, and SRSF6 alone can compensate the function of SRSF1. Interestingly, differentiated muscle reduces the expression of splicing suppressors, rather than enhancers, to generate a functional Wnt-sensitive MuSK isoform to promote neuromuscular signal transmission. Finally, we developed splice-switching antisense oligonucleotides, which could be used to selectively modulate the expression of MUSK isoforms toward a beneficial outcome for therapeutic intervention.

The genetic network of sex determination in the model organism Drosophila melanogaster was investigated in great detail. Such knowledge not only advances our understanding of the evolution and regulation of sexual dimorphism in insects, but also serves as a basis for developing genetic control strategies for pest species. In this study, we isolated the sex determination gene transformer (Dstra) from a global fruit pest, the spotted-wing Drosophila (Drosophila suzukii), and characterized its gene organization. By comparing the deduced protein sequence of Dstra with its orthologs from 22 species, we found that tra genes from Dipteran species are divergent. Our research demonstrated that Dstra undergoes sex-specific splicing, and we validated its developmental expression profile. We engineered a piggyBac-based transformation vector expressing the complete Dstra coding sequence under the control of the Tetracycline-Off system. Through germ-line transformation, we generated 4 independent transgenic lines, producing pseudo-females from chromosomal males in the absence of tetracycline. This observation indicated ectopic expression of Dstra, confirmed by the detection of female Dstra transcripts in transgenic males. The pseudo-females exhibited altered wing patterns, feminized abdomen, abnormal reproductive organs, and disrupted sexual behavior. Ectopic expression of Dstra affected the sex-specific splicing pattern of the downstream gene fruitless, but not doublesex. In conclusion, our study provides comprehensive genetic, morphological, and behavioral evidence that Dstra controls sexual development in D. suzukii. We discuss the potential applications of this research for genetic control strategies targeting Dstra or using its gene elements.

The alternative splicing of a gene results in distinct transcript isoforms that can result in proteins that differ in function. Alternative splicing processes are prevalent in the brain, have varying incidence across brain regions, and can present sexual dimorphism. Exposure to opiates and other substances of abuse can also alter the type and incidence of the splicing process and the relative abundance of the isoforms produced. The disruption of alternative splicing patterns associated with sex differences and morphine exposure in the prefrontal cortex of a pig model was studied. The numbers of genes presenting one or more significant (FDR-adjusted p-value < 0.05) alternative splicing events were 933 and 1,368 genes when comparing females relative to males and morphine- relative to saline-treated animals, respectively. The sex-dependent opioid effect was most extreme in the contrast between morphine- versus saline-treated males with 1,934 significantly differentially spliced genes. The most frequent and significant alternative splicing type was skipped exon (∼56 % event), followed by retained intron (∼15 % events). The pathways encompassing a significant number of differentially spliced genes included axon guidance, glutamatergic synapses, circadian rhythm, and lysine degradation. Genes in these pathways included ROBO1, SEMA6C, GRIN3A, GRM2, ARNTL, CLOCK, HYKK, and DOT1L. Transcription factors ETV7 and DMAP1 presented a significant number of differentially spliced target genes. The distribution of the genes presenting differential alternative splicing in the axon guidance and circadian rhythm pathways indicates that this regulatory mechanism impacts hubs and peripheral genes. The identification of sexual dimorphism in the effect of morphine across multiple pathways confirms the necessity to explore the effects of drugs of abuse within sex. Altogether, our findings advance the understanding of the response to factors that can impact the activity of excitatory synapses by modulating transcriptional mechanisms that support the plasticity of the prefrontal cortex.

Amylose has a major influence over starch properties and end-use quality in wheat. The granule-bound starch synthase I, encoded by Wx-1, is the single enzyme responsible for amylose synthesis. Natural null mutants of Wx-1 appear at extremely low frequencies, particularly in the Wx-D1 locus, where only four spontaneous null variants have been identified, with different geographic origins. The current study identified an induced Wx-D1 null mutant (M4-9484) from the M4 generation of an ethyl methanesulfonate-mutagenized population of wheat cv. 'SM126'.

The sequencing showed that the complete Wx-D1 ORF sequences of 'SM126' and M4-9484 were 2862 bp long and that there was one SNP mutation between them. The mutation was located at the RNA splice site within the junction of exon 8 and intron 8, which led to abnormal transcription of Wx-D1, with five different aberrant transcripts being identified in the mutant. The Wx-D1 null allele resulted in amylose and total starch content being decreased in M4-9484 in comparison with the wild-type 'SM126', with higher swelling capacity and being fully pasted at higher temperatures than the wild-type parent.

The mutation of the Wx-D1 null gene affects the formation of amylose directly, resulting in significantly altered starch properties. This discovery offers valuable insights for enhancing wheat starch quality and contributes to the diversification of starch characteristics. It also deepens our understanding of the genetic and molecular mechanisms underlying amylose synthesis, thereby supporting breeding programs. © 2024 Society of Chemical Industry.

The Rbfox proteins regulate alternative pre-mRNA splicing by binding to the RNA element GCAUG. In the nucleus, most of Rbfox is bound to the large assembly of splicing regulators (LASR), a complex of RNA-binding proteins that recognize additional RNA motifs. However, it remains unclear how the different subunits of the Rbfox/LASR complex act together to bind RNA and regulate splicing. We used a nuclease protection assay to map the transcriptome-wide footprints of Rbfox1/LASR on nascent cellular RNA. In addition to GCAUG, Rbfox1/LASR binds RNA motifs for LASR subunits hnRNPs M, H/F, and C and Matrin3. These elements are often arranged in tandem, forming multipart modules of RNA motifs. To distinguish contact sites of Rbfox1 from the LASR subunits, we analyzed a mutant Rbfox1(F125A) that has lost RNA binding but remains associated with LASR. Rbfox1(F125A)/LASR complexes no longer interact with GCAUG but retain binding to RNA elements for LASR. Splicing analyses reveal that in addition to activating exons through adjacent GCAUG elements, Rbfox can also stimulate exons near binding sites for LASR subunits. Minigene experiments demonstrate that these diverse elements produce a combined regulatory effect on a target exon. These findings illuminate how a complex of RNA-binding proteins can decode combinatorial splicing regulatory signals by recognizing groups of tandem RNA elements.

The rapid and accurate diagnosis of rare diseases is paramount in directing clinical management. In recent years, the integration of multi-omics approaches has emerged as a potential strategy to overcome diagnostic hurdles. This review examines the application of multi-omics technologies, including genomics, epigenomics, transcriptomics, proteomics, and metabolomics, in relation to the diagnostic journey of rare diseases. We explore how these combined approaches enhance the detection of pathogenic genetic variants and decipher molecular mechanisms. This review highlights the groundbreaking potential of multi-omics in advancing the precision medicine paradigm for rare diseases, offering insights into future directions and clinical applications. IMPACT: This review discusses using current tests and emerging technologies to diagnose pediatric rare diseases. We describe the next steps after inconclusive molecular testing and a structure for using multi-omics in further investigations. The use of multi-omics is expanding, and it is essential to incorporate it into clinical practice to enhance individualized patient care.

In recent years, there has been a growing appreciation for how regulatory events that occur either co- or post-transcriptionally contribute to the control of gene expression. Messenger RNAs (mRNAs) are extensively regulated throughout their metabolism in a precise spatiotemporal manner that requires sophisticated molecular mechanisms for cell-type-specific gene expression, which dictates cell function. Moreover, dysfunction at any of these steps can result in a variety of human diseases, including cancers, muscular atrophies, and neurological diseases. This review summarizes the steps of the central dogma of molecular biology, focusing on the post-transcriptional regulation of gene expression.

Since the discovery of RNA splicing and its role in gene expression, researchers have sought a set of rules, an algorithm or a computational model that could predict the splice isoforms, and their frequencies, produced from any transcribed gene in a specific cellular context. Over the past 30 years, these models have evolved from simple position weight matrices to deep-learning models capable of integrating sequence data across vast genomic distances. Most recently, new model architectures are moving the field closer to context-specific alternative splicing predictions, and advances in sequencing technologies are expanding the type of data that can be used to inform and interpret such models. Together, these developments are driving improved understanding of splicing regulatory mechanisms and emerging applications of the splicing code to the rational design of RNA- and splicing-based therapeutics.

SF3B1 is a core component of the spliceosome involved in branch point recognition and 3' splice site selection. SF3B1 mutation is common in myelodysplastic syndrome and other blood disorders. The most common mutation in SF3B1 is K700E, a lysine to glutamic acid change within the pre-mRNA interacting heat repeat domain. A hallmark of SF3B1 mutation is an increased use of cryptic 3' splice sites; however, the properties distinguishing SF3B1-sensitive splice junctions from other alternatively spliced junctions are unknown. We identify a subset of 192 core splice junctions that are mis-spliced with SF3B1 K700E mutation. We use our core set to test whether SF3B1-sensitive splice sites are different from control cryptic 3' splice sites via RNA structural accessibility. As a comparison, we define a set of SF3B1-resistant splice junctions with cryptic splice site use that does not change with SF3B1 K700E mutation. We find sequence differences between SF3B1-sensitive and SF3B1-resistant junctions, particularly at the cryptic sites. SF3B1-sensitive cryptic 3' splice sites are within an extended polypyrimidine tract and have lower splice site strength scores. We develop experimental RNA structure data for 83 SF3B1-sensitive junctions and 39 SF3B1-resistant junctions. We find that the pattern of structural accessibility at the NAG splicing motif in cryptic and canonical 3' splice sites is similar. In addition, this pattern can be found in both SF3B1-resistant and SF3B1-sensitive junctions. However, SF3B1-sensitive junctions have cryptic splice sites that are less structurally distinct from the canonical splice sites. In addition, SF3B1-sensitive splice junctions are overall more flexible than SF3B1-resistant junctions. Our results suggest that the SF3B1-sensitive splice junctions have unique structure and sequence properties, containing poorly differentiated, weak splice sites that lead to altered 3' splice site recognition in the presence of SF3B1 mutation.

U2AF1 is a core component of spliceosome and controls cell-fate specific alternative splicing. U2AF1 mutations have been frequently identified in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) patients, and mutations in U2AF1 are associated with poor prognosis in hematopoietic malignant diseases. Here, by forced expression of mutant U2AF1 (U2AF1 S34F) in hematopoietic and leukemic cell lines, we find that U2AF1 S34F causes increased reactive oxygen species (ROS) production. In hematopoietic cell line, a defect in mitochondrial function and DNA damage response deficiency are found in U2AF1 S34F expressing 32D cells. In leukemic cell line Molm13 cells, U2AF1 mutation leads to resistance to DNA damaging agents. Accumulation of DNA damage is also found in U2AF1 S34F expressing leukemic cells when treated with DNA damage agent. Finally, in our established hematopoietic-specific U2af1 S34F knock-in mice model, U2AF1 mutation leads to the development of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) and causes DNA damage accumulation in hematopoietic cells. Our study provides evidence that U2AF1 mutation causes DNA damage response deficiency and DNA damage accumulation in hematopoietic cells, and suggests that mutant U2AF1 induced higher ROS production, resistance to DNA damaging agents and increased genomic instability may contribute to poor prognosis of AML patients with U2AF1 mutations.

Identifying the sequence composition of different splicing modes is a challenge in current research. This study explored the dispersion distributions of 6-mer subsets in human acceptor splicing regions. Without differentiating acceptor splicing modes, obvious differences were observed across the upstream, core, and downstream regions of splicing sites for 16 dispersion distributions. These findings indicate that the dispersion value of each subset can effectively characterize the compositional properties of splicing sequences. When acceptor splicing sequences were classified into common, constitutive, and alternative modes, the differences in dispersion distributions for most of the XY1 6-mer subsets were significant among the three splicing modes. Furthermore, the alternative splicing mode was classified into normal, exonic, and intronic sub-modes, the differences in dispersion distributions for most of the XY1 6-mer subsets were also significant among the three splicing sub-modes. Our results indicate that dispersion values of XY1 6-mer subsets not only revealed the sequence composition patterns of acceptor splicing regions but also effectively identified the differences in base correlation among various acceptor splicing modes. Our research provides new insights into revealing and predicting different splicing modes.

Premature ovarian insufficiency (POI) is a reproductive endocrine disorder characterized by infertility and the perimenopausal syndrome. Its genetic etiology is highly heterogeneous and not yet fully understood. Limited by short-read sequencing, the profile and structural variation of the full-length transcript for POI have remained elusive. Therefore, this study included peripheral blood samples from 5 POI patients and 5 controls, characterizing full-length transcripts of POI using Oxford Nanopore sequencing firstly. Ultimately, we identified 26,122 transcripts, including 7,724 novel gene loci and 13,593 novel transcripts. A total of 382 differentially expressed transcripts were identified, including 366 down-regulated and 16 up-regulated transcripts. Based on transcript structure variant analysis, 8,834 alternative splicing events, 65,254 alternative polyadenylation sites and 32 motifs were further identified, revealing the diversity sources of transcript isoforms, proteins and genetic complexity. Enrichment analysis of differentially AS genes suggested that the ferroptosis pathway may play an important role in the pathogenesis of POI.Additionally, 494 high-confidence lncRNAs, 1,768 transcription factors, and novel gene-coding regions were predicted based on full-length transcript sequence. Analysis of immune cell subtypes revealed the expression of CD8 + T cells and monocytes were down-regulated in POI, which was significantly positively correlated with AMH, suggesting that CD8 + T cells and monocytes could serve as potential diagnostic markers and immunotherapy targets for POI. Conclusively, this study provides new perspectives on the pathogenesis, post-transcriptional regulation mechanisms, and immune targets of POI.

How master splicing regulators cross talk with each other and to what extent transcription regulators are differentially spliced remain unclear in the developing brain. Here, cell-type-specific RNA-Seq analyses of the developing neocortex uncover variable expression of the Rbfox1/2/3 genes and enriched alternative splicing events in transcription regulators, altering protein isoforms or inducing nonsense-mediated mRNA decay. Transient expression of Rbfox proteins in radial glial progenitors induces neuronal splicing events preferentially in transcription regulators such as Meis2 and Tead1 Surprisingly, Rbfox proteins promote the inclusion of a mammal-specific alternative exon and a previously undescribed poison exon in Ptbp1 Simultaneous ablation of Rbfox1/2/3 in the neocortex downregulates neuronal isoforms and disrupts radial neuronal migration. Furthermore, the progenitor isoform of Meis2 promotes Tgfb3 transcription, while the Meis2 neuron isoform promotes neuronal differentiation. These observations indicate that transcription regulators are differentially spliced between cell types in the developing neocortex. (The sex has not been reported to affect cortical neurogenesis in mice, and embryos of both sexes were studied without distinguishing one or the other.).

Junctional epidermolysis bullosa (JEB) is characterized by mucocutaneous fragility. We enrolled 69 cases of recessive JEB, with 13.0% of these cases remained genetically undiagnosed following an initial exome sequencing. Among cases carried COL17A1 variants, this proportion can reach 31.6%. We employed genome sequencing to genetically diagnosis these cases. Four deep intronic variants (c.4156+117 G > A, c.2039-104 G > A and c.1267+237dupC in the COL17A1 gene and c.-38 + 2 T > C in the LAMB3 gene) were identified in six cases. The c.4156+117 G > A variant was found in three of the five cases, suggesting it may be a common deep intronic variant in Chinese JEB. Splicing analysis revealed that these variants caused splicing defect by inducing exon skipping, or pseudoexon insertion into the transcript in HaCaT cells, not in HEK293 cells. Our results emphasize the importance of selecting the right cell line for mRNA analysis.

The human angiotensin converting enzyme 2 (ACE2) gene encodes a type I transmembrane protein, which is homologous to angiotensin I-converting enzyme (ACE) and belongs to the angiotensin-converting enzyme family of dipeptidyl carboxypeptidases. As highlighted by the COVID-19 pandemic, ACE2 is not only crucial for the renin-angiotensin-aldosterone system (RAAS), but also displays great affinity with the SARS-CoV-2 spike protein, representing the major receptor of the virus. Given the significance of ACE2 in COVID-19, especially among cancer patients, the present study aims to explore the transcriptional landscape of ACE2 in human cancer and non-cancerous cell lines through the design and implementation of a custom targeted long-read sequencing approach. Bioinformatics analysis of the massive parallel sequencing data led to the identification of novel ACE2 mRNA splice variants (ACE2 sv.7-sv.12) that demonstrate previously uncharacterized exon-skipping events as well as 5' and/or 3' alternative splice sites. Demultiplexing of the sequencing data elucidated the differential expression profile of the identified splice variants in multiple human cell types, whereas in silico analysis suggests that some of the novel splice variants could produce truncated ACE2 isoforms with altered functionalities, potentially influencing their interaction with the SARS-CoV-2 spike protein. In summary, our study sheds light on the complex alternative splicing landscape of the ACE2 gene in cancer cell lines, revealing novel splice variants that could have significant implications for SARS-CoV-2 susceptibility in cancer patients. These findings contribute to the increased understanding of ACE2's role in COVID-19 and highlight the importance of considering alternative splicing as a key factor in viral pathogenesis. Undoubtably, further research is needed to explore the functional roles of these variants and their potential as therapeutic targets in the ongoing fight against COVID-19.

In this study, we identified and characterized 23 genes encoding serine/arginine-rich (SR) protein in hot pepper (Capsicum annuum), named CaSR here. These CaSR proteins are grouped into seven subfamilies. Phylogenetic analysis revealed a high degree of similarity between CaSRs and their homologous proteins in other plants. Promoter regions of SR proteins are enriched with elements relating to light response, stress, hormone signaling, and plant growth. Notably, transcription levels of several proteins, including CaSR33, CaSR34, and CaSR34a, were upregulated by salt, drought, and cold stresses, suggesting potential roles of these proteins in stress tolerance. We also observed an increase of CaSR transcript population resulting from alternative splicing (AS) regulation, mainly intron retention. AS patterns of CaSR genes varied among tissues. Higher AS intensity was found in the RS subfamily, while some genes in the RSZ subfamily showed no AS regulation under the conditions used here. Interestingly, a cross-species comparative study with Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) showed that many AS events impact the region which codes the RNA recognition motif (RRM) domain of the protein, indicating a conserved regulatory mechanism of SR proteins in plants. Our findings reveal the functional diversity and evolutionary conservation of SR proteins in hot pepper and highlight AS as a mechanism enhancing plant adaptability, providing insights for future stress-resistant crop development.

Oncogenes are typically overexpressed in tumor tissues and often linked to poor prognosis. However, recent advancements in bioinformatics have revealed that many highly expressed genes in tumors are associated with better patient outcomes. These genes, which act as tumor suppressors, are referred to as "paradoxical genes." Analyzing The Cancer Genome Atlas (TCGA) confirmed the widespread presence of paradoxical genes, and KEGG analysis revealed their role in regulating tumor metabolism. Mechanistically, discrepancies between gene and protein expression-affected by pre- and post-transcriptional modifications-may drive this phenomenon. Mechanisms like upstream open reading frames and alternative splicing contribute to these inconsistencies. Many paradoxical genes modulate the tumor immune microenvironment, exerting tumor-suppressive effects. Further analysis shows that the stage- and tumor-specific expression of these genes, along with their environmental sensitivity, influence their dual roles in various signaling pathways. These findings highlight the importance of paradoxical genes in resisting tumor progression and maintaining cellular homeostasis, offering new avenues for targeted cancer therapy.

Alternative splicing plays a fundamental role in gene expression and protein complexity. Aberrant splicing impairs cell homeostasis and is closely associated with aging and cellular senescence. Significant changes to alternative splicing, including dysregulated splicing events and the abnormal expression of splicing factors, have been detected during the aging process or in age-related disorders. Here, we highlight the possibility of suppressing aging and cellular senescence by controlling alternative splicing. In this review, we will summarize the latest research progress on alternative splicing in aging and cellular senescence, discuss the roles and regulatory mechanisms of alternative splicing during aging, and then excavate existing and potential approaches to anti-aging by controlling alternative splicing. Novel therapeutic breakthroughs concerning aging and senescence entail a further understanding of regulating alternative splicing mechanically and accurately.

The polyomavirus family consists of a highly diverse group of small DNA viruses isolated from various species, including humans. Some family members have been used as model systems to understand the fundamentals of modern biology. After the discovery of the first two human polyomaviruses (JC virus and BK virus) during the early 1970s, their current number reached 14 today. Some family members cause considerably severe human diseases, including polyomavirus-associated nephropathy (PVAN), progressive multifocal leukoencephalopathy (PML), trichodysplasia spinulosa (TS) and Merkel cell carcinoma (MCC). Polyomaviruses encode universal regulatory and structural proteins, but some members express additional virus-specific proteins and microRNA, which significantly contribute to the viral biology, cell transformation, and perhaps progression of the disease that they are associated with. In the current review, we summarized the recent advances in discovery, and functional and structural analysis of those viral proteins and microRNA.

Dysregulation of human DNA polymerase delta (Polδ) subunits is associated with genome instability and pathological disorders. Genome databases suggest the expression of several spliced variants of subunits which may alter Polδ function. Here, we analyzed the protein-encoding variants of the Polδ subunit p12 and their association with cancer. p12 isoform-2 (p12*) encodes a 79 aa protein with a C-terminal tail distinct from the previously characterized p12. Like p12, p12* dimerizes and interacts with p125 and p50 subunits and is thus an integral component of Polδ. Further, we observed dysregulated p12* expression in low-grade glioma, renal, thyroid, and pancreatic carcinomas. This study identifies a previously unrecognized Polδ complex and highlights a possible regulatory role of p12 variants in cellular phenotypes.

Alternative splicing (AS) is a key element of eukaryotic gene expression that increases transcript and proteome diversity in cells, thereby altering their responses to external stimuli and stresses. While AS has been intensively researched in plants and animals, its frequency, conservation, and putative impact on virulence, are relatively still understudied in plant pathogenic fungi. Here, we profiled the AS events occurring in genes of Cladosporium fulvum isolates Race 5 and Race 4, during nearly a complete compatible infection cycle on their tomato host. Our studies revealed extensive heterogeneity in the transcript isoforms assembled from different isolates, infections, and infection timepoints, as over 80% of the transcript isoforms were singletons that were detected in only a single sample. Despite that, nearly 40% of the protein-coding genes in each isolate were predicted to be recurrently AS across the disparate infection timepoints, infections, and the two isolates. Of these, 37.5% were common to both isolates and 59% resulted in multiple protein isoforms, thereby putatively increasing proteome diversity in the pathogen by 31% during infections. An enrichment analysis showed that AS mostly affected genes likely to be involved in the transport of nutrients, regulation of gene expression, and monooxygenase activity, suggesting a role for AS in finetuning adaptation of C. fulvum on its tomato host during infections. Tracing the location of the AS genes on the fungal chromosomes showed that they were mostly located in repeat-rich regions of the core chromosomes, indicating a causal connection between gene location on the genome and propensity to AS. Finally, multiple cases of differential isoform usage in AS genes of C. fulvum were identified, suggesting that modulation of AS at different infection stages may be another way by which pathogens refine infections on their hosts.

Prostate cancer (PCa) is the most prevalent type of cancer and the second leading cause of mortality in males, with a marked increase in incidence observed across the globe. In the present study, whole-transcriptome analysis was conducted to identify differentially expressed circular RNAs (DE-circRNAs). The coding abilities of the DE-circRNAs were analyses, and it was found that hsa_circ_0085121 (circRNF19A) not only exhibited overexpression in PCa cells and tumor samples, but also encoded a 490 amino acid polypeptide designated circRNF19A-490aa. The knockdown of circRNF19A was observed to notably inhibit the proliferation, invasion, migration and docetaxel resistance of PCa cells. In contrast, mutation of the IRES significantly impaired the tumor-promoting function of circRNF19A, indicating that circRNF19A-490aa is the primary form that regulates the malignant behaviors of PCa cells. Mechanistically, circRNF19A-490aa was demonstrated to interact with HSP90AA1, thereby enhancing AR activity and facilitating the activation of the Akt/mTOR and PLK1 pathways. Furthermore, circRNF19A-490aa was observed to interact with HNRNPF, facilitating the recruitment of HNRNPF to the splicing site of AR-V7 and enhancing its alternative splicing. Finally, the androgen receptor (AR) was observed to bind to the promoter region of the RNF19A gene, subsequently regulating the expression of circRNF19A and circRNF19A-490aa. These data indicate that circRNF19A plays a pivotal role in AR activation and AR-V7 generation by encoding a novel protein, circRNF19A-490aa, and targeting circRNF19A may prove an effective strategy for impeding the progression of CRPC.

Effective cancer immunotherapy relies on enhancing the host's immune response, particularly by boosting T cell-mediated cytotoxicity against tumor cells. In this study, we identify CWF19-like cell cycle control factor 1 (CWF19L1) as a novel splicing regulator that enhances T cell-mediated cytotoxicity. CWF19L1 interacts prominently with key splicing factors within the nucleus, including components of the U5 small nuclear ribonucleoprotein and the pre-mRNA processing factor 19 (PRPF19) complex. Deficiency of CWF19L1 disrupts alternative splicing of immune-related genes, resulting in diminished expression of cytotoxic molecules. Furthermore, CWF19L1 plays a critical role in promoting T cell-mediated antitumor responses by upregulating the expression of effector cytokines. Our findings unveil previously undocumented functions of CWF19L1 in alternative splicing and its involvement in the regulation of antitumor immunity, highlighting its potential as a therapeutic target for novel cancer immunotherapies.

Spermatogenesis is a highly coordinated process that requires the precise expression of specific subsets of genes in different types of germ cells, controlled both temporally and spatially. Among these genes, those that can exert an indispensable influence in spermatogenesis via participating in alternative splicing make up the overwhelming majority. mRNA alternative-splicing (AS) events can generate various isoforms with distinct functions from a single DNA sequence, based on specific AS codes. In addition to enhancing the finite diversity of the genome, AS can also regulate the transcription and translation of certain genes by directly binding to their cis-elements or by recruiting trans-elements that interact with consensus motifs. The testis, being one of the most complex tissue transcriptomes, undergoes unparalleled transcriptional and translational activity, supporting the dramatic and dynamic transitions that occur during spermatogenesis. Consequently, AS plays a vital role in producing an extensive array of transcripts and coordinating significant changes throughout this process. In this review, we summarize the intricate functional network of alternative splicing in spermatogenesis based on the integration of current research findings.

In recent years, significant strides in both conceptual understanding and technological capabilities have bolstered our comprehension of the factors underpinning cancer initiation and progression. While substantial insights have unraveled the molecular mechanisms driving carcinogenesis, there has been an overshadowing of the critical contribution made by epigenetic pathways, which works in concert with genetics. Mounting evidence demonstrates cancer as a complex interplay between genetics and epigenetics. Notably, epigenetic elements play a pivotal role in governing alternative pre-mRNA splicing, a primary contributor to protein diversity. In this review, we have provided detailed insights into the bidirectional communication between epigenetic modifiers and alternative splicing, providing examples of specific genes and isoforms affected. Notably, succinct discussion on targeting epigenetic regulators and the potential of the emerging field of epigenome editing to modulate splicing patterns is also presented. In summary, this review offers valuable insights into the intricate interplay between epigenetics and alternative splicing in cancer, paving the way for novel approaches to understanding and targeting this critical process.

Ribonucleic acid (RNA) undergoes dynamic changes in its structure and function under various intracellular and extracellular conditions over time. However, there is a lack of research on the concept of the RNA age to describe its diverse fates. This study proposes a definition of RNA age to address this issue. RNA age was defined as a sequence of numbers wherein the elements in the sequence were the nucleotide ages of the ribonucleotide residues in the RNA. Mean nucleotide age was used to represent RNA age. This definition describes the temporal properties of RNAs that have undergone diverse life histories and reflects the dynamic state of each ribonucleotide residue, which can be expressed mathematically. Notably, events (including base insertions, base deletions, and base substitutions) are likely to cause RNA to become younger or older when using mean nucleotide ages to represent the RNA age. Although information, including the presence of added markers in RNA, chemical modification structure of the RNA, and the excision of introns in the mRNA in cells, may provide a basis for identifying RNA age, little is known about determining the RNA age of extracellular RNA in the wild. Nonetheless, we believe that RNA age has an important relationship with the diverse biological properties of RNA under intracellular and extracellular conditions. Therefore, our proposed definition of RNA age offers new perspectives for studying dynamic changes in RNA function, RNA aging, ancient RNA, environmental RNA, and the ages of other biomolecules.

RNA processing involves steps such as capping, splicing, polyadenylation, modification, and nuclear export. These steps are essential for transforming genetic information in DNA into proteins and contribute to RNA diversity and complexity. Many biochemical methods have been developed to profile and quantify RNAs, as well as to identify the interactions between RNAs and RNA-binding proteins (RBPs), especially when coupled with high-throughput sequencing technologies. With the rapid accumulation of diverse data, it is crucial to develop computational methods to convert the big data into biological knowledge. In particular, machine learning and deep learning models are commonly utilized to learn the rules or codes governing the transformation from DNA sequences to intriguing RNAs based on manually designed or automatically extracted features. When precise enough, the RNA codes can be incredibly useful for predicting RNA products, decoding the molecular mechanisms, forecasting the impact of disease variants on RNA processing events, and identifying driver mutations. In this review, we systematically summarize the biochemical and computational methods for deciphering five important RNA codes related to alternative splicing, alternative polyadenylation, RNA localization, RNA modifications, and RBP binding. For each code, we review the main types of experimental methods used to generate training data, as well as the key features, strategic model structures, and advantages of representative tools. We also discuss the challenges encountered in developing predictive models using large language models and extensive domain knowledge. Additionally, we highlight useful resources and propose ways to improve computational tools for studying RNA codes.