Eukaryotic RNA transcripts undergo extensive processing before becoming functional messenger RNAs, with splicing being a critical and highly regulated step that occurs both co-transcriptionally and post-transcriptionally. Recent analyses have revealed, with unprecedented spatial and temporal resolution, that up to 40% of mammalian introns are retained after transcription termination and are subsequently removed largely while transcripts remain chromatin-associated. Post-transcriptional splicing has emerged as a key layer of gene expression regulation during development, stress response and disease progression. The control of post-transcriptional splicing regulates protein production through delayed splicing and nuclear export, or nuclear retention and degradation of specific transcript isoforms. Here, we review current methodologies for detecting post-transcriptional splicing, discuss the mechanisms controlling the timing of splicing and examine how this temporal regulation affects gene expression programmes in healthy cells and in disease states.

Alternative splicing (AS), a key post-transcriptional regulatory mechanism, is frequently dysregulated in cancer, driving both tumor progression and immune modulation. Aberrant AS influences antigen presentation, T cell activation, immune checkpoint regulation, and cytokine signaling, contributing to immune evasion but also presenting unique therapeutic vulnerabilities. Targeting AS has emerged as a promising strategy in cancer immunotherapy. Splicing-derived neoantigens have been identified as potent inducers of CD8⁺ T cell responses, offering potential for personalized treatment. AS modulators such as PRMT5 inhibitor GSK3326595 enhance immunotherapy efficacy by upregulating MHC class II expression and promoting T cell infiltration, while RBM39 inhibitor indisulam induces tumor-specific neoantigens. Furthermore, combining AS-targeting drugs with immune checkpoint inhibitors (ICIs) has demonstrated synergistic effects, improved response rates and overcoming resistance in preclinical models. Despite these advances, challenges remain in optimizing drug specificity and minimizing toxicity. Future efforts should focus on refining AS-targeting therapies, identifying predictive biomarkers, and integrating these approaches into clinical applications. This review highlights the therapeutic potential of AS modulation in cancer immunotherapy and its implications for advancing precision oncology.

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.

Messenger RNA (mRNA) splicing is a fundamental and tightly regulated process in eukaryotes, where the spliceosome removes non-coding sequences from pre-mRNA to produce mature mRNA for protein translation. Alternative splicing enables the generation of multiple RNA isoforms and protein products from a single gene, regulating both isoform diversity and abundance. While splicing is widespread in eukaryotes, only ∼3% of genes in Saccharomyces cerevisiae undergo splicing, with most containing a single intron. However, intron-containing genes, primarily ribosomal protein genes, are highly expressed and constitute about one-third of the total mRNA pool. These genes are transcriptionally regulated by Repressor Activator Protein 1 ( RAP1 ), prompting us to investigate whether RAP1 influences mRNA splicing. Using RNA sequencing, we identified a novel role for RAP1 in alternative splicing, particularly in intron retention (IR) while minor effects were observed on alternative 3' and 5' splice site usage. Many IR-containing transcripts introduced premature termination codons, likely leading to degradation via nonsense-mediated decay (NMD). Consistent with previous literature, genes with predicted NMD in our study also had reduced overall expression levels suggesting that RAP1 plays an important role in this understudied mechanism of gene expression regulation.

Climate change has caused drastic declines in corals. As sessile organisms, corals acclimate to environmental shifts through genome-wide changes in gene expression, epigenetic modifications, and alterations in microbiome composition. However, alternative splicing (AS), a conserved mechanism of stress response in many organisms, has been under-explored in corals. Using short-term acute thermal stress assays, we investigated patterns of AS in the scleractinian coral Acropora cervicornis during response to low (33°C), medium (35°C), and high (37°C) heat stress and subsequent overnight recovery. Our findings demonstrate reproducible dynamic shifts in AS of at least 40 percent of all genes during response to heat treatment and the recovery phase. The relative proportion of AS increased in response to heat stress and was primarily dominated by intron retention in specific classes of transcripts, including those related to splicing regulation itself. While AS returned to baseline levels post-exposure to low heat, AS persisted even after reprieve from higher levels of heat stress, which was associated with irreversible loss of photosynthetic efficiency of the symbiont. Our findings demonstrate that, although animals, corals are more plant-like in their likely usage of AS for regulating thermal stress response and recovery.

The maternal-fetal interface represents a critical site of immunological interactions that can greatly influence pregnancy outcomes. The unique cellular composition and cell-cell interactions taking place within these tissues has spurred substantial research efforts focused on the maternal-fetal interface. With the recent advent of single-cell technologies, multiple investigators have applied such methods to gain an unprecedented level of insight into maternal-fetal communication. Here, we provide an overview of the dynamic cellular composition and cell-cell communications at the maternal-fetal interface as reported by single-cell investigations. By primarily focusing on data from pregnancies in the second and third trimesters, we aim to showcase how single-cell technologies have bolstered the foundational understanding of each cell's contribution to physiologic gestation. Indeed, single-cell technologies have enabled the examination of classical placental cells, such as the trophoblast, as well as uncovered new roles for structural cells now recognized as active participants in pregnancy and parturition, such as decidual and fetal stromal cells, which are reviewed herein. Furthermore, single-cell data investigating the ontogeny, function, differentiation, and interactions among immune cells present at the maternal-fetal interface, namely macrophages, T cells, dendritic cells, neutrophils, mast cells, innate lymphoid cells, natural killer cells, and B cells are discussed in this review. Moreover, a key output of single-cell investigations is the inference of cell-cell interactions, which has been leveraged to not only dissect the intercellular communications within specific tissues but also between compartments such as the decidua basalis and placental villi. Collectively, this review emphasizes the ways by which single-cell technologies have expanded the understanding of cell composition and cellular processes underlying pregnancy in mid-to-late gestation at the maternal-fetal interface, which can prompt their continued application to reveal new pathways and targets for the treatment of obstetrical disease.

Alternative splicing is a critical post-transcriptional regulatory mechanism in eukaryotes. While infection with Ralstonia solanacearum GMI1000 significantly alters plant alternative splicing patterns, the underlying molecular mechanisms remain unclear. Herein, the effect of the GMI1000 Type III secretion system effectors on alternative splicing in the tomato cultivar Heinz 1706 was investigated. The RNA-seq analysis confirmed genome-wide alternative splicing changes induced by the Type III secretion system in tomato, including 1386 differential alternatively spliced events across 1023 genes, many of which are associated with plant defense. Seven nucleus-localized Type III effectors were transiently expressed in an RLPK splicing reporter system transgenic tobacco, identifying RipP2 as an effector that modulates alternative splicing levels. Sequence analysis, protein-protein interaction assays, and AlphaFold2 structural predictions revealed that RipP2 interacted with the tomato splicing factor SR34a. Furthermore, RipP2 acetylated a conserved lysine at position 132 within the SWQDLKD motif of SR34a, regulating its splicing pattern in defense-related genes and modulating plant immunity. This study elucidates how the "RipP2-SR34a module" influences plant immune responses by regulating the alternative splicing of immune-related genes, providing new insights into pathogen-plant interactions and splicing regulation.

Adhesion G protein-coupled receptors (aGPCRs) are expressed in all organs and are involved in various mechanobiological processes. They are heavily alternatively spliced, forecasting an extraordinary molecular structural diversity. Here, we uncovered the existence of unconventional single-transmembrane (1TM)-containing ADGRL/Cirl proteins devoid of the conventional GPCR layout (i.e., the 7TM signaling unit) in Drosophila. These 1TM proteins are made as a result of intron retention and provide an N-terminal fragment that acts as an interactor to allow Gαo-dependent signaling through conventional 7TM-containing Cirl isoforms encoded by the same gene. This molecular mechanism determines sensory precision of neurons in response to mechanical stimulation in vivo. This action mode of aGPCR provides a promising entry point for experimental and therapeutic approaches to intervene in aGPCR signaling and implicates alternative splicing as a physiological strategy to express a given aGPCR together with its molecular interactor.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Single-cell RNA sequencing (scRNA-seq) provides gene expression profiles at the single-cell level. Hence, we evaluated gene expression in the peripheral blood of patients with COPD.

Peripheral blood samples from seven healthy controls and eight patients with COPD were obtained in this study. The 10X Genomics Chromium Instrument and cDNA synthesis kit were utilized to generate a barcoded cDNA library for single cell RNA-sequencing. We compared the scRNA-seq data between the COPD and control groups using computational analysis. Functional analyses were performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses.

scRNA-seq was used to analyze the transcriptome of peripheral blood mononuclear cells from seven normal controls and eight patients with COPD. We found an increased number of monocyte/macrophages in the COPD group compared to the normal control group. Among the differentially expressed genes (DEGs) in monocyte/macrophages, we identified 15 upregulated genes (EGR1, NR4A1, CCL3, CXCL8, PTGS2, CD83, BCL2A1, SGK1, IL1B, BTG2, NFKBIZ, DUSP2, MAFB, PLAUR and CCL3L1) and 7 downregulated genes (FOLR3, RPS4Y1, HLA-DRB5, NAMPT, CD52, TMEM176A and TMEM176B) in the COPD group compared to the normal control group.

Using scRNA-seq, we found differences in cell type distribution, especially in monocyte/ macrophages. Several upregulated and downregulated genes were found in the monocyte/macrophages of the COPD group.

Alternative polyadenylation (APA) is emerging as a post-transcriptional regulatory mechanism, similar as that of alternative splicing (AS), and plays a prominent role in regulating gene expression and increasing the complexity of the transcriptome and proteome. We use polyadenylation selected long-read isoform sequencing to obtain full-length transcript sequences in porcine muscles at five developmental stages. We identify numerous novel transcripts unannotated in the existing pig genome, including transcripts mapping to known and unknown gene loci, and widespread transcript diversity in porcine muscles. The top 100 most isoformic genes are mainly enriched in Gene Ontology terms related to muscle growth and development. It is revealed that intron retention/exon inclusion and the usage of distal polyadenylation site (PAS) are associated with ageing through analyzing changes of AS and PAS during muscle development. We also identify developmental changes in major transcripts and major PASs. Furthermore, genes/transcripts important for muscle development are identified. The results confirm the importance of AS and APA in pig muscles, substantially increasing transcriptional diversity and showing an important mechanism underlying gene regulation in muscles.

Alternative splicing has long been recognized as a powerful tool to expand the diversity of the transcriptome and the proteome. The study of hematopoiesis, from hematopoietic stem cell maintenance and differentiation into committed progenitors to maturation into functional blood cells, has led the field of stem cell research and cellular differentiation for decades. The importance of aberrant splicing due to mutations in cis has been exemplified in thalassemias, resulting from aberrant expression of β-globin. The simultaneous development of increasingly sophisticated technologies, in particular the combination of multicolor flow cytometric cell sorting with bulk and single-cell sequencing, has provided sophisticated insights into the complex regulation of the blood system. The recognition that mutations in key splicing factors drive myeloid malignancies, in particular myelodysplastic syndromes, has galvanized research into alternative splicing in hematopoiesis and its diseases. In this review, we will update the audience on the exciting novel technologies, highlight alternative splicing events and their regulators with essential functions in hematopoiesis, and provide a high-level overview how splicing factor mutations contribute to hematologic malignancies.

Hematopoietic stem cells (HSCs) are vital for the differentiation of all mature blood cells, with their homeostasis being tightly regulated by intrinsic and extrinsic factors. Alternative splicing, mediated by the spliceosome complex, plays a crucial role in regulating HSC homeostasis by increasing protein diversity. This study focuses on the ATP-dependent RNA helicase DHX16, a key spliceosome component, and its role in HSC regulation. Using conditional knockout mice, we demonstrate that loss of Dhx16 in the hematopoietic system results in significant depletion of hematopoietic stem and progenitor cells, bone marrow failure, and rapid mortality. Dhx16-deficient HSCs exhibit impaired quiescence, G2-M phase cell cycle arrest, reduced protein synthesis, abnormal ribosome assembly, increased apoptosis, and decreased self-renewal capacity. Multi-omics analysis identified intron 4 retention in Emg1 mRNA in Dhx16 knockout HSCs, leading to reduced EMG1 protein expression, disrupted ribosome assembly, and nucleolar stress, activating the p53 pathway. Overexpression of Emg1 in Dhx16-deficient HSCs partially restored ribosome assembly and HSC function, suggesting Emg1 as a potential therapeutic target for ribosomopathies. Our findings reveal the critical role of Dhx16 in HSC homeostasis through the regulation of alternative splicing and ribosome assembly, providing insights into the molecular mechanisms underlying hematopoietic diseases and potential therapeutic strategies.

Alternative splicing (AS) and intron retention (IR) implicated in multiple pathophysiological processes, have rarely been reported in systemic sclerosis (SSc).

We integrated bulk RNA-seq and 4D label-free mass spectrometry to perform a multi-omics analysis of AS and IR in SSc skin tissue and fibroblasts. RMATS and iREAD were used to identify AS and IR, which were validated by real-time PCR. Spearman correlation and the LASSO method were employed to assess correlations among clinical features, introns, splicing factors (regulators of AS) and proteins.

AS profiles showed distinct alterations in SSc skin tissue, with the most pronounced changes occurring in IR. AS and IR were associated with total modified Rodnan skin score (mRSS) and local skin score. Upon TGF-β stimulation, fibroblasts exhibited significant alterations in IR profiles, affecting genes related to fibroblast proliferation and collagen fibril organization. A comprehensive integrated analysis of introns, exons, and proteome profiles revealed that IR exerted a negative impact on protein expression, with certain changes being under intronic control. RT-PCR confirmed the presence of intron and exon-derived sequences of CTTN, OGA, MED16 and PHYKPL. Additionally, notable changes were observed in the regulatory network of splicing factors in SSc skin tissues. These factors are also involved in fibrosis pathways and correlated with clinical features.

Totally, abnormal AS, IR profiles and splicing factors were identified in SSc, altered IRs and splicing factors participated in fibrosis-related pathways. IR exerted a negative impact on protein expression in TGF-β-stimulated fibroblasts. Clarification of the IR mechanisms will provide new insights into the pathophysiology of SSc.

Intron retention (IR) constitutes a less explored form of alternative splicing, wherein introns are retained within mature mRNA transcripts. This investigation demonstrates that the cell division cycle (CDC)-like kinase 2 (CLK2) undergoes liquid-liquid phase separation (LLPS) within nuclear speckles in response to heat shock (HS). The formation of CLK2 condensates depends on the intrinsically disordered region (IDR) located within the N-terminal amino acids 1-148. Phosphorylation at residue T343 sustains CLK2 kinase activity and promotes overall autophosphorylation, which inhibits the LLPS activity of the IDR. These CLK2 condensates initiate the reorganization of nuclear speckles, transforming them into larger, rounded structures. Moreover, these condensates facilitate the recruitment of splicing factors into these compartments, restricting their access to mRNA for intron splicing and promoting the IR. The retained introns lead to the sequestration of transcripts within the nucleus. These findings extend to the realm of glioma stem cells (GSCs), where a physiological state mirroring HS stress inhibits T343 autophosphorylation, thereby inducing the formation of CLK2 condensates and subsequent IR. Notably, expressing the CLK2 condensates hampers the maintenance of GSCs. In conclusion, this research unveils a mechanism by which IR is propelled by CLK2 condensates, shedding light on its role in coping with cellular stress.

Peripheral inflammation is often associated with depressive disorders, and immunological biomarkers of depression remain a focus of investigation.

We performed RNA-seq analysis of RNA transcripts of human peripheral blood mononuclear cells from a case-control study including subjects with self-reported depression in the pre-symptomatic state of major depressive disorder and analyzed differentially expressed genes (DEGs) and the frequency of intron retention (IR) using rMATS.

Among the statistically significant DEGs identified, the 651 upregulated DEGs were particularly enriched in the term "bacterial infection and phagocytosis", whereas the 820 downregulated DEGs were enriched in the terms "antigen presentation" and "T-cell proliferation and maturation". We also analyzed 158 genes for which the IR was increased (IncIR) and 211 genes for which the IR was decreased (DecIR) in the depressed subjects. Although the Gene Ontology terms associated with IncIR and DecIR were very similar to those of the up- and downregulated genes, respectively, IR genes appeared to be particularly enriched in genes with sensor functions, with a preponderance of the term "ciliary assembly and function". The observation that IR genes specifically interact with innate immunity genes suggests that immune-related genes, as well as cilia-related genes, may be excellent markers of depression. Re-analysis of previously published RNA-seq data from patients with MDD showed that common IR genes, particularly our predicted immune- and cilia-related genes, are commonly detected in populations with different levels of depression, providing validity for using IR to detect depression.

Depression was found to be associated with activation of the innate immune response and relative inactivation of T-cell signaling. The DEGs we identified reflect physiological demands that are controlled at the transcriptional level, whereas the IR results reflect a more direct mechanism for monitoring protein homeostasis. Accordingly, an alteration in IR, namely IncIR or DecIR, is a stress response, and intron-retained transcripts are sensors of the physiological state of the cytoplasm. The results demonstrate the potential of relative IR as a biomarker for the immunological stratification of depressed patients and the utility of IR for the discovery of novel pathways involved in recovery from depression.

We characterized the regulatory mechanisms and role in human myeloid cell survival and differentiation of PRPF40A, a splicing factor lacking a canonical RNA Binding Domain. Upon PRPF40A knockdown, HL-60 cells displayed increased cell death, decreased proliferation and slight differentiation phenotype with upregulation of immune activation genes. Suggestive of both redundant and specific functions, cell death but not proliferation was rescued by overexpression of its paralog PRPF40B. Transcriptomic analysis revealed the predominant role of PRPF40A as an activator of cassette exon inclusion of functionally relevant splicing events. Mechanistically, the exons exclusively upregulated by PRPF40A are flanked by short and GC-rich introns which tend to localize to nuclear speckles in the nucleus center. These PRPF40A regulatory features are shared with other splicing regulators such as SRRM2, SON, PCBP1/2, and to a lesser extent TRA2B and SRSF2, as a part of a functional network that regulates splicing partly via co-localization in the nucleus.

We performed long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors to discover new transcript and protein isoforms expressed during immune responses to diverse pathogens. Long-read transcriptome profiling reveals novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. Widespread loss of intron retention occurs as a common result of all pathogen stimulations. We highlight novel transcripts of NFKB1 and CASP1 that may indicate novel immunological mechanisms. RNA expression differences did not result in differences in the amounts of secreted proteins. Clustering analysis of secreted proteins revealed a correlation between chemokine (receptor) expression on the RNA and protein levels in C. albicans- and poly(I:C)-stimulated PBMCs. Isoform aware long-read sequencing of pathogen-stimulated immune cells highlights the potential of these methods to identify novel transcripts, revealing a more complex transcriptome landscape than previously appreciated.

Due to insufficient knowledge about key molecular events, Hepatocellular carcinoma (HCC) lacks effective treatment targets. Spliceosome-related genes were significantly altered in HCC. Oncofetal proteins are ideal tumor therapeutic targets. Screening of differentially expressed Spliceosome-related oncofetal protein in embryonic liver development and HCC helps discover effective therapeutic targets for HCC.

Differentially expressed spliceosome genes were analysis in fetal liver and HCC through bioinformatics analysis. Small nuclear ribonucleoprotein polypeptide E (SNRPE) expression was detected in fetal liver, adult liver and HCC tissues. The role of SNRPE in HCC was performed multiple assays in vitro and in vivo. SNRPE-regulated alternative splicing was recognized by RNA-Seq and confirmed by multiple assays.

We herein identified SNRPE as a crucial oncofetal splicing factor, significantly associated with the adverse prognosis of HCC. SOX2 was identified as the activator for SNRPE reactivation. Efficient knockdown of SNRPE resulted in the complete cessation of HCC tumorigenesis and progression. Mechanistically, SNRPE knockdown reduced FGFR4 mRNA expression by triggering nonsense-mediated RNA decay. A partial inhibition of SNRPE-induced malignant progression of HCC cells was observed upon FGFR4 knockdown.

Our findings highlight SNRPE as a novel oncofetal splicing factor and shed light on the intricate relationship between oncofetal splicing factors, splicing events, and carcinogenesis. Consequently, SNRPE emerges as a potential therapeutic target for HCC treatment. Model of oncofetal SNRPE promotes HCC tumorigenesis by regulating the AS of FGFR4 pre-mRNA.

The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However, the systems-level effects of AS-NMD in this context are poorly understood.

We developed an R package, factR2, which offers a comprehensive suite of AS-NMD analysis functions. Using this tool, we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably, this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms.

Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes.

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.

Erythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation.

In vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371- HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371- HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371- HPCs were performed to assay the signals changed by ELK1.

Here, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371- hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371- HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment.

ALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy.

The adult mammalian brain retains some capacity to replenish neurons and glia, holding promise for brain regeneration. Thus, understanding the mechanisms controlling adult neural stem cell (NSC) differentiation is crucial. Paradoxically, adult NSCs in the subependymal zone transcribe genes associated with both multipotency maintenance and neural differentiation, but the mechanism that prevents conflicts in fate decisions due to these opposing transcriptional programmes is unknown. Here we describe intron detention as such control mechanism. In NSCs, while multiple mRNAs from stemness genes are spliced and exported to the cytoplasm, transcripts from differentiation genes remain unspliced and detained in the nucleus, and the opposite is true under neural differentiation conditions. We also show that m6A methylation is the mechanism that releases intron detention and triggers nuclear export, enabling rapid and synchronized responses. m6A RNA methylation operates as an on/off switch for transcripts with antagonistic functions, tightly controlling the timing of NSCs commitment to differentiation.

In the intricate orchestration of the central dogma, pre-mRNA splicing plays a crucial role in the post-transcriptional process that transforms DNA into mature mRNA. Widely acknowledged as a pivotal RNA processing step, it significantly influences gene expression and alters the functionality of gene product proteins. Although U2-dependent spliceosomes efficiently manage the removal of over 99% of introns, a distinct subset of essential genes undergo splicing with a different intron type, denoted as minor introns, using U12-dependent spliceosomes. Mutations in spliceosome component genes are now recognized as prevalent genetic abnormalities in cancer patients, especially those with hematologic malignancies. Despite the relative rarity of minor introns, genes containing them are evolutionarily conserved and play crucial roles in functions such as the RAS-MAPK pathway. Disruptions in U12-type minor intron splicing caused by mutations in snRNA or its regulatory components significantly contribute to cancer progression. Notably, recurrent mutations associated with myelodysplastic syndrome (MDS) in the minor spliceosome component ZRSR2 underscore its significance. Examination of ZRSR2-mutated MDS cells has revealed that only a subset of minor spliceosome-dependent genes, such as LZTR1, consistently exhibit missplicing. Recent technological advancements have uncovered insights into minor introns, raising inquiries beyond current understanding. This review comprehensively explores the importance of minor intron regulation, the molecular implications of minor (U12-type) spliceosomal mutations and cis-regulatory regions, and the evolutionary progress of studies on minor, aiming to provide a sophisticated understanding of their intricate role in cancer biology.

Intron retention is a mechanism of post-transcriptional gene regulation, including genes involved in erythropoiesis. Erythropoietin (EPO) is a hormone without evidence of intracellular vesicle storage that regulates erythropoiesis. We hypothesize that EPO uses intron retention as a mechanism of post-transcriptional regulation in response to hypoxia and ischemia. Cell models of hypoxia and ischemia for kidney, liver, and brain cells were examined for intron retention by real time quantitative PCR. EPO expression increased in most cells except for blood brain barrier and liver cells. The intron retained transcript ratio decreased in brain cells, except for Astrocytes, but showed no change in kidney or liver after 24 h of ischemia. The shift in intron ratio was maintained when using poly (A) enriched cDNA, suggesting that intron retention is not due to immature transcripts. The expression of EPO was elevated at variable time points amongst cell models with the intron ratio also changing over a time course of 2 to 16 h after ischemia. We conclude that intron retention is a mechanism regulating EPO expression in response to ischemia in a tissue specific manner.

Ovarian cancer (OC) is the most lethal gynecologic malignant tumour. The mechanism promoting OC initiation and progression remains unclear. SET domain bifurcated histone lysine methyltransferase 1(SETDB1) acts as an oncogene in a variety of tumours. This study aims to explore the role of SETDB1 in OC.

GEO, TCGA, CSIOVDB and CPTAC databases jointly analysed SETDB1 mRNA and protein expression. Effect of SETDB1 expression on the clinical prognosis of OC patients was analysed through online Kaplan‒Meier plotter and CSIOVDB database. Then, the effect of SETDB1 in OC cells progression and mobility was examined using MTT, EdU, colony formation and transwell assay. Additionally, Cistrome DB database was used to visualize the binding of SETDB1 protein and splicing factor 3b subunit 4 (SF3B4) promoter, and dual-luciferase reporter gene assay was performed to confirm the interaction. Finally, bioinformatics analysis was employed to reveal the relationship between SETDB1 and the microenvironment of OC.

In the present study, we found that SETDB1 was obviously upregulated in OC and its overexpression predicted poor prognosis of OC patients. Then, we verified that SETDB1 promoted the progression and motility of OC cells in vitro. Knockdown of SETDB1 had the opposite effect. Further research showed that SETDB1 acted as a transcription factor to activate SF3B4 expression. SF3B4 knockdown impaired the effect of SETDB1 to promote the proliferative capacity and motility of OC cells. Finally, the results of bioinformatics analysis confirmed that SETDB1 regulated the immune microenvironment of ovarian cancer.

SETDB1 promoted ovarian cancer progression by upregulating the expression of SF3B4 and inhibiting the tumour immunity. SETDB1 may be a promising prognostic and therapeutic marker for OC.

Ubiquitination plays an important role in proliferating and invasive characteristic of glioblastoma (GBM), similar to many other cancers. Tripartite motif 25 (TRIM25) is a member of the TRIM family of proteins, which are involved in tumorigenesis through substrate ubiquitination.

Difference in TRIM25 expression levels between nonneoplastic brain tissue samples and primary glioma samples was demonstrated using publicly available glioblastoma database, immunohistochemistry, and western blotting. TRIM25 knockdown GBM cell lines (LN229 and U251) and patient derived GBM stem-like cells (GSCs) GBM#021 were used to investigate the function of TRIM25 in vivo and in vitro. Co-immunoprecipitation (Co-IP) and mass spectrometry analysis were performed to identify NONO as a protein that interacts with TRIM25. The molecular mechanisms underlying the promotion of GBM development by TRIM25 through NONO were investigated by RNA-seq and validated by qRT-PCR and western blotting.

We observed upregulation of TRIM25 in GBM, correlating with enhanced glioblastoma cell growth and invasion, both in vitro and in vivo. Subsequently, we screened a panel of proteins interacting with TRIM25; mass spectrometry and co-immunoprecipitation revealed that NONO was a potential substrate of TRIM25. TRIM25 knockdown reduced the K63-linked ubiquitination of NONO, thereby suppressing the splicing function of NONO. Dysfunctional NONO resulted in the retention of the second intron in the pre-mRNA of PRMT1, inhibiting the activation of the PRMT1/c-MYC pathway.

Our study demonstrates that TRIM25 promotes glioblastoma cell growth and invasion by regulating the PRMT1/c-MYC pathway through mediation of the splicing factor NONO. Targeting the E3 ligase activity of TRIM25 or the complex interactions between TRIM25 and NONO may prove beneficial in the treatment of GBM.

Exitrons are exonic introns. This subclass of intron retention alternative splicing does not contain a Pre-Terminating stop Codon. Therefore, when retained, they are always a part of a protein. Intron retention is a frequent phenomenon predominantly found in plants, which results in either the degradation of the transcripts or can serve as a stable intermediate to be processed upon induction by specific signals or the cell status. Interestingly, exitrons have coding ability and may confer additional attributes to the proteins that retain them. Therefore, exitron-containing and exitron-spliced isoforms will be a driving force for creating protein diversity in the proteome of an organism. This review establishes a basic understanding of exitron, discussing its genesis, key features, identification methods and functions. We also try to depict its other potential roles. The present review also aims to provide a fundamental background to those who found such exitronic sequences in their gene(s) and to speculate the future course of studies.

Intron splicing is a key regulatory step in gene expression in eukaryotes. Three sequence elements required for splicing-5' and 3' splice sites and a branchpoint-are especially well-characterized in Saccharomyces cerevisiae, but our understanding of additional intron features that impact splicing in this organism is incomplete, due largely to its small number of introns. To overcome this limitation, we constructed a library in S. cerevisiae of random 50-nt (N50) elements individually inserted into the intron of a reporter gene and quantified canonical splicing and the use of cryptic splice sites by sequencing analysis. More than 70% of approximately 140,000 N50 elements reduced splicing by at least 20%. N50 features, including higher GC content, presence of GU repeats, and stronger predicted secondary structure of its pre-mRNA, correlated with reduced splicing efficiency. A likely basis for the reduced splicing of such a large proportion of variants is the formation of RNA structures that pair N50 bases-such as the GU repeats-with other bases specifically within the reporter pre-mRNA analyzed. However, multiple models were unable to explain more than a small fraction of the variance in splicing efficiency across the library, suggesting that complex nonlinear interactions in RNA structures are not accurately captured by RNA structure prediction methods. Our results imply that the specific context of a pre-mRNA may determine the bases allowable in an intron to prevent secondary structures that reduce splicing. This large data set can serve as a resource for further exploration of splicing mechanisms.

Alternative splicing changes are closely linked to aging, though it remains unclear if they are drivers or effects. As organisms age, splicing patterns change, varying gene isoform levels and functions. These changes may contribute to aging alterations rather than just reflect declining RNA quality control. Three main splicing types-intron retention, cassette exons, and cryptic exons-play key roles in age-related complexity. These events modify protein domains and increase nonsense-mediated decay, shifting protein isoform levels and functions. This may potentially drive aging or serve as a biomarker. Fluctuations in splicing factor expression also occur with aging. Somatic mutations in splicing genes can also promote aging and age-related disease. The interplay between splicing and aging has major implications for aging biology, though differentiating correlation and causation remains challenging. Declaring a splicing factor or event as a driver requires comprehensive evaluation of the associated molecular and physiological changes. A greater understanding of how RNA splicing machinery and downstream targets are impacted by aging is essential to conclusively establish the role of splicing in driving aging, representing a promising area with key implications for understanding aging, developing novel therapeutical options, and ultimately leading to an increase in the healthy human lifespan.

Human transcriptome can undergo RNA mis-splicing due to spliceopathies contributing to the increasing number of genetic diseases including muscular dystrophy (MD), Alzheimer disease (AD), Huntington disease (HD), myelodysplastic syndromes (MDS). Intron retention (IR) is a major inducer of spliceopathies where two or more introns remain in the final mature mRNA and account for many intronic expansion diseases. Potential removal of such introns for therapeutic purposes can be feasible when utilizing bioinformatics, catalytic RNAs, and nano-drug delivery systems. Overcoming delivery challenges of catalytic RNAs was discussed in this review as a future perspective highlighting the significance of utilizing synthetic biology in addition to high throughput deep sequencing and computational approaches for the treatment of mis-spliced transcripts.

The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anaemia. TTD-associated mutations typically cause unstable mutant proteins involved in various steps of gene expression, severely reducing steady-state mutant protein levels. However, to date, no such link to instability of gene-expression factors for TTD-associated mutations in MPLKIP/TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry-based interaction proteomics, we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue.

Hematopoiesis is an essential process for organismal development and homeostasis. Epigenetic regulation of gene expression is critical for stem cell self-renewal and differentiation in normal hematopoiesis. Increasing evidence shows that disrupting the balance between self-renewal and cell fate decisions can give rise to hematological diseases such as bone marrow failure and leukemia. Consequently, next-generation sequencing studies have identified various aberrations in histone modifications, DNA methylation, RNA splicing, and RNA modifications in hematologic diseases. Favorable outcomes after targeting epigenetic regulators during disease states have further emphasized their importance in hematological malignancy. However, these targeted therapies are only effective in some patients, suggesting that further research is needed to decipher the complexity of epigenetic regulation during hematopoiesis. In this review, an update on the impact of the epigenome on normal hematopoiesis, disease initiation and progression, and current therapeutic advancements will be discussed.

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1mut erythroid progenitor cells. We uncovered distinct cryptic 3' splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 3' splice site usage in SF3B1mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.

Methyltransferase-like 3 (METTL3), a key component of the m6A methyltransferase complex, regulates the splicing, nuclear transport, stability, and translation of its target genes. However, the mechanism underlying the regulation of METTL3 expression by alternative splicing (AS) remains unknown. We analyzed the expression pattern of METTL3 after AS in human tissues and confirmed the expression of an isoform retaining introns 8 and 9 (METTL3-IR). We confirmed the different intracellular localizations of METTL3-IR and METTL3 proteins using immunofluorescence microscopy. Furthermore, the endogenous expression of METTL3-IR at the protein level was different from that at the mRNA level. We found that 3'-UTR generation by intron retention (IR) inhibited the export of METTL3-IR mRNA to the cytoplasm, which in turn suppressed protein expression. To the best of our knowledge, this is the first study to confirm the regulation of METTL3 gene expression by AS, providing evidence that the suppression of METTL3 protein expression by IR is an integral part of the mechanism by which 3'-UTR generation regulates protein expression via inhibition of RNA export to the cytoplasm. [BMB Reports 2023; 56(9): 514-519].