Environmental control of flowering through alternative splicing of FLC and COOLAIR.

From annotation to regulation: Iso-seq reveals novel isoforms and alternative splicing features underlying salinity responses in Arabidopsis roots.

Pulmonary hypertension (PH) is a fatal disease characterized by pulmonary vascular remodeling, primarily driven by excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs). Although noncoding RNAs, such as circular RNAs, have been implicated in PH, the role of tRNA-derived small RNA remains poorly understood. Among them, i-tRF (internal tRNA-derived fragment)-Asp represents a novel tRNA-derived small RNA whose function in hypoxic PH is unknown. This study aims to investigate whether i-tRF-Asp contributes to PASMC proliferation and vascular remodeling in hypoxic PH. To elucidate the functional role of i-tRF-Asp, loss-of-function experiments were conducted in vitro using hypoxic mouse PASMCs, alongside in vivo studies using both SuHx (Sugen5416+hypoxia) induced PH models in both adult male C57BL/6 mice and Sprague-Dawley rats. These models employed antisense oligonucleotide inhibitors and adeno-associated virus-mediated silencing. The underlying mechanisms were further explored via RNA immunoprecipitation, liquid-liquid phase separation assays, cell proliferation assessment, Western blot, alternative splicing profiling, and molecular dynamics simulation. Inhibition of i-tRF-Asp ameliorated hypoxia-induced pulmonary vascular remodeling, primarily through suppressing excessive proliferation of mouse PASMCs. Further analysis indicated that i-tRF-Asp facilitates liquid-liquid phase separation of hnRNPU (heterogeneous nuclear ribonucleoprotein U). This process mediates selective recruitment of Tcf7l2 (transcription factor 7-like 2) pre-mRNA and promotes alternative splicing, resulting in a pro-proliferative Tcf7l2 transcript variant that drives mouse PASMC proliferation. Furthermore, we have identified that the endonuclease ERN1 (endoribonuclease IRE1) can upregulate the expression of i-tRF-Asp. The endoribonuclease ERN1 upregulates the expression of i-tRF-Asp, which in turn facilitates mouse PASMC proliferation and pulmonary vascular remodeling. This process is driven by hnRNPU liquid-liquid phase separation-dependent alternative splicing of the long Tcf7l2 transcript isoform.

Internode morphology critically determines bamboo wood quality. Phyllostachys edulis 'Heterocycla' (GJ) is a natural dwarf mutant of P. edulis (WT), yet regulatory mechanisms remain unclear. Here, we show that impaired parenchyma cell elongation is the primary cellular basis for dwarfism in GJ. This phenotype correlates with a marked reduction in gibberellin (GA) and an increase in abscisic acid (ABA) levels. Through weighted gene co-expression network analysis, we identified PeGA20ox1 as a critical regulator of internode elongation. In GJ, PeGA20ox1 undergoes alternative splicing (AS), producing a transcript, PeGA20ox1-GJ, with a premature termination codon. Moreover, we characterized that a bifunctional transcription factor PeRAP2-1 mediates GA/ABA homeostasis, which is an activator of PeABA3 and an inhibitor of PeGA20ox1 by binding to different cis-acting elements. Intriguingly, this AS event results in the loss of function of PeGA20ox1-GJ, which disrupts the PeRAP2-1-mediated hormonal balance, ultimately inhibiting internode elongation. This study suggests that the posttranscriptional regulation induced by AS may adapt to the rapid growth of bamboo plants and provides important genetic resources for the molecular breeding of bamboo with improved culm traits.

Aberrant alternative polyadenylation (APA) and alternative splicing (AS) contribute to numerous diseases, including cancer; however, their coordinated roles in ovarian cancer remain poorly understood. Here, we investigated CPSF7, an APA factor markedly upregulated in ovarian cancer and associated with poor prognosis. Silencing CPSF7 suppressed proliferation, migration, and invasion of ovarian cancer cells, while antisense oligonucleotides (ASOs) targeting CPSF7 reduced tumor growth in a patient‑derived xenograft (PDX) model. Mechanistically, knockdown of the splicing factor SNRPD2 induced exon 4 skipping in CPSF7 pre‑mRNA. Loss of exon 4 disrupted the RNA recognition motif (RRM) domain essential for CPSF7‑mediated pre‑mRNA cleavage and polyadenylation, and introduced premature termination codons (PTCs) that generated noncoding transcripts subject to nonsense‑mediated decay (NMD), thereby reducing CPSF7 expression. Thus, efficient splicing mediated by SNRPD2 is crucial for sustaining high CPSF7 levels in ovarian cancer cells. Functional assays showed that CPSF7 knockdown reduced proliferation and metastatic potential in cells with elevated SNRPD2, suggesting that CPSF7 is a key mediator of SNRPD2-driven oncogenesis. Moreover, CPSF7 governed specific APA events to maintain transcript stability, with UBE2K identified as a critical downstream target. CPSF7 preferentially bound distal polyadenylation signals (PASs) within the predominant UBE2K transcript (UBE2K-201), thereby increasing its mRNA stability and maintaining high functional UBE2K expression. Collectively, these findings reveal that AS and APA are interconnected in ovarian cancer via the SNRPD2-CPSF7-UBE2K axis, which drives disease progression and represents a promising target for therapeutic intervention.

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) severely limits clinical efficacy. Myeloid zinc finger 1 (MZF1) inhibited EGFR phosphorylation and internalization by maintaining zinc homeostasis, which suggested its potential as a therapeutic target for resensitization. The two MZF1 splice variants exhibited functional divergence. MZF1S demonstrated significantly reduced Zn2+-binding capacity compared with MZF1L owing to C2H2 domain deletion. Although MZF1S was specifically upregulated in resistant patients, MZF1L predominated in treatment-sensitive cohorts, the mechanisms regulating their differential expression remained uncharacterized. We identified NSUN7/YBX1-mediated RNA m5C methylation as a critical driver of resistance. This modification promoted competitive binding of SRSF1 over SRSF3 to MZF1 pre-mRNA and enhanced MZF1S production. Overlapping loci co-regulated by RNA m5C and DNA 5-methylcytosine methylation in the MZF1 5'-untranslated region suggested coordinated epigenetic control of alternative splicing. Characterization of these loci revealed a UHRF1/DNMT1-NSUN7/YBX1 axis that synergistically regulated splice variant selection. Targeting this axis suppressed MZF1S while restoring MZF1L expression, which resensitized resistant cells to EGFR-TKIs. Our findings established a dual-layer epigenetic mechanism governing alternative splicing in drug resistance and proposed methylation-regulated splice variants as biomarkers and therapeutic targets for overcoming EGFR-TKI resistance in non-small-cell lung cancer.

Alternative splicing of immune checkpoints: Classifications, mechanisms, and therapeutic implications for overcoming immune checkpoint blockade resistance.

RNA binding proteins: Post-transcriptional regulation in craniofacial bone, tooth, and periodontium.

Alternative RNA splicing is a fundamental mechanism for enhancing proteomic diversity, and its dysregulation is a hallmark of cancer progression. However, the dynamic regulatory networks controlling oncogenic splicing events remain poorly understood. Our previous work identified the inclusion of CLSTN1 exon 11 as critical for the epithelial-to-mesenchymal transition (EMT). Here, we demonstrate that this splicing event promotes breast cancer metastasis by enhancing cell migration, invasion, and the generation of circulating tumor cells (CTCs) in vivo. To translate this finding into a therapeutic strategy, we developed splice-switching antisense oligonucleotides (ASOs) that effectively reverse exon 11 inclusion and suppress cancer cell migration. Furthermore, through systematic screening, we identified the RNA-binding protein TIA1 as a key suppressor of exon 11 inclusion. TIA1 inhibits EMT and metastasis, but its function is antagonized during EMT by phosphorylation mediated by the kinase DAPK3, which is upregulated in this process. This work defines a novel DAPK3-TIA1-CLSTN1 splicing axis that drives breast cancer metastasis, revealing new layers of post-transcriptional regulation and presenting promising therapeutic avenues for targeting pro-metastatic splicing.

Alternative splicing generates a novel CARD9 isoform.

Myotonic dystrophy type 1 is the most prevalent adult-onset muscular dystrophy and is characterized by progressive muscle weakness, myotonia, cardiac conduction defects, endocrine dysfunction, and central nervous system involvement. Myotonic dystrophy type 1is caused by an unstable CTG repeat expansion in the 3' untranslated region of the DMPK gene, which produces toxic CUG-expanded transcripts that sequester RNA-binding proteins such as Muscleblind-like, induce widespread alternative splicing defects, and drive an RNA gain-of-function mechanism rather than simple DMPK haploinsufficiency. Despite major advances in understanding the molecular pathogenesis of myotonic dystrophy type 1, there is still no approved cure or disease-modifying therapy. This review summarizes the molecular basis of myotonic dystrophy type 1 and provides an in-depth overview of emerging therapeutic strategies that directly target the underlying pathogenic cascade at the DNA and RNA levels. Gene therapy-based approaches, including CRISPR-mediated genome editing, aim to reduce or eliminate the expanded CTG repeats or expanded DMPK allele and its toxic transcripts. In parallel, a broad spectrum of RNA-directed interventions is being developed, encompassing antisense oligonucleotides, antibody-penetrating and cell-penetrating peptide-conjugated antisense oligonucleotides to enhance skeletal and cardiac muscle delivery, small interfering RNAs, and microRNA-based tools such as antagomiRs. Additional strategies exploit engineered RNA-binding proteins and peptide decoys to disrupt toxic ribonuclear aggregates, polyadenylation signal-driven premature transcriptional termination to selectively silence mutant DMPK, and small molecules that modulate RNA metabolism, dissolve CUG RNA foci, or correct downstream mis-splicing. By integrating data from preclinical models and ongoing clinical trials, including recent advances with muscle‑targeted antisense oligonucleotide conjugates and gene therapy, this review outlines the current status, strengths, and limitations of these mechanism-based therapies for myotonic dystrophy type 1. The discussion highlights key translational challenges such as efficient delivery to skeletal muscle, the heart, and brain, long-term safety, and robust pharmacodynamic biomarkers as well as opportunities for combination and next-generation approaches aimed at converting molecular correction into durable clinical benefit for patients with myotonic dystrophy type 1.

Evolution of steroid receptor coactivator Taiman in arthropods.

Ketohexokinase: A Central Mediator of Fructose-Associated Pathogenesis and Promising Therapeutic Target.

Heart failure (HF) involves pathological cardiac remodeling, including cardiomyocyte loss and dysfunction. While the RNA-binding protein CPEB4 has been linked to cardiomyocyte activation, its role in HF remains unclear. We established in vivo and in vitro models of cardiac injury using isoproterenol (ISO). An HF mouse model was induced by chronic ISO administration (10mg/kg/day, 3 weeks), while cellular injury was modeled by treating HL-1 atrial cardiomyocytes (HL-1) with ISO (10 µM, 48h). To investigate the molecular mechanisms, we employed transcriptome sequencing, qRT-PCR, and functional assays following siRNA-mediated knockdown of Cpeb4. Key endpoints included cell viability (CCK-8), apoptosis (Annexin V/7-AAD flow cytometry), and the alternative splicing of Eif4a2. In the HF mouse model, we observed significant cardiac hypertrophy and inflammatory infiltration, which correlated with a marked upregulation of Cpeb4 expression in myocardium. Notably, in vitro, siRNA-mediated knockdown of Cpeb4 significantly attenuated ISO-induced apoptosis and enhanced cell viability in HL-1 cells. Mechanistically, Cpeb4 depletion corrected the ISO-induced dysregulation of Eif4a2 alternative splicing, restoring the expression of its major isoforms and thereby ameliorating cellular injury. Additional co-knockdown experiments indicated that Eif4a2 contributes to the protective phenotype observed upon Cpeb4 inhibition. Our study identifies the Cpeb4-Eif4a2 axis as a key regulator in heart failure, with Cpeb4 associated with aberrant Eif4a2 splicing and cardiomyocyte apoptosis, suggesting a promising therapeutic target.

Gastric cancer (GC) is a highly malignant tumor with significant morbidity and mortality rates globally. Recent studies have shown that RUNX2, a member of the RUNX family known for its role in osteoblast differentiation and bone morphogenesis, is associated with the pathogenesis and progression of GC, while the underlying mechanisms of the pathogenesis and progression of GC are largely unknown. In this study, we investigated the role of RUNX2 in GC progression by analyzing its effects on gene expression and alternative splicing (AS) in HGC-27 cells. We silenced RUNX2 by small interfering RNA (siRUNX2), and then analyzed the globally regulated transcriptome profile by sequencing method (RNA-seq) to identify the differentially expressed genes (DEGs) and alternative splicing (AS) genes. The downstream targets of RUNX2 were identified by performing CUT&Tag and sequencing experiment in HGC-27 cells. Using RNA-seq, we identified 314 DEGs and 1120 regulated AS events (RASEs) in HGC-27 cells upon RUNX2 knockdown. The DEGs were enriched in pathways related to cell cycle regulation and apoptosis, while the RASEs were predominantly involved in cell cycle processes. These findings suggest that RUNX2 not only modulates gene expression but also extensively influences AS, which is crucial for cellular processes such as proliferation and survival. Notably, we found that RUNX2 regulates the expression of the splicing factor SF3B6 by binding to its promoter region. Our results showed that SF3B6 expression was significantly decreased in siRUNX2 samples, and its downregulation was associated with altered AS profiles of genes involved in the cell cycle. Our findings demonstrate that RUNX2 modulates the transcriptome and AS profile in GC cells, with a particular focus on its regulation of SF3B6. This study highlights the multifaceted role of RUNX2 in GC progression and suggests that targeting the RUNX2-SF3B6 axis could be a promising therapeutic strategy for GC.

Epigenetic regulation of neuronal calcium signaling during postnatal cortical development is critical for voluntary movement. Protein arginine methyltransferase 5 (PRMT5) acts as an epigenetic regulator that is involved in movement disorders, yet its underlying mechanism remains poorly understood. Here, we report that conditional knockout of Prmt5 in excitatory neurons (cKONex) during motor cortex development leads to severe hyperactivity and shortened lifespan in mice. Transcriptomics analysis reveals that PRMT5 deficiency upregulated Cacna1d expression without affecting its alternative splicing. We demonstrate that PRMT5 directly binds to the Cacna1d promoter, and represses its expression via catalyzing H4R3me2s histone methylation. Electrophysiological studies showed deletion of Prmt5 increased calcium influx and neuronal excitability, resulting from elevated L-type voltage-gated calcium channel (Cav1.3) activity. The enhanced calcium level further leads to oxidative phosphorylation defects in neurons. Importantly, administration of the L-type channel blocker, nimodipine, rescues hyperactivity and neuronal hyper-excitability in vivo, as well as markedly extends the lifespan of cKONex mice. Our findings establish a novel epigenetic mechanism wherein PRMT5-Cacna1d axis regulates neuronal excitability and motor development. These data provide new insights into the calcium channelopathy of movement disorders.

Evaluation of Ogataea polymorpha DUR31 TPP riboswitch as a tool to downregulate gene expression in the yeast Komagataella phaffii.

Histone variants are critical components of neuronal chromatin that are emerging as key regulators of long-term memory formation. We previously showed that depleting the macrodomain-containing histone variant macroH2A1 (mH2A1) in the mouse hippocampus impairs long-term memory, establishing this histone as essential for memory consolidation. However, mH2A1 undergoes alternative splicing to generate two isoforms, mH2A1.1 and mH2A1.2, which differ by a single exon within the macrodomain. Though mH2A1.1 and mH2A1.2 have been reported to regulate unique molecular processes in non-neuronal cells, distinct functional contributions of hippocampal mH2A1.1 and mH2A1.2 to long-term memory formation in the adult brain are unknown. Here, we characterized genomic localization of mH2A1 splice isoforms in the mouse hippocampus and evaluated how isoform-specific knockdown impacts hippocampal transcription and memory. Although both isoforms localize to and regulate memory-relevant genes, their depletion affected largely non-overlapping gene sets, and only loss of mH2A1.1 impaired long-term memory. Notably, mH2A1.1 depletion increased expression of several genes that negatively regulate memory formation, including the well-established memory suppressor calcineurin. Thus, under normal conditions, mH2A1.1 may promote memory by repressing transcriptional programs that constrain plasticity. Together, these findings reveal isoform-specific functions of mH2A1 in the hippocampus and identify alternative splicing of mH2A1 as a key epigenetic mechanism that fine-tunes neural chromatin composition to enable long-term memory formation.

Polygala tenuifolia Willd. (P. tenuifolia) is one of the source plants of the traditional Chinese medicine 'Yuanzhi' and is widely used in clinical practice. As a meso-xerophytic medicinal plant with strong environmental adaptability, it is listed as a nationally protected wild medicinal species in China. However, the molecular mechanisms underlying its drought tolerance remain unclear. Therefore, a genome-wide identification of the ERF subfamily inP. tenuifoliawas conducted to provide candidate genes for drought adaptation research and functional validation. Ethylene-responsive factors (ERFs) play important roles in plant responses to drought stress. In this study, 78 PtERF genes were systematically identified for the first time in P. tenuifolia. Phylogenetic analysis classified these genes into five subgroups (B1-B5), with members within the same subgroup exhibiting similar gene structures and conserved motifs. Collinearity analysis revealed that segmental duplication was the primary driving forceunderlying PtERF family evolution. Cis-acting element analysis revealed that PtERF promoter regions were enriched with drought-responsive cis-elements, including abscisic acid and methyl jasmonate (MeJA)-responsive elements. Transcriptome analysis combined with RT-qPCR verification identified five core drought-responsive genes, among which PtERF33 expression exhibited the highest level of upregulation. Further investigation showed that PtERF33 produced two alternatively spliced transcripts: the full-length PtERF33.1 transcript and the intron-retained PtERF33.2 transcript. These two transcripts exhibited opposite expression patterns under drought stress, suggesting that they may regulate drought responses through functional antagonism. These findings provide a systematic basis for identifying drought-resistant ERF genes in P. tenuifolia and establish a foundation for clarifying the role of alternative splicing in drought adaptation in this species.

Superoxide dismutases (SODs) are involved in cellular reactive oxygen species (ROS) homeostasis under diverse conditions. Among different plant SODs, CuZnSODs (CSDs) are most abundant and localized to multiple compartments, including chloroplast. Rice (Oryza sativa) genome encodes a chloroplastic CuZnSOD (OsCSD2, LOC_Os08g44770), which undergoes alternative splicing (AS), generating two splice variants (SVs), OsCSD2-SV1 (constitutive) and OsCSD2-SV2 (alternative). The AS-event remove 12 nucleotides in OsCSD2-SV2, causing loss of four amino acids (29GPTT32) in OsCSD2-SV2. The two OsCSD2 isoforms were analyzed at RNA and protein levels. The OsCSD2-SV1 and OsCSD2-SV2 transcripts showed comparable stability but differed in expression pattern in certain tissues and stress conditions. For assessment of 'GPTT loss', OsCSD2 isoforms were over-expressed in E. coli, purified, and compared for biochemical-biophysical characteristics. Both isoforms were found to be homodimers (subunit molecular weight: ~19 kDa), and enzymatically active; however, OsCSD2-SV2 (lacking GPTT) showed substantially lower SOD activity than OsCSD2-SV1. Both isoforms showed comparable pH optima, but differed in SOD activity and stability at higher pH and temperature (OsCSD2-SV1 > OsCSD2-SV2), sensitivity to H2O2 (OsCSD2-SV1 > OsCSD2-SV2), and Cu cofactor content. Circular dichroism (CD) showed discernible differences between the isoforms, while differential scanning fluorimetry (DSF) revealed lower thermostability of OsCSD2-SV2, likely due to reduced H-bonds/other interactions. OsCSD2 isoforms showed heteromeric interaction with each other and with the SOD-specific chaperone. Analysis of purified homo/heterodimers indicated that heteromeric interaction reduces the SOD activity of OsCSD2. The study showed several new insights into AS-mediated modulation of rice OsCSD2, which might be important for its chloroplastic functions.