pre-mRNA Splicing Research Articles

Synthesis, Biophysical and Biological Evaluation of Splice-Switching Oligonucleotides with Multiple LNA-Phosphothiotriester Backbones.

Polyanionic antisense oligonucleotides hold great promise as RNA targeting drugs but issues with bioavailability hinder their development. Uncharged phosphorus-based backbones are promising alternatives but robust methods to produce them are limited. We report the synthesis and properties of oligonucleotides containing charge-neutral LNA alkyl phosphothiotriester backbones combined with 2'-O-methyl phosphorothioate nucleotides for therapeutic applications. The nature of the triester alkyl group dictates the success of solid-phase synthesis; tertiary alkyl groups are lost during the P(III) oxidation step, whereas primary alkyl groups are partially cleaved during deprotection. In contrast, oligonucleotides containing secondary phosphothiotriester linkages are stable, and large numbers of triesters can be incorporated. The modified oligonucleotides have excellent duplex stability with complementary RNA and exhibit strong nuclease resistance. To expand synthetic flexibility, oligonucleotides containing multiple internal alkynyl phosphothiotriesters can be conjugated to lipids, carbohydrates, or small molecules through CuAAC click chemistry. Oligonucleotides containing LNA-THP phosphothiotriesters exhibit high levels of pre-mRNA splice switching in eukaryotic cells.

GhMAC3e is involved in plant growth and defense response to Verticillium dahliae.

GhMAC3e expression was induced by various stresses and hormones. GhMAC3e may regulate plant growth by influencing auxin distribution, and play important roles in Verticillium wilt resistance via mediating SA signaling. The MOS4-Associated Complex (MAC) is a highly conserved protein complex involved in pre-mRNA splicing and spliceosome assembly, which plays a vital role in plant immunity. It comprises key components such as MOS4, CDC5, and PRL1. MAC3A/B, as U-box E3 ubiquitin ligases, are crucial for various plant processes including development, stress responses, and disease resistance. However, their roles in cotton remain largely unknown. In this study, we first cloned the GhMAC3e gene from cotton and explored its biological functions by using virus-induced gene silencing (VIGS) in cotton and transgenic overexpression in Arabidopsis. The results showed that GhMAC3e is ubiquitously expressed in cotton tissues and could be induced by salt stress, Verticillium dahliae (VD) infection, PEG, ABA, ETH, GA3, MeJA, and SA. Silencing GhMAC3e retarded primary stem growth and reduced biomass of cotton coupled with the reduced auxin content in the petioles and veins. Silencing GhMAC3e up-regulated expression of cell growth-related genes GhXTH16 and Gh3.6, while down-regulated GhSAUR12 expression. Ectopic expression of GhMAC3e in Arabidopsis significantly enhanced its resistance to Verticillium wilt (VW) in terms of decreased pathogen biomass and lowered plant mortality. Overexpression of GhMAC3e dramatically upregulated AtPR1 by around 15 fold and more than 262 fold under basal and VD inoculation condition, respectively. This change was not associated with the expression of GhNPR1. In conclusion, GhMAC3e may not only regulate plant growth by influencing auxin distribution and growth-related gene expression, but also play important roles in VW resistance via mediating SA signaling independent of NPR1 transcription level.

Recurrent Neurodevelopmentally Associated Variants of the Pre-mRNA Splicing Factor U2AF2 Alter RNA Binding Affinities and Interactions.

De novo mutations affecting the pre-mRNA splicing factor U2AF2 are associated with developmental delays and intellectual disabilities, yet the molecular basis is unknown. Here, we demonstrated by fluorescence anisotropy RNA binding assays that recurrent missense mutants (Arg149Trp, Arg150His, or Arg150Cys) decreased the binding affinity of U2AF2 for a consensus splice site RNA. Crystal structures at 1.4 Å resolutions showed that Arg149Trp or Arg150His disrupted hydrogen bonds between U2AF2 and the terminal nucleotides of the RNA site. Reanalysis of publicly available RNaseq data confirmed that U2AF2 depletion altered splicing of transcripts encoding RNA binding proteins (RBPs). These results confirmed that the impaired RNA interactions of Arg149Trp and Arg150His U2AF2 variants could contribute to dysregulating an RBP-governed neurodevelopmental program of alternative splicing.

Alternative splicing of PBRM1 mediates resistance to PD-1 blockade therapy in renal cancer.

Alternative pre-mRNA splicing (AS) is a biological process that results in proteomic diversity. However, implications of AS alterations in cancer remain poorly understood. Herein, we performed a comprehensive AS analysis in cancer driver gene transcripts across fifteen cancer types and found global alterations in inclusion rates of the PBAF SWI/SNF chromatin remodeling complex subunit Polybromo 1 (PBRM1) exon 27 (E27) in most types of cancer tissues compared with those in normal tissues. Further analysis confirmed that PBRM1 E27 is excluded by the direct binding of RBFOX2 to intronic UGCAUG elements. In addition, the E27-included PBRM1 isoform upregulated PD-L1 expression via enhanced PBAF complex recruitment to the PD-L1 promoter. PBRM1wild-type patients with clear cell renal cell carcinoma were resistant to PD-1 blockade therapy when they expressed low RBFOX2 mRNA levels. Overall, our study suggests targeting of RBFOX2-mediated AS of PBRM1 as a potential therapeutic strategy for immune checkpoint blockade.

Mechanical Loading Modulates AMPK and mTOR Signaling in Muscle Cells.

Skeletal muscle adaptation to exercise involves various phenotypic changes that enhance the metabolic and contractile functions. One key regulator of these adaptive responses is the activation of AMPK, which is influenced by exercise intensity. However, the mechanistic understanding of AMPK activation during exercise remains incomplete. In this study, we utilized an in vitro model to investigate the effects of mechanical loading on AMPK activation and its interaction with the mTOR signaling pathway. Proteomic analysis of muscle cells subjected to static loading (SL) revealed distinct quantitative protein alterations associated with RNA metabolism, with 10% SL inducing the most pronounced response compared to lower intensities of 5% and 2% as well as the control. Additionally, 10% SL suppressed RNA and protein synthesis while activating AMPK and inhibiting the mTOR pathway. We also found that SRSF2, necessary for pre-mRNA splicing, is regulated by AMPK and mTOR signaling, which, in turn, is regulated in an intensity-dependent manner by SL with the highest expression in 2% SL. Further examination showed that the ADP/ATP ratio was increased after 10% SL compared to the control and that SL induced changes in mitochondrial biogenesis. Furthermore, Seahorse assay results indicate that 10% SL enhances mitochondrial respiration. These findings provide novel insights into the cellular responses to mechanical loading and shed light on the intricate AMPK-mTOR regulatory network in muscle cells.

The RNA helicase LOS4 regulates pre-mRNA splicing of key genes (EIN2, ERS2, CTR1) in the ethylene signaling pathway.

The Arabidopsis RNA helicase LOS4 plays a key role in regulating pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. The plant hormone ethylene plays diverse roles in plant growth, development, and responses to stress. Ethylene is perceived by the membrane-bound ethylene receptors complex, and then triggers downstream components, such as EIN2, to initiate signal transduction into the nucleus, leading to the activation of ethylene-responsive genes. Over the past decades, substantial information has been accumulated regarding gene cloning, protein-protein interactions, and downstream gene expressions in the ethylene pathway. However, our understanding of mRNA post-transcriptional processing and modification of key genes in the ethylene signaling pathway remains limited. This study aims to provide evidence demonstrating the involvement of the Arabidopsis RNA helicase LOS4 in pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. Various genetic approaches including RNAi gene silencing, CRISPR-Cas9 gene editing, and amino acid mutations were employed in this study. When LOS4 was silenced or knocked down, the ethylene sensitivity of etiolated seedlings was significantly enhanced. Further investigation revealed errors in the EIN2 pre-mRNA splicing when LOS4 was knocked down. In addition, aberrant pre-mRNA splicing was observed in the ERS2 and CTR1 genes in the pathway. Biochemical assays indicated that the los4-2 (E94K) mutant protein exhibited increased ATP binding and enhanced ATP hydrolytic activity. Conversely, the los4-1 (G364R) mutant had reduced substrate RNA binding and lower ATP binding activities. These findings significantly advanced our comprehension of the regulatory functions and molecular mechanisms of RNA helicase in ethylene signaling.

RNA splicing as a therapeutic target in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematological disorders and are more commonly found in people over the age of 60. MDS patients exhibit peripheral blood cytopenias and carry an increased risk of disease progression to acute myeloid leukemia (AML). Splicing factor mutations (including genes SF3B1, SRSF2, U2AF1 and ZRSR2) are early events identified in more than 50% of MDS cases. These mutations cause aberrant pre-mRNA splicing and impact MDS pathophysiology. Emerging evidence shows that splicing factor-mutant cells are more sensitive to perturbations targeting the spliceosome, aberrantly spliced genes and/or their regulated molecular pathways. This review summarizes current therapeutic strategies and ongoing efforts targeting splicing factor mutations for the treatment of MDS.

Defective Integrator activity shapes the transcriptome of patients with multiple sclerosis.

HP1α/CBX5 is an epigenetic regulator with a suspected role in multiple sclerosis (MS). Here, using high-depth RNA sequencing on monocytes, we identified a subset of MS patients with reduced CBX5 expression, correlating with progressive stages of the disease and extensive transcriptomic alterations. Examination of rare non-coding RNA species in these patients revealed impaired maturation/degradation of U snRNAs and enhancer RNAs, indicative of reduced activity of the Integrator, a complex with suspected links to increased MS risk. At protein-coding genes, compromised Integrator activity manifested in reduced pre-mRNA splicing efficiency and altered expression of genes regulated by RNA polymerase II pause-release. Inactivation of Cbx5 in the mouse mirrored most of these transcriptional defects and resulted in hypersensitivity to experimental autoimmune encephalomyelitis. Collectively, our observations suggested a major contribution of the Integrator complex in safeguarding against transcriptional anomalies characteristic of MS, with HP1α/CBX5 emerging as an unexpected regulator of this complex's activity. These findings bring novel insights into the transcriptional aspects of MS and provide potential new criteria for patient stratification.

A novel cryptic splice donor due to synonymous variant in VPS13A as an underlying cause of a chorea-acanthocytosis in a large family

Chorea-acanthocytosis (ChAc) is a rare inherited disease of the nervous system. In this disease the neurological manifestations are associated with acanthocytosis of the red blood cells. The clinical features appear in the third to fourth decades of life. Generalized weakness, choreiform movement disorder, decline in cognition, and psychiatric symptoms are the characteristic features of the disease. The differential diagnosis between Huntington's disease and ChAc is difficult because both the diseases share similar neurological features. Herein, we recruited a large family with multiple individuals initially diagnosed as having Huntington's disease. Analysis of the DNA samples of affected individuals by exome sequencing detected a synonymous variant (NM_001018037.2; c.5040C > T) in the VPS13A. Multiple splice site detection tools were used to predict the potential pathogenicity of the novel synonymous variant. The variant, identified in this study, was predicted to be a cryptic splice donor site that may lead to aberrant pre-mRNA splicing. Reverse transcriptase PCR analyses of patient blood-derived RNA showed activation of a cryptic mid‐exon splice donor, leading to frameshift. The variant was confirmed in all other affected and unaffected individuals using Sanger sequencing.This is the first report of synonymous variants of VPS13A as an underlying cause of ChAc. Our results provide the first direct evidence of the involvement of a synonymous variant of VPS13A in ChAc. Additionally, this study emphasized the importance of considering VPS13A gene mutations in the screening of Huntington's patients.

RNA-Binding Protein-Mediated Alternative Splicing Regulates Abiotic Stress Responses in Plants.

The alternative splicing of pre-mRNA generates distinct mRNA variants from a pre-mRNA, thereby modulating a gene's function. The splicing of pre-mRNA depends on splice sites and regulatory elements in pre-mRNA, as well as the snRNA and proteins that recognize these sequences. Among these, RNA-binding proteins (RBPs) are the primary regulators of pre-mRNA splicing and play a critical role in the regulation of alternative splicing by recognizing the elements in pre-mRNA. However, little is known about the function of RBPs in stress response in plants. Here, we summarized the RBPs involved in the alternative splicing of pre-mRNA and their recognizing elements in pre-mRNA, and the recent advance in the role of RBP-mediated alternative splicing in response to abiotic stresses in plants. This review proposes that the regulation of pre-mRNA alternative splicing by RBPs is an important way for plants to adapt to abiotic stresses, and the regulation of alternative splicing by RBPs is a promising direction for crop breeding.

The role of the U1 snRNP complex in the regulation of gene expression: recent reports

U1 snRNP (U1 small nuclear ribonucleoprotein) is a nuclear ribonucleoprotein complex involved mainly in pre-mRNA splicing, which is a key regulatory process in the eukaryotic gene expression pathway, but also in the process of preventing premature transcription termination (telescripting). U1 snRNP interacts directly with RNA polymerase II, thereby influencing the synthesis and maturation of transcripts in the cell nucleus, including the formation of the 3' end of mRNA and polyadenylation. At the level of cell physiology, it regulates the functioning of mitochondria and energy metabolism. The core of the U1 snRNP complex is U1 snRNA, encoded by many copies of genes that differ in sequence and expression level, and the expression of some of them leads to the formation of defective products. According to current reports, U1 snRNA can be used for therapeutic purposes to regulate gene expression and improve mRNA splicing defects, which are the cause of many diseases. Here we present selected recent discoveries and achievements related to U1 snRNP.

Npac Regulates Pre-mRNA Splicing in Mouse Embryonic Stem Cells.

As a reader of tri-methylated lysine 36 on histone H3 (H3K36me3), Npac has been shown to have a significant role in gene transcription elongation. However, its potential implication in RNA splicing remains unknown. Here, we characterized the phenotypes of Npac knockout in mES cells. We discovered that loss of Npac disrupts pluripotency and identity in mESCs. We also found that Npac is associated with many cellular activities, including cell proliferation, differentiation, and transcription regulation. Notably, we uncovered that Npac is associated with RNA splicing machinery. Furthermore, we found that Npac regulates alternative splicing through its interaction with the splicing factors, including Srsf1. Our research thus highlights the important role of Npac in maintaining ESC identity through the regulation of pre-mRNA splicing.

SNRPB2 promotes triple-negative breast cancer progression by controlling alternative splicing of MDM4 pre-mRNA.

Alternative splicing generates cancer-specific transcripts and is now recognized as a hallmark of cancer. However, the critical oncogenic spliceosome-related proteins involved in triple-negative breast cancer (TNBC) remain elusive. Here, we explored the expression pattern of spliceosome-related proteins in TNBC, non-TNBC, and normal breast tissues from The Cancer Genome Atlas breast cancer (TCGA-BRCA) cohort, revealing higher expression of nearly half of spliceosome-related proteins in TNBC than their counterparts. Among these TNBC-specific spliceosome-related proteins, the expression of SNRPB2 was associated with poor prognosis in patients with TNBC. In TNBC cells, the knockdown of SNRPB2 strongly suppressed cell proliferation and invasion and induced cell cycle arrest. Mechanistically, transcriptome data showed that SNRPB2 knockdown inactivated E2F1 signaling, which regulated the cell cycle. We further validated the downregulation of several cell cycle genes in SNRPB2 knockdown cells. Moreover, the analysis showed that SNRPB2 knockdown triggered the alteration of many alternative splicing events, most of which were skipping of exon. In TNBC cells, it was found that SNRPB2 knockdown led to the skipping of exon 6 in MDM4 pre-mRNA, generating MDM4-S transcript and downregulating MDM4 protein expression. More importantly, downregulation of MDM4 decreased retinoblastoma 1 (Rb1) protein expression, which is a target of MDM4 and a regulator of E2F1 signaling. In summary, the current study revealed an SNRPB2/MDM4/Rb axis in promoting the progression of TNBC, providing novel insights and novel targets for combating TNBC.

An early Transcriptomic Investigation in Adult Patients with Spinal Muscular Atrophy Under Treatment with Nusinersen

Spinal muscular atrophy (SMA) is a rare degenerative disorder with loss of motor neurons caused by mutations in the SMN1 gene. Nusinersen, an antisense oligonucleotide, was approved for SMA treatment to compensate the deficit of the encoded protein SMN by modulating the pre–mRNA splicing of SMN2, the centromeric homologous of SMN1, thus inducing the production of a greater amount of biologically active protein. Here, we reported a 10-month transcriptomics investigation in 10 adult SMA who received nusinersen to search for early genetic markers for clinical monitoring. By comparing their profiles with age-matched healthy controls (HC), we also analyzed the changes in miRNA/mRNAs expression and miRNA-target gene interactions possibly associated with SMA. A multidisciplinary approach of HT-NGS followed by bioinformatics/biostatistics analysis was applied. Within the study interval, those SMA patients who showed some clinical improvements were characterized by having the SMN2/SMN1 ratio slightly increased over the time, while in the stable ones the ratio decreased, suggesting that the estimation of SMN2/SMN1 expression may be an early indicator of nusinersen efficacy. On the other hand, the expression of 38/147 genes/genetic regions DE at T0 between SMA and HC like TRADD and JUND resulted “restored” at T10. We also confirmed the dysregulation of miR-146a(-5p), miR-324-5p and miR-423-5p in SMA subjects. Of interest, miR-146a-5p targeted SMN1, in line with experimental evidence showing the key role of astrocyte-produced miR-146a in SMA motor neuron loss. Molecular pathways such as NOTCH, NF-kappa B, and Toll-like receptor signalings seem to be involved in the SMA pathogenesis.

PTEN controls alternative splicing of autism spectrum disorder-associated transcripts in primary neurons.

Phosphatase and tensin homologue (PTEN) is the main antagonist of the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signalling pathway and mutated in 10-20% of individuals with autism spectrum disorder (ASD) exhibiting macrocephaly. Hyperactive mTOR signalling is responsible for some aspects during PTEN-ASD progression, e.g. neuronal hypertrophy and -excitability, but PI3K/mTOR-independent processes have additionally been described. There is emerging evidence that PTEN regulates gene transcription, spliceosome formation and pre-mRNA splicing independently of PI3K/mTOR. Altered splicing is a hallmark of brains from individuals with idiopathic and PTEN-ASD, however, molecular mechanisms are yet to be identified. We performed RNA-Seq followed by analysis of altered transcript splicing in Pten-deficient primary cortical mouse neurons, which we compared with published data from PTEN-deficient human neuronal stem cells. This analysis identified that transcripts were globally mis-spliced in a developmentally regulated fashion and cluster in synaptic and gene expression regulatory processes. Strikingly, splicing defects following Pten-deficiency represent a significant number of other known ASD-susceptibility genes. Furthermore, we show that exons with strong 3' splice sites are more frequently mis-spliced under Pten-deficient conditions. Our study indicates that PTEN-ASD is a multifactorial condition involving the dysregulation of other known ASD-susceptibility genes.

C2H2-zinc-finger transcription factors bind RNA and function in diverse post-transcriptional regulatory processes

Cys2-His2 zinc-finger proteins (C2H2-ZNFs) constitute the largest class of DNA-binding transcription factors (TFs) yet remain largely uncharacterized. Although certain family members, e.g., GTF3A, have been shown to bind both DNA and RNA, the extent to which C2H2-ZNFs interact with-and regulate-RNA-associated processes is not known. Using UV crosslinking and immunoprecipitation (CLIP), we observe that 148 of 150 analyzed C2H2-ZNFs bind directly to RNA in human cells. By integrating CLIP sequencing (CLIP-seq) RNA-binding maps for 50 of these C2H2-ZNFs with data from chromatin immunoprecipitation sequencing (ChIP-seq), protein-protein interaction assays, and transcriptome profiling experiments, we observe that the RNA-binding profiles of C2H2-ZNFs are generally distinct from their DNA-binding preferences and that they regulate a variety of post-transcriptional processes, including pre-mRNA splicing, cleavage and polyadenylation, and m6A modification of mRNA. Our results thus define a substantially expanded repertoire of C2H2-ZNFs that bind RNA and provide an important resource for elucidating post-transcriptional regulatory programs.

Alternative splicing of Mff regulates AMPK-mediated phosphorylation, mitochondrial fission and antiviral response

Mitochondrial morphology and function change dynamically in response to intracellular signaling and the surrounding environment. The mitochondrial fission factor Mff, which localizes to the outer mitochondrial membrane, mediates not only mitochondrial fission by recruiting the dynamin-related GTPase Drp1 to mitochondrial fission sites but also the double-stranded RNA-induced antiviral response on mitochondria through mitochondrial antiviral signaling (MAVS). Mff is reported to be regulated by AMP-activated protein kinase (AMPK)-mediated protein phosphorylation and alternative pre-mRNA splicing; however, the relationships among RNA splicing, phosphorylation, and multiple functions of Mff have not been fully understood. Here, we showed that mouse Mff has a tissue-specific splicing pattern, and at least eight Mff splice isoforms were expressed in mouse embryonic fibroblasts (MEFs). We introduced single Mff isoforms into Mff knockout MEFs and found that insertion of exon 6 just after the phosphorylation site, by the alternative splicing, reduced its phosphorylation by AMPK and its functions in mitochondrial fission and the antiviral response. In addition, the underlying mechanism repressing these functions was independent of phosphorylation. These results indicate that multiple functions of Mff on mitochondria are regulated by AMPK-mediated phosphorylation and alternative splicing, under the control of energy metabolism and cellular differentiation.

Identification of Entinostat as a Novel Modifier of STAT3 Pre-mRNA Alternative Splicing.

Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic factor involved in multiple vital biological processes and a key mediator of gene transcription in response to cytokines, growth factors and aberrant activation of oncogenic signaling. STAT3 has two splicing isoforms, STAT3α and STAT3β, derived from alternative splicing of exon 23 within pre-mRNA. STAT3β differs from STAT3α by replacement of 55 amino-acid residues in the C-terminal transactivation domain with 7 specific amino acids. Thus, a shorter STAT3β was originally regarded as a dominant negative isoform of STAT3α. Recently accumulating evidence from independent studies have shown STAT3 splicing isoforms confer distinct and overlapping functions in many fundamental cellular regulatory steps such as cell differentiation, inflammatory responses, and cancer progression. However, relatively little is known about the mechanisms of STAT3 pre-mRNA splicing, and it remains undiscovered which chemical compounds or bioactive substances can induce the STAT3β expression. In this study, we generated a potent reporter for detection of alternative splicing of STAT3 pre-mRNA optimized for the screening of function-known chemical library, and successfully identified entinostat, a histone deacetylase inhibitor, as a novel inducer of STAT3β through modulating mRNA splicing. Our findings demonstrate that alternative splicing of STAT3 can be regulated by a compound, providing an important clue for understanding the regulation mechanisms of the expression balance of STAT3 isoforms in a chemical biology approach. Entinostat is likely to be a promising seed compound for elucidating how the higher ratio of STAT3β expression impacts on biological responses associated with Janus kinase (JAK)/STAT3 signaling pathway.

SLC12A1 variant c.1684+1 G>A causes Bartter syndrome type 1 by promoting exon 13 skipping.

Bartter syndrome type 1, an autosomal recessive genetic disorder, is caused by pathogenic loss-of-function variants in the SLC12A1 gene. It is characterized by metabolic alkalosis and prenatal-onset polyuria leading to polyhydramnios. We identified pathogenic gene in a 12-day-old newborn boy with Bartter syndrome type 1 using whole-exome sequencing. Sanger sequencing validated the identified variants. A minigene assay was performed to investigate the effect of a novel splice site variant on pre-mRNA splicing. We found a compound heterozygous variants in the SLC12A1 gene, consisting of a known pathogenic missense mutation (NM_000338: c.769 G>A; p.Gly257Ser) and a novel splice site variant (c.1684+1 G>A). In silico predictions and an in vitro minigene splicing assay demonstrated that the splicing variant c.1684+1 G>A abolished a consensus splice donor site of SLC12A1 intron 13, resulting in complete exon 13 skipping, translational frameshift, and premature termination codon, ultimately leading to loss of SLC12A1 function. Using a cell-based in vitro assay, we revealed the aberrant effect of the pathogenic splicing variant SLC12A1 c.1684+1 G>A on pre-mRNA splicing. Our findings expand the gene mutation spectrum of Bartter syndrome type 1, providing a basis for genetic diagnosis and the development of genetic medicines.

Rare occurrence of cryptic 5' splice sites by downstream 3' splice site/exon boundary mutations in a heavy-ion-induced egy1-4 allele of Arabidopsis thaliana.

Pre-mRNA splicing is a fundamental process in eukaryotic gene expression, and the mechanism of intron definition, involving the recognition of the canonical GU (5'-splice site) and AG (3'-splice site) dinucleotides by splicing factors, has been postulated for most cases of splicing initiation in plants. Splice site mutations have played crucial roles in unraveling the mechanism of pre-mRNA splicing in planta. Typically, splice site mutations abolish splicing events or activate one or more cryptic splice sites surrounding the mutated region. In this report, we investigated the splicing pattern of the EGY1 gene in an Ar-ion-induced egy1-4 allele of Arabidopsis thaliana. egy1-4 has an AG-to-AC mutation in the 3'-end of intron 3, along with 4-bp substitutions and a 5-bp deletion in adjacent exon 4. RT-PCR, cDNA cloning, and amplicon sequencing analyses of EGY1 revealed that while most wild-type EGY1 mRNAs had a single splicing pattern, egy1-4 mRNAs had multiple splicing defects. Almost half of EGY1 transcripts showed 'intron retention' at intron 3, while the other half exhibited activation of 3' cryptic splice sites either upstream or downstream of the original 3'-splice site. Unexpectedly, around 8% of EGY1 transcripts in egy1-4 exhibited activation of cryptic 5'-splice sites positioned upstream of the authentic 5'-splice site of intron 3. Whole genome resequencing of egy1-4 indicated that it has no other known impactful mutations. These results may provide a rare, but real case of activation of cryptic 5'-splice sites by downstream 3'-splice site/exon mutations in planta.