GO:0000398 mRNA splicing, via spliceosome: Mechanism, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0000398 (mRNA splicing, via spliceosome) is the biological process that joins exons and removes introns from primary mRNA transcripts through a spliceosomal mechanism.
• The spliceosome is a dynamic ribonucleoprotein machine that assembles on pre-mRNA in stepwise fashion and catalyzes two transesterification reactions.
• Spliceosome activity is regulated by phosphorylation, notably through the CDK7-CDK11 axis, linking transcription and splicing.
• Mis-splicing caused by mutations in spliceosome components such as U2AF1 and SF3B3 drives cancer progression and chemotherapy resistance.
• Spliceosome-exosome coupling provides nuclear mRNA surveillance, degrading aberrant transcripts to protect cellular integrity.
• CRISPR-based knockout, point mutation, knock-in, and overexpression models enable causal dissection of splicing factor function in disease.
Description
mRNA splicing, via spliceosome (GO:0000398) is the essential biological process that removes introns from primary messenger RNA transcripts and ligates exons to produce mature mRNA. This process is carried out by the spliceosome, a highly dynamic ribonucleoprotein complex that recognizes splice sites and catalyzes two sequential transesterification reactions. Because most human genes contain introns, spliceosome-mediated splicing is required for the expression of nearly all protein-coding genes and expands proteome diversity through alternative splicing. Dysregulation of this process is increasingly recognized as a driver of human disease, including cancer and hematological malignancies. Understanding the molecular mechanisms, regulatory inputs, and disease relevance of GO:0000398 is therefore critical for both basic biology and therapeutic development.
mRNA splicing, via spliceosome At A Glance
| GO ID | GO:0000398 |
|---|---|
| GO term | mRNA splicing, via spliceosome |
| Ontology | biological_process |
| Synonym | mRNA splicing; nuclear mRNA splicing, via spliceosome; nuclear mRNA splicing via U12-type spliceosome; nuclear mRNA splicing via U2-type spliceosome; pre-mRNA splicing; splicing AT-AC intron; splicing GT-AG intron |
| Major function | Removal of introns and joining of exons from pre-mRNA to produce mature mRNA |
| Cellular location | Nucleus, particularly nuclear speckles |
| Key machinery | Spliceosome (snRNPs U1, U2, U4/U6, U5 and associated proteins) |
| Regulatory kinases | CDK7 and CDK11 regulate spliceosome assembly and catalysis |
| Surveillance pathway | Spliceosome-exosome coupling for nuclear mRNA quality control |
What Is GO:0000398?
GO:0000398 describes the joining together of exons from one or more primary transcripts of messenger RNA and the excision of intron sequences via a spliceosomal mechanism, so that mRNA consisting only of the joined exons is produced. This definition encompasses both U2-type and U12-type spliceosomal splicing and includes the processing of GT-AG and AT-AC introns.
Why Is mRNA splicing, via spliceosome Important in Cell Biology?
mRNA splicing, via spliceosome is fundamental to gene expression because it converts pre-mRNA into mature mRNA and enables alternative splicing, which vastly expands proteomic diversity. Defects in this process cause widespread mis-splicing that contributes to cancer, chemotherapy resistance, and other diseases. Moreover, the spliceosome is a target of regulatory signaling pathways, including the CDK7-CDK11 axis, making it a dynamic hub for cellular adaptation. Nuclear mRNA surveillance through spliceosome-exosome coupling further highlights its role in maintaining transcriptome fidelity.
• Essential for maturation of nearly all protein-coding transcripts in eukaryotes.
• Enables alternative splicing, generating multiple protein isoforms from a single gene.
• Mutations in spliceosome components such as U2AF1 cause mis-splicing and chemotherapy resistance in acute myeloid leukemia.
• SF3B3-regulated mTOR alternative splicing promotes colorectal cancer progression and metastasis.
• Spliceosome phase separation, regulated by USP42, supports tumorigenesis through nuclear speckle dynamics.
• RBM25-dependent ACLY RNA splicing restrains inflammation via metabolic rewiring.
• CDK7-CDK11 axis coordinates transcription and splicing, linking cell cycle control to RNA processing.
• Spliceosome-exosome pathway provides nuclear mRNA surveillance and degrades aberrant transcripts.
• Serves as a therapeutic target for splicing modulators in oncology and genetic disorders.
• Conserved from yeast to humans, enabling mechanistic studies in model organisms.
What Happens During mRNA splicing, via spliceosome?
Spliceosome Assembly on Pre-mRNA
In simple terms: The spliceosome is built piece by piece on the pre-mRNA, like assembling a machine around the part that needs cutting.
Spliceosome assembly begins with recognition of the 5' splice site by U1 snRNP and the branch point by SF1/BBP, followed by U2 snRNP recruitment to the branch point. This early complex (complex A) is stabilized by protein factors including U2AF and SF3B3, which help define exon-intron boundaries. In Saccharomyces cerevisiae, similar stepwise assembly ensures fidelity of splice site selection. The assembly process is regulated by phosphorylation events, including those mediated by CDK7 and CDK11.
Catalytic Activation and Transesterification
In simple terms: Once fully assembled, the spliceosome cuts the intron at two points and stitches the exons together in two chemical steps.
The catalytically active spliceosome (complex B) undergoes rearrangements that bring the 5' splice site, branch point, and 3' splice site into proximity. The first transesterification reaction forms a lariat intron intermediate, and the second reaction joins the exons and releases the intron lariat. These reactions are mediated by the RNA components of snRNPs and associated proteins, with dynamic conformational changes driven by ATP-dependent helicases. CDK11 has been implicated in regulating catalytic steps of splicing.
Spliceosome Disassembly and Exon Ligation
In simple terms: After the intron is removed and exons are joined, the spliceosome falls apart and the mature mRNA is released.
Following catalysis, the spliceosome disassembles in an ATP-dependent manner, releasing the mRNA and intron lariat. The exon junction complex is deposited on the spliced mRNA, marking it for downstream processes such as export and translation. Disassembly factors including Prp43 and Ntr1/Ntr2 are conserved from yeast to humans. Proper disassembly is essential for recycling of spliceosomal components.
Nuclear mRNA Surveillance and Quality Control
In simple terms: If splicing goes wrong, a surveillance system catches the faulty mRNA and destroys it.
The spliceosome is functionally coupled to the exosome, a 3'-5' exonuclease complex that degrades aberrant or unprocessed transcripts in the nucleus. This spliceosome-exosome pathway is evolutionarily conserved and ensures that only correctly spliced mRNAs reach the cytoplasm. Defects in this surveillance can lead to accumulation of mis-spliced transcripts with pathogenic potential. This quality control mechanism is critical for maintaining transcriptome integrity.
Regulation by Phase Separation and Signaling
In simple terms: Splicing factors can cluster together in liquid-like droplets, and signaling enzymes can turn splicing on or off.
USP42 drives nuclear speckle mRNA splicing by directing dynamic phase separation, promoting tumorigenesis. The CDK7-CDK11 axis regulates spliceosome assembly and catalytic activity through phosphorylation of splicing factors. RBM25 modulates ACLY RNA splicing to rewire metabolism and restrain inflammation. These regulatory layers allow splicing to respond to cellular signals and metabolic states.
Key Genes Involved in GO:0000398 mRNA splicing, via spliceosome
The following genes and proteins are core components or regulators of mRNA splicing, via spliceosome, with established roles in spliceosome assembly, catalysis, regulation, and disease.
| Gene | Major Role | Research Relevance |
|---|---|---|
| U2AF1 | Recognizes 3' splice site and recruits U2 snRNP | Mutations cause mis-splicing and chemotherapy resistance in AML |
| SF3B3 | Component of SF3b complex in U2 snRNP | Regulates mTOR alternative splicing in colorectal cancer |
| RBM25 | RNA-binding protein regulating alternative splicing | Controls ACLY splicing and inflammation via metabolic rewiring |
| USP42 | Deubiquitinase driving phase separation | Promotes nuclear speckle splicing and tumorigenesis |
| CDK7 | Cyclin-dependent kinase phosphorylating RNA Pol II and splicing factors | Regulates spliceosome assembly and transcription-splicing coupling |
| CDK11 | Cyclin-dependent kinase regulating splicing | Part of CDK7-CDK11 axis in spliceosome regulation |
| U1 snRNP | Recognizes 5' splice site | Core spliceosomal component |
| U2 snRNP | Recognizes branch point | Core spliceosomal component |
| U4/U6 snRNP | Forms tri-snRNP with U5 | Core spliceosomal component |
| U5 snRNP | Catalytic core component | Core spliceosomal component |
| Prp43 | ATP-dependent helicase for spliceosome disassembly | Conserved disassembly factor |
| Ntr1/Ntr2 | Disassembly factors | Conserved from yeast to humans |
| Exosome complex | 3'-5' exonuclease for mRNA surveillance | Coupled to spliceosome for quality control |
| SF1/BBP | Branch point recognition | Early spliceosome assembly factor |
| U2AF | 3' splice site recognition | Essential splicing factor |
| Spliceosomal ATPases | Drive conformational changes | Energy for spliceosome dynamics |
| Nuclear speckle proteins | Organize splicing factors | Phase separation and splicing regulation |
How Is mRNA splicing, via spliceosome Regulated?
mRNA splicing, via spliceosome is regulated at multiple levels. The CDK7-CDK11 axis controls spliceosome assembly and catalytic activity through phosphorylation of splicing factors and RNA polymerase II. USP42 directs dynamic phase separation to organize nuclear speckles and promote splicing. RBM25 regulates alternative splicing of ACLY to rewire metabolism and restrain inflammation. Additionally, the spliceosome is coupled to the exosome for nuclear mRNA surveillance, ensuring degradation of aberrant transcripts. These regulatory mechanisms allow splicing to respond to cellular signals and maintain transcriptome fidelity.
mRNA splicing, via spliceosome and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| U2AF1 | Acute myeloid leukemia, chemotherapy resistance | Point mutation knock-in in AML cell lines |
| SF3B3 | Colorectal cancer progression and metastasis | Knockout or knockdown in CRC cell lines |
| USP42 | Tumorigenesis via phase separation | Overexpression and knockout in cancer cells |
| RBM25 | Inflammation and metabolic rewiring | Knockout in immune or metabolic cell models |
| Exosome components | Nuclear mRNA surveillance defects | Knockout in yeast or human cells |
Spliceosome Mutations in Cancer
Mutations in spliceosome components such as U2AF1 cause mis-splicing of mRNA translation genes, conferring resistance to chemotherapy in acute myeloid leukemia. SF3B3-regulated mTOR alternative splicing promotes colorectal cancer progression and metastasis. These findings establish spliceosome dysregulation as a driver of cancer pathogenesis and a potential therapeutic target.
Spliceosome Phase Separation and Tumorigenesis
USP42 drives nuclear speckle mRNA splicing via dynamic phase separation to promote tumorigenesis. This highlights how aberrant regulation of splicing factor condensation can contribute to cancer. Targeting phase separation machinery may offer new therapeutic avenues.
Inflammation and Metabolic Rewiring
RBM25 is required to restrain inflammation via ACLY RNA splicing-dependent metabolism rewiring. This links splicing regulation to inflammatory responses and metabolic pathways. Dysregulation of this axis may contribute to inflammatory diseases.
Nuclear mRNA Surveillance Defects
The evolutionarily conserved spliceosome-exosome pathway in nuclear mRNA surveillance degrades aberrant transcripts. Defects in this pathway can lead to accumulation of mis-spliced mRNAs with pathogenic potential. Understanding this surveillance mechanism is important for diseases linked to RNA processing defects.
From mRNA splicing, via spliceosome-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of a splicing factor affect cell viability? | CRISPR knockout in cancer cell lines |
| Does a specific mutation in U2AF1 cause mis-splicing? | Point mutation knock-in in AML cells |
| How does a splicing factor fusion affect splicing? | Knock-in of tagged fusion protein |
| Does overexpression of a splicing regulator promote tumorigenesis? | Overexpression in cell lines and xenografts |
| What is the role of phase separation in splicing? | Tagged knock-in with fluorescent reporters |
| How does spliceosome-exosome coupling work? | Knockout of exosome components in yeast |
How to Study the mRNA splicing, via spliceosome Process
| Method | What It Measures | Typical Application |
|---|---|---|
| RNA-seq | Splicing events and isoform expression | Detect mis-splicing in cancer cells |
| CRISPR screen | Essential splicing factors | Identify therapeutic targets |
| Affinity proteomics | Spliceosome composition | Study assembly and regulation |
| Fluorescence microscopy | Nuclear speckle and condensate dynamics | Analyze phase separation |
| Ribo-seq | Translation efficiency of spliced mRNAs | Link splicing to protein output |
| CLIP-seq | RNA binding sites of splicing factors | Map RBM25 targets |
| Yeast genetics | Conserved splicing mechanisms | Study spliceosome-exosome coupling |
| Phosphoproteomics | Kinase substrates in splicing | Analyze CDK7-CDK11 axis |
RNA Sequencing and Splice Isoform Analysis
RNA-seq enables genome-wide detection of splicing events and quantification of isoform usage. It is used to identify mis-splicing caused by spliceosome mutations. Alternative splicing changes can be analyzed using computational tools.
CRISPR Screens for Splicing Factors
CRISPR library screening can identify splicing factors required for cell growth or drug resistance. Pooled screens with splicing reporters enable functional dissection of spliceosome components. This approach is powerful for discovering novel regulators.
Proteomics and Interactomics
Affinity purification coupled with mass spectrometry identifies spliceosome-associated proteins. Proteomics can reveal dynamic changes in spliceosome composition during assembly. This method is useful for studying regulatory phosphorylation.
Imaging and Phase Separation Assays
Fluorescence microscopy visualizes nuclear speckles and phase-separated condensates. Live-cell imaging tracks spliceosome dynamics in real time. These assays are essential for studying USP42-mediated phase separation.
How CRISPR Can Be Used to Study GO:0000398 mRNA splicing, via spliceosome
Knockout
CRISPR knockout of splicing factors such as SF3B3 or RBM25 enables loss-of-function studies to determine their role in splicing and disease. Knockout cell models are used to assess effects on proliferation, metastasis, and inflammation. These models are essential for validating candidate genes from screens.
Point Mutation
Point mutation knock-in of U2AF1 mutations recapitulates mis-splicing and chemotherapy resistance observed in AML patients. This approach allows precise modeling of disease-associated mutations. It is valuable for testing targeted therapies.
Knock-in
Tagged knock-in of splicing factors, such as fluorescently labeled USP42, enables live-cell imaging of phase separation and nuclear speckle dynamics. Knock-in of epitope tags facilitates proteomic analysis of spliceosome complexes. This strategy is useful for studying dynamic assembly.
Overexpression
Overexpression of splicing regulators like USP42 promotes tumorigenesis and can be used to study gain-of-function effects. Overexpression models help identify downstream splicing changes and oncogenic pathways. They complement knockout studies for bidirectional analysis.
How EDITGENE Supports mRNA splicing, via spliceosome Research
Researchers studying mRNA splicing, via spliceosome-related genes often need to determine whether a candidate gene is causally involved in splicing regulation, disease progression, or therapeutic response. EDITGENE provides comprehensive CRISPR-based services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for mRNA splicing, via spliceosome research.
Frequently Asked Questions About mRNA splicing, via spliceosome
What is mRNA splicing, via spliceosome?
It is the biological process GO:0000398 that removes introns and joins exons from pre-mRNA using the spliceosome to produce mature mRNA.
What genes are involved in mRNA splicing, via spliceosome?
Key genes include U2AF1, SF3B3, RBM25, USP42, CDK7, CDK11, and snRNP components.
How is the spliceosome assembled?
The spliceosome assembles stepwise on pre-mRNA, with U1 and U2 snRNPs recognizing splice sites, followed by tri-snRNP recruitment and catalytic activation.
What diseases are linked to spliceosome dysfunction?
Spliceosome mutations are linked to acute myeloid leukemia, colorectal cancer, and other malignancies.
How does CDK7-CDK11 regulate splicing?
CDK7 and CDK11 phosphorylate splicing factors and RNA polymerase II to coordinate transcription and splicing.
What is the role of phase separation in splicing?
Phase separation organizes splicing factors into nuclear speckles, and USP42 drives this process to promote tumorigenesis.
How does RBM25 regulate inflammation?
RBM25 controls ACLY RNA splicing to rewire metabolism and restrain inflammation.
What is spliceosome-exosome coupling?
It is a conserved surveillance pathway where the spliceosome is coupled to the exosome to degrade aberrant transcripts.
How can CRISPR help study splicing?
CRISPR knockout, point mutation, knock-in, and overexpression models enable functional dissection of splicing factors in disease.
What methods are used to study splicing?
RNA-seq, CRISPR screens, proteomics, and imaging are commonly used to study splicing mechanisms and regulation.
Conclusion
mRNA splicing, via spliceosome (GO:0000398) is a central biological process required for the maturation of nearly all protein-coding transcripts. Its dysregulation contributes to cancer, chemotherapy resistance, and inflammatory diseases. Advances in CRISPR modeling and high-throughput methods continue to illuminate the mechanisms and therapeutic potential of targeting the spliceosome.
References
- 1. Liu S et al.. 2021. USP42 drives nuclear speckle mRNA splicing via directing dynamic phase separation to promote tumorigenesis.. Cell Death Differ 28(8):2482-2498 PMID: 33731873
- 2. Matera AG et al.. 2014. A day in the life of the spliceosome.. Nat Rev Mol Cell Biol 15(2):108-21 PMID: 24452469
- 3. Rájecký M et al.. 2025. CDK7-CDK11 axis in spliceosome regulation and pre-mRNA splicing.. Nucleic Acids Res 53(22) PMID: 41428738
- 4. Zhang Y et al.. 2024. RBM25 is required to restrain inflammation via ACLY RNA splicing-dependent metabolism rewiring.. Cell Mol Immunol 21(11):1231-1250 PMID: 39251781
- 5. Senn KA et al.. 2024. Mechanisms and regulation of spliceosome-mediated pre-mRNA splicing in Saccharomyces cerevisiae.. Wiley Interdiscip Rev RNA 15(4):e1866 PMID: 38972853
- 6. Xu T et al.. 2024. SF3B3-regulated mTOR alternative splicing promotes colorectal cancer progression and metastasis.. J Exp Clin Cancer Res 43(1):126 PMID: 38671459
- 7. Jin P et al.. 2024. Mutant U2AF1-Induced Mis-Splicing of mRNA Translation Genes Confers Resistance to Chemotherapy in Acute Myeloid Leukemia.. Cancer Res 84(10):1583-1596 PMID: 38417135
- 8. Abbas DK et al.. 2026. Evolutionarily conserved spliceosome-exosome pathway in nuclear mRNA surveillance.. Genes Dev 40(13-14):1119-1132 PMID: 42140674
