GO:0071013 catalytic step 2 spliceosome: Components, Assembly and Research Methods, Genes, Functions and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0071013 catalytic step 2 spliceosome is the spliceosomal complex that carries out the second catalytic step of pre-mRNA splicing, after the first cleavage at the 5' splice site has occurred.
• It contains three snRNPs, including U5, bound to the splicing intermediate, and its protein composition differs substantially from the catalytic step 1 (activated) spliceosome.
• Cryo-EM structures of yeast and human catalytic step 2 spliceosomes have revealed the architecture of the RNA catalytic core and the conformational changes required for exon ligation.
• CDK11-mediated phosphorylation of SF3B1 regulates pre-mRNA splicing and influences early spliceosome activation and proofreading by DHX15.
• The catalytic step 2 spliceosome is a central node in gene expression, and its dysfunction is linked to splicing-related diseases including cancer and developmental disorders.
• CRISPR knockout, point-mutation, knock-in, overexpression and library screening enable functional dissection of catalytic step 2 spliceosome components in disease models.
Description
The catalytic step 2 spliceosome (GO:0071013) is a cellular-component term describing the spliceosomal complex that executes the second catalytic step of pre-mRNA splicing, the exon-ligation reaction that joins two exons and releases the intron lariat. It is defined as a spliceosomal complex containing three snRNPs, including U5, bound to a splicing intermediate in which the first catalytic cleavage of the 5' splice site has already occurred. Its precise subunit composition differs significantly from that of the catalytic step 1, or activated, spliceosome, and includes many proteins in addition to those found in the associated snRNPs. Understanding this complex is essential because the second catalytic step is the committed, irreversible event that determines the final mRNA sequence, and errors in this step can produce aberrant transcripts with direct consequences for cell physiology. Recent cryo-EM studies of yeast and human spliceosomes have provided near-atomic views of the catalytic step 2 spliceosome, revealing how the RNA catalytic core is positioned and how protein factors stabilize the reaction intermediate. Complementary biochemical work in purified systems has dissected the requirements for step 2 catalysis, showing that specific RNA and protein elements are needed for efficient exon ligation. Together, these structural and biochemical advances make GO:0071013 a tractable target for functional genomics, CRISPR modeling and therapeutic hypothesis testing.
catalytic step 2 spliceosome At A Glance
| GO ID | GO:0071013 |
|---|---|
| GO term | catalytic step 2 spliceosome |
| Ontology | cellular_component |
| Synonym | mammalian spliceosomal complex C; mammalian spliceosomal complex C1; yeast spliceosomal complex A2-2 |
| Major function | Executes the second catalytic step of pre-mRNA splicing (exon ligation) after 5' splice site cleavage |
| snRNP content | Contains three snRNPs, including U5, bound to the splicing intermediate |
| Composition distinction | Subunit composition differs significantly from the catalytic step 1 (activated) spliceosome and includes many additional proteins |
| Structural knowledge | Near-atomic cryo-EM structures available for yeast and human catalytic step 2 spliceosomes |
| Regulatory input | CDK11-dependent phosphorylation of SF3B1 and DHX15-mediated proofreading influence spliceosome activation and progression |
What Is GO:0071013?
In plain terms, GO:0071013 describes the molecular machine that performs the second cut-and-join reaction of splicing. According to the QuickGO definition, it is a spliceosomal complex that contains three snRNPs, including U5, bound to a splicing intermediate in which the first catalytic cleavage of the 5' splice site has occurred. The precise subunit composition differs significantly from that of the catalytic step 1, or activated, spliceosome, and includes many proteins in addition to those found in the associated snRNPs. Synonyms include mammalian spliceosomal complex C, mammalian spliceosomal complex C1, and yeast spliceosomal complex A2-2. This complex represents a distinct functional state rather than a static structure, and its assembly and catalytic competence depend on prior activation steps and on regulatory phosphorylation events.
Why Is catalytic step 2 spliceosome Important in Cell Biology?
The catalytic step 2 spliceosome is important because it catalyzes the exon-ligation reaction that defines the protein-coding output of most human genes, and its assembly state is a checkpoint for splicing fidelity. Structural and biochemical studies show that the complex must undergo precise conformational rearrangements to position the 3' splice site and the branch-point adenosine for catalysis, and that protein factors such as SF3B1 and DHX15 modulate this process. Because splicing errors are associated with human disease, including cancer and developmental disorders, understanding GO:0071013 provides a mechanistic basis for interpreting disease variants and for designing targeted interventions.
• Defines the committed step of exon ligation, which determines mature mRNA sequence and protein identity.
• Provides a structural framework for understanding RNA catalysis in the spliceosome, including the role of U5 snRNP and the RNA catalytic core.
• Serves as a hub for regulatory inputs such as CDK11-mediated SF3B1 phosphorylation and DHX15 helicase proofreading.
• Links splicing fidelity to disease mechanisms, including cancer-associated splicing factor mutations and U6 RNA-related disease associations.
• Enables functional genomics studies using CRISPR knockout, point mutation, knock-in and overexpression models of spliceosome components.
• Supports drug-discovery efforts targeting splicing regulation, because step 2 catalysis is a discrete, structurally characterized state.
• Facilitates interpretation of transcriptomic and proteomic data by providing a defined complex for pathway enrichment and co-expression analyses.
• Connects to RNA biogenesis pathways, including U6 RNA biogenesis, which is relevant to disease association studies.
What Happens During catalytic step 2 spliceosome?
Transition from the activated spliceosome to the catalytic step 2 state
In simple terms: The spliceosome first cuts one end of the intron, then rearranges itself into a new shape that is ready to join the exons.
The catalytic step 2 spliceosome forms after the first catalytic cleavage of the 5' splice site has occurred, and its subunit composition differs significantly from that of the catalytic step 1, or activated, spliceosome. Structural studies of the human spliceosome activated for step 2 of splicing show that this transition involves remodeling of the RNA network and repositioning of the U5 snRNP and associated proteins. Cryo-EM analysis of the yeast catalytic step I spliceosome provides a reference point for understanding the preceding state and the rearrangements that lead to step 2. The precise subunit composition of the step 2 complex includes many proteins in addition to those found in the associated snRNPs, reflecting its specialized catalytic role.
Exon ligation and intron release
In simple terms: In this step, the two exons are stitched together and the intron is released as a lariat.
The catalytic step 2 spliceosome executes the second catalytic step of splicing, in which the 3' splice site is attacked and the two exons are ligated, releasing the intron lariat. Biochemical dissection of step 2 catalysis in a purified yeast system has defined the RNA and protein requirements for efficient exon ligation. The reaction depends on the correct positioning of the branch-point adenosine and the 3' splice site within the catalytic core, which is stabilized by the U5 snRNP and additional proteins. Because this step is essentially irreversible, it represents a key fidelity checkpoint in mRNA maturation.
Conformational equilibrium and proofreading
In simple terms: The spliceosome can wobble between slightly different shapes, and quality-control factors help it choose the correct one.
Structural analysis of the catalytic spliceosome has revealed a conformational equilibrium between distinct states, which is thought to influence catalytic activity and substrate selection. CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1, and this modification affects early spliceosome activation. DHX15 helicase has been implicated in proofreading during early spliceosome activation, providing an additional quality-control layer that can influence progression toward step 2. Together, these findings indicate that the catalytic step 2 spliceosome is not a static endpoint but a dynamic state whose stability is actively regulated.
Coupling to upstream assembly and snRNP biogenesis
In simple terms: The step 2 machine depends on earlier assembly steps and on the availability of properly made snRNP components.
Progression to the catalytic step 2 spliceosome requires prior assembly events, including the function of U2 snRNP components such as Snu17p, which is required for the first catalytic step and for progression of spliceosome assembly. U6 RNA biogenesis is also relevant because U6 is a core catalytic snRNA, and defects in its biogenesis have disease associations. These dependencies mean that perturbations in upstream snRNP assembly or modification can indirectly affect the abundance and activity of the catalytic step 2 spliceosome.
Key Genes Involved in GO:0071013 catalytic step 2 spliceosome
The following genes and proteins are established components or regulators of the catalytic step 2 spliceosome and its associated snRNPs, based on structural, biochemical and functional studies.
| Gene | Major Role | Research Relevance |
|---|---|---|
| SF3B1 | Core U2 snRNP protein; target of CDK11 phosphorylation that regulates splicing | Frequently mutated in cancer; central to step 2 activation and inhibitor studies |
| CDK11 | Kinase that phosphorylates SF3B1 and regulates pre-mRNA splicing | Regulatory node for spliceosome activation; candidate drug target |
| DHX15 | Helicase implicated in proofreading during early spliceosome activation | Quality-control factor influencing progression to step 2 |
| U5 snRNP components | Structural and catalytic components of the step 2 spliceosome | Essential for exon ligation and catalytic core stability |
| U2 snRNP components | Contribute to branch-point recognition and step 1 catalysis | Required for progression of spliceosome assembly |
| U6 snRNA | Catalytic snRNA of the spliceosome | Biogenesis defects have disease associations |
| Snu17p (yeast) | U2 snRNP protein required for first catalytic step and assembly progression | Model for studying step 1 to step 2 transition |
| Prp8 | Core U5 protein forming part of the catalytic center | Key structural determinant of the catalytic step 2 state |
| Prp16 | ATPase/helicase involved in step 2 fidelity | Model for proofreading and conformational equilibrium |
| Prp22 | Helicase involved in mRNA release after exon ligation | Relevant to post-catalytic complex disassembly |
| SF3A1 | U2 snRNP component contributing to spliceosome assembly | Potential modifier of step 2 progression |
| SF3B2 | U2 snRNP component associated with SF3B complex | Candidate for functional studies of step 2 regulation |
| SF3B3 | U2 snRNP component associated with SF3B complex | Relevant to splicing inhibitor response |
| SF3B5 | U2 snRNP component associated with SF3B complex | Potential target for splicing modulation |
| SF3B6 | U2 snRNP component associated with SF3B complex | Candidate for CRISPR perturbation studies |
| CWC22 | Protein associated with step 2 catalysis in yeast and human | Model for exon ligation requirements |
| Prp18 | Protein required for step 2 catalysis in purified systems | Biochemical model for exon ligation |
| Slu7 | Protein required for step 2 catalysis in purified systems | Biochemical model for exon ligation |
How Is catalytic step 2 spliceosome Regulated?
The catalytic step 2 spliceosome is regulated by phosphorylation and by ATP-dependent helicases. CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1, and this modification influences early spliceosome activation. Structural and biochemical evidence indicates that DHX15 helicase contributes to proofreading during early spliceosome activation, thereby influencing progression toward the catalytic step 2 state. The catalytic spliceosome exists in a conformational equilibrium between distinct states, and this equilibrium is thought to be modulated by protein factors and ATP-dependent remodeling. In addition, upstream assembly steps, including U2 snRNP function and U6 RNA biogenesis, set the stage for step 2 catalysis and can therefore indirectly regulate its efficiency. These layers of regulation ensure that exon ligation occurs only after proper splice-site recognition and branch-point positioning.
catalytic step 2 spliceosome and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| SF3B1 | Cancer-associated splicing factor mutations and altered splicing | Knock-in of disease-associated point mutations in cell lines |
| CDK11 | Regulation of splicing and potential cancer dependency | Knockout and point-mutation models to dissect kinase function |
| DHX15 | Proofreading of spliceosome activation | Knockout and helicase-dead knock-in models |
| U6 snRNA pathway genes | U6 RNA biogenesis and disease association | Knockout and overexpression models of biogenesis factors |
| U2 snRNP genes (e.g., Snu17p homologs) | Spliceosome assembly progression and step 1 catalysis | Knockout and tagged knock-in models for assembly studies |
Cancer and splicing factor mutations
Splicing factors that function in or regulate the catalytic step 2 spliceosome, such as SF3B1, are recurrently mutated in human cancers, and CDK11-mediated phosphorylation of SF3B1 is a key regulatory event in pre-mRNA splicing. Because the catalytic step 2 spliceosome executes exon ligation, mutations that alter its assembly or activity can change the mRNA isoform landscape of tumor cells. Structural knowledge of the step 2 state provides a framework for interpreting how cancer-associated mutations affect catalysis and for designing splicing-modulatory therapeutics.
Developmental and splicing-related disorders
Proper spliceosome assembly and catalysis are required for normal development, and defects in core splicing components can lead to developmental disorders. U6 RNA biogenesis and disease association studies highlight how disruption of core snRNA pathways can affect spliceosome function. U2 snRNP proteins such as Snu17p are required for the first catalytic step and for progression of spliceosome assembly, indicating that perturbations in early assembly can indirectly impair step 2 catalysis.
Splicing fidelity and transcriptome instability
The catalytic step 2 spliceosome is a fidelity checkpoint, and its conformational equilibrium and proofreading mechanisms help ensure accurate exon ligation. DHX15-mediated proofreading during early spliceosome activation provides one layer of quality control that can influence downstream step 2 catalysis. When fidelity is compromised, aberrant transcripts can accumulate, with potential consequences for cellular homeostasis and disease.
From catalytic step 2 spliceosome-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is a candidate splicing factor required for step 2 catalysis? | CRISPR knockout cell line followed by RT-PCR and RNA-seq |
| Does a disease-associated point mutation alter step 2 efficiency? | Point-mutation knock-in cell line |
| Where does a protein localize within the catalytic step 2 spliceosome? | Tagged knock-in with affinity purification and proteomics |
| Does overexpression of a splicing factor change isoform usage? | Overexpression cell model with RNA-seq |
| Which genes buffer loss of a step 2 component? | CRISPR library screening with fitness readouts |
| How does a regulatory kinase affect step 2 progression? | Kinase knockout or inhibitor-treated cells with splicing assays |
How to Study the catalytic step 2 spliceosome Process
| Method | What It Measures | Typical Application |
|---|---|---|
| RNA-seq | Global changes in splicing and gene expression | Phenotyping CRISPR knockout or point-mutation cells |
| RT-PCR | Specific exon ligation and intron retention events | Validating step 2 catalysis defects |
| Cryo-EM | Three-dimensional structure of spliceosomal complexes | Mapping catalytic core and protein interactions |
| In vitro splicing assay | Catalytic activity of purified spliceosomes | Testing requirements for step 2 catalysis |
| Affinity purification-mass spectrometry | Protein composition of the catalytic step 2 spliceosome | Defining subunit composition and interactors |
| Phosphoproteomics | Phosphorylation events on splicing factors | Identifying regulatory modifications such as SF3B1 phosphorylation |
| CRISPR library screening | Fitness effects of gene perturbations | Identifying modifiers of step 2 spliceosome function |
| Fluorescence imaging | Localization and dynamics of spliceosome components | Studying assembly and disassembly in cells |
RNA-seq and splicing assays
RNA-seq is widely used to measure changes in splicing patterns after perturbation of catalytic step 2 spliceosome components, including exon skipping and intron retention events. Targeted RT-PCR assays can specifically monitor the second catalytic step by detecting ligated exons and released intron lariats. These methods are typically applied to CRISPR knockout or point-mutation cell models to link genotype to splicing phenotype.
Structural biology (cryo-EM)
Cryo-EM has been used to determine near-atomic structures of yeast and human spliceosomes, including the catalytic step 2 state, revealing the architecture of the RNA catalytic core and associated proteins. These structures provide a template for interpreting disease mutations and for designing experiments that test the role of specific residues. Structural analysis of the conformational equilibrium of the catalytic spliceosome has also highlighted dynamic states relevant to regulation.
Biochemical reconstitution and catalysis assays
Purified in vitro splicing systems allow dissection of step 2 catalysis requirements, including the roles of specific proteins and RNA elements. Such assays can be combined with mutant proteins or modified RNAs to test mechanistic hypotheses. Biochemical reconstitution complements structural and cellular approaches by isolating the minimal components needed for exon ligation.
Proteomics and interactomics
Affinity purification coupled to mass spectrometry can define the protein composition of the catalytic step 2 spliceosome and identify associated factors. Tagged knock-in models enable endogenous-complex purification, reducing artifacts from overexpression. Proteomic data can also reveal phosphorylation events, such as CDK11-dependent SF3B1 phosphorylation, that regulate spliceosome function.
How CRISPR Can Be Used to Study GO:0071013 catalytic step 2 spliceosome
Knockout
CRISPR knockout of genes encoding catalytic step 2 spliceosome components or regulators, such as SF3B1 or CDK11, can reveal essentiality and splicing phenotypes. Knockout cell lines are typically validated by RT-PCR and RNA-seq to detect changes in exon ligation and intron retention. Because many spliceosome components are essential, inducible or conditional knockout strategies may be required to study their roles.
Point Mutation
Point-mutation knock-in can model disease-associated variants in splicing factors and test their effects on step 2 catalysis. For example, mutations affecting SF3B1 phosphorylation sites can be introduced to dissect CDK11-dependent regulation. Such models are valuable for linking specific residues to catalytic efficiency and fidelity.
Knock-in
Tagged knock-in of endogenous spliceosome genes enables affinity purification and imaging of the catalytic step 2 spliceosome without overexpression artifacts. Knock-in of reporter cassettes can also be used to monitor splicing of specific substrates in live cells. These models support structural and proteomic studies of the native complex.
Overexpression
Overexpression of splicing factors or regulatory kinases can be used to test gain-of-function effects on step 2 catalysis and isoform usage. Overexpression models are particularly useful for studying dominant effects of disease-associated variants. Combining overexpression with RNA-seq provides a global view of splicing changes.
How EDITGENE Supports catalytic step 2 spliceosome Research
Researchers studying catalytic step 2 spliceosome-related genes often need to determine whether a candidate gene is causally involved in splicing regulation, disease-associated isoform changes, or cellular fitness. Establishing causality requires precise genetic models that can isolate the contribution of a single gene or variant within the complex. EDITGENE provides end-to-end CRISPR services to generate such models and to support downstream functional and bioinformatic analysis.
Contact EDITGENE today to design your custom CRISPR model for catalytic step 2 spliceosome research.
Frequently Asked Questions About catalytic step 2 spliceosome
What is GO:0071013 catalytic step 2 spliceosome?
GO:0071013 is a cellular-component term describing the spliceosomal complex that contains three snRNPs, including U5, bound to a splicing intermediate after the first catalytic cleavage of the 5' splice site, and that carries out the second catalytic step of pre-mRNA splicing.
What genes are involved in the catalytic step 2 spliceosome?
Key genes and proteins include SF3B1, CDK11, DHX15, U5 and U2 snRNP components, U6 snRNA, Prp8, Prp16, Prp22, and step 2 factors such as CWC22, Prp18 and Slu7.
How does the catalytic step 2 spliceosome differ from the step 1 spliceosome?
Its precise subunit composition differs significantly from that of the catalytic step 1, or activated, spliceosome, and it includes many proteins in addition to those found in the associated snRNPs.
What reaction does the catalytic step 2 spliceosome catalyze?
It catalyzes the second catalytic step of splicing, in which the 3' splice site is attacked and the two exons are ligated, releasing the intron lariat.
How is the catalytic step 2 spliceosome regulated?
It is regulated by phosphorylation events such as CDK11-mediated SF3B1 phosphorylation and by ATP-dependent helicases such as DHX15 that contribute to proofreading during early spliceosome activation.
Which diseases are linked to catalytic step 2 spliceosome dysfunction?
Splicing factor mutations, including those in SF3B1, are linked to cancer, and defects in core splicing components and U6 RNA biogenesis have disease associations.
What methods are used to study the catalytic step 2 spliceosome?
Common methods include cryo-EM, in vitro splicing assays, RNA-seq, RT-PCR, affinity purification-mass spectrometry, phosphoproteomics and CRISPR screening.
Can CRISPR be used to study catalytic step 2 spliceosome genes?
Yes, CRISPR knockout, point-mutation knock-in, tagged knock-in, overexpression and library screening are all used to dissect the function of step 2 spliceosome components and regulators.
What is the role of CDK11 in the catalytic step 2 spliceosome?
CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1, influencing early spliceosome activation and progression toward step 2.
What is the role of DHX15 in spliceosome proofreading?
DHX15 helicase has been implicated in proofreading during early spliceosome activation, providing a quality-control layer that can influence step 2 catalysis.
Conclusion
GO:0071013 catalytic step 2 spliceosome defines the spliceosomal state that performs exon ligation, a committed and highly regulated step in mRNA maturation. Structural, biochemical and functional studies have revealed its composition, dynamics and regulatory inputs, including CDK11-mediated SF3B1 phosphorylation and DHX15-dependent proofreading. Because splicing defects are linked to cancer and other diseases, precise CRISPR models of step 2 spliceosome components are valuable for mechanistic and translational research. EDITGENE supports this work with knockout, point-mutation, knock-in, overexpression and library screening services tailored to splicing biology.
References
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- 2. Wan R et al.. 2016. Structure of a yeast catalytic step I spliceosome at 3.4 Å resolution.. Science 353(6302):895-904 PMID: 27445308
- 3. Bertram K et al.. 2017. Cryo-EM structure of a human spliceosome activated for step 2 of splicing.. Nature 542(7641):318-323 PMID: 28076346
- 4. Wilkinson ME et al.. 2021. Structural basis for conformational equilibrium of the catalytic spliceosome.. Mol Cell 81(7):1439-1452.e9 PMID: 33705709
- 5. Mroczek S et al.. 2013. U6 RNA biogenesis and disease association.. Wiley Interdiscip Rev RNA 4(5):581-92 PMID: 23776162
- 6. Gottschalk A et al.. 2001. A novel yeast U2 snRNP protein, Snu17p, is required for the first catalytic step of splicing and for progression of spliceosome assembly.. Mol Cell Biol 21(9):3037-46 PMID: 11287609
- 7. Zhang Z et al.. 2026. Structural basis of the regulation by CDK11 kinase of early spliceosome activation and evidence for its proofreading by DHX15 helicase.. Nat Commun 17(1) PMID: 42399621
- 8. Ohrt T et al.. 2013. Molecular dissection of step 2 catalysis of yeast pre-mRNA splicing investigated in a purified system.. RNA 19(7):902-15 PMID: 23685439
