GO:0000386 second spliceosomal transesterification activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0000386 describes the second transesterification reaction of spliceosomal mRNA splicing, in which the free 3'-hydroxyl group of the 5' exon attacks the 3' splice site to ligate exons and release the intron lariat.
• The reaction is catalyzed by the step II catalytically activated spliceosome, a large ribonucleoprotein complex containing Prp8, Prp16, Prp22, Cwc2, U6 snRNA and other factors.
• Prp16 ATPase drives the transition between the first and second steps by destabilizing Cwc2, U6 snRNA and Prp8 interactions.
• Prp22 is a RNA helicase required for the second step and for release of the spliced mRNA.
• The second step can be distinguished from the first step using adenosine phosphorothioate analogs and quantitative in vitro splicing assays.
• Dysregulation of second-step splicing factors has been linked to cancer biology, including Mcl1 regulation in neuroblastoma.
Description
GO:0000386, second spliceosomal transesterification activity, is a molecular function that catalyzes the second chemical step of pre-mRNA splicing. In this reaction, the free 3'-hydroxyl group of the 5' exon acts as a nucleophile and attacks the 3' splice site, joining the two exons and releasing the intron as a lariat structure. This step is essential for producing mature messenger RNA in eukaryotes and is carried out by the step II catalytically activated spliceosome. Researchers study this activity to understand how spliceosomal rearrangements and ATP-dependent helicases control the fidelity and timing of exon ligation. The second transesterification step is mechanistically distinct from the first step and can be selectively probed with nucleotide analogs and high-throughput in vitro assays. Because exon ligation determines the final mRNA sequence, defects in this step can alter gene expression programs relevant to cancer and other diseases.
second spliceosomal transesterification activity At A Glance
| GO ID | GO:0000386 |
|---|---|
| GO term | second spliceosomal transesterification activity |
| Ontology | molecular_function |
| Synonym | 3'-splice site cleavage; exon ligation; lariat formation; 5'-splice site cleavage |
| Major function | Catalysis of exon ligation and intron lariat release during spliceosomal mRNA splicing |
| Catalytic complex | Step II catalytically activated spliceosome |
| Key cofactor | ATP-dependent RNA helicases such as Prp16 and Prp22 |
| Substrate | Pre-mRNA exon-intron intermediate with a free 5' exon 3'-OH |
| Product | Ligated exons and intron lariat |
What Is GO:0000386?
GO:0000386 is defined as the catalysis of the second transesterification reaction of spliceosomal mRNA splicing. During this reaction, the free 3'-hydroxyl group of the 5' exon attacks the 3' splice site, leading to ligation of the two exons and detachment of the non-expressed intronic sequences. In cis splicing, the intron is released in a lariat structure.
Why Is second spliceosomal transesterification activity Important in Cell Biology?
The second transesterification reaction is the committed step that defines the final exon-exon junction of mature mRNA. Its correct execution is required for accurate gene expression, and its misregulation can change the coding sequence or abundance of transcripts that control cell growth and survival. Because the reaction is catalyzed by a dynamic ribonucleoprotein machine, it also serves as a model for understanding RNA-catalyzed chemistry and ATP-dependent remodeling in the spliceosome.
• Defines the final exon-exon junction of mature mRNA.
• Required for release of the intron lariat and spliced mRNA.
• Controlled by ATP-dependent helicases Prp16 and Prp22.
• Can be selectively inhibited or probed with nucleotide analogs.
• Measurable by quantitative high-throughput in vitro splicing assays.
• Linked to cancer cell survival through splicing of Mcl1 in neuroblastoma.
• Provides a target for understanding spliceosome catalysis and fidelity.
• Relevant to diseases caused by splicing factor mutations or dysregulation.
What Happens During second spliceosomal transesterification activity?
Transition from step I to step II
In simple terms: The spliceosome rearranges after the first cut to get ready for the second cut.
After the first transesterification reaction, the spliceosome must be remodeled into the step II catalytically activated state. This transition is driven in part by the ATPase Prp16, which destabilizes interactions among Cwc2, U6 snRNA and Prp8 at the boundary between the first and second steps. Structural studies of the yeast step II catalytically activated spliceosome have revealed the architecture required for the second reaction.
Nucleophilic attack by the 5' exon
In simple terms: The free end of the first exon attacks the start of the second exon.
In the second transesterification reaction, the free 3'-hydroxyl group of the 5' exon acts as the nucleophile and attacks the 3' splice site. This chemistry is distinct from the first step, in which the branch point adenosine attacks the 5' splice site. The reaction results in ligation of the two exons and detachment of the intron in a lariat structure.
Exon ligation and intron release
In simple terms: The two exons are joined and the intron is set free as a loop.
Following the second transesterification, the exons are covalently ligated and the intron is released as a lariat. The RNA helicase Prp22 is required for the second step and for subsequent release of the spliced mRNA from the spliceosome. Functional domains of Prp22 have been mapped and shown to be important for its role in splicing.
ATP dependence and helicase dynamics
In simple terms: Energy from ATP is used to rearrange the splicing machine.
RNA helicase dynamics are essential for pre-mRNA splicing, including the second step. Prp16 ATPase activity is required for the transition between the first and second steps, and its action destabilizes specific interactions within the spliceosome. Prp22 also functions as an ATP-dependent helicase in the second step and in mRNA release.
Distinguishing the second step experimentally
In simple terms: Scientists can tell the first and second steps apart using special chemical probes.
Adenosine phosphorothioates such as ATP alpha S and ATP tau S differentially affect the two steps of mammalian pre-mRNA splicing, allowing the second step to be distinguished from the first. Quantitative high-throughput in vitro splicing assays have also been developed to identify inhibitors of spliceosome catalysis, including the second step.
Key Genes Involved in GO:0000386 second spliceosomal transesterification activity
The following genes and proteins are core components or regulators of the second spliceosomal transesterification activity, based on published biochemical and structural studies.
| Gene | Major Role | Research Relevance |
|---|---|---|
| PRP8 | Core spliceosomal protein at the catalytic center; interacts with Cwc2 and U6 snRNA | Mutations can affect step II catalysis and splicing fidelity |
| PRP16 | ATPase that drives the transition between the first and second steps | Studied for its role in destabilizing Cwc2, U6 snRNA and Prp8 interactions |
| PRP22 | RNA helicase required for the second step and mRNA release | Functional domains mapped for splicing activity |
| CWC2 | Spliceosomal protein whose interactions are destabilized by Prp16 at the step I-II transition | Used to study step II activation |
| U6 snRNA | Catalytic RNA component of the spliceosome | Interactions with Prp8 and Cwc2 are regulated during step II |
| MCL1 | Anti-apoptotic protein whose splicing is regulated by spliceosome components | Spliceosome components regulate Mcl1 activity in neuroblastoma |
| ATP | Energy source for helicase-driven rearrangements | Nucleotide analogs differentially affect the two splicing steps |
| Prp8 | Yeast ortholog of PRP8; part of the step II catalytically activated spliceosome | Structural studies of yeast step II spliceosome |
| Prp16 | Yeast ATPase required for step II transition | Biochemical characterization of step I-II transition |
| Prp22 | Yeast RNA helicase required for second step | Domain analysis of splicing factor function |
| Cwc2 | Yeast protein interacting with U6 snRNA and Prp8 | Destabilized by Prp16 at the step I-II transition |
| U6 | Yeast U6 snRNA involved in catalysis | Studied in step II activation |
| Mcl1 | Mouse/human ortholog; anti-apoptotic Bcl-2 family member | Spliceosome components regulate Mcl1 activity in neuroblastoma |
How Is second spliceosomal transesterification activity Regulated?
The second transesterification activity is regulated by ATP-dependent RNA helicases. Prp16 ATPase activity controls the transition between the first and second steps by destabilizing interactions among Cwc2, U6 snRNA and Prp8. Prp22 is required for the second step and for release of the spliced mRNA. Nucleotide analogs such as ATP alpha S and ATP tau S differentially affect the two steps of mammalian pre-mRNA splicing, indicating that the second step has distinct nucleotide requirements. Quantitative in vitro splicing assays can be used to measure inhibitors that affect spliceosome catalysis.
second spliceosomal transesterification activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| MCL1 | Neuroblastoma cell survival and apoptosis | Knockout or overexpression in neuroblastoma cell lines |
| PRP8 | Splicing fidelity and step II catalysis | Point mutation or knockout in yeast or human cells |
| PRP16 | Step I-II transition and splicing regulation | ATPase-dead point mutant |
| PRP22 | Second step and mRNA release | Domain deletion or point mutation |
Cancer and spliceosome components
Multiple components of the spliceosome regulate Mcl1 activity in neuroblastoma, linking second-step splicing factors to cancer cell survival. Mcl1 is an anti-apoptotic protein, and its regulation by spliceosome components can influence apoptotic resistance in tumor cells.
Splicing factor dysregulation
Because the second transesterification step is essential for exon ligation, mutations or dysregulation of core factors such as PRP8, PRP16 or PRP22 could alter splicing outcomes. However, specific human disease associations for these factors require further study.
From second spliceosomal transesterification activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is a splicing factor required for the second step? | Knockout cell line or yeast strain |
| Does a specific residue affect catalysis? | Point mutation knock-in |
| Where does a factor localize during step II? | Tagged knock-in with fluorescent or affinity tag |
| Does overexpression alter splicing? | Overexpression cell model |
| Can small molecules inhibit the second step? | Quantitative in vitro splicing assay |
| Does a nucleotide analog affect step II? | In vitro splicing with ATP analogs |
How to Study the second spliceosomal transesterification activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| In vitro splicing assay | Second transesterification activity and inhibitor sensitivity | High-throughput screening |
| ATP analog sensitivity | Differential effects on step I vs step II | Step-specific analysis |
| Cryo-EM | Structure of step II catalytically activated spliceosome | Mechanistic studies |
| Mutagenesis | Functional domains of Prp22 and Prp16 | Domain mapping |
| RNA helicase assays | ATP-dependent remodeling | Helicase dynamics |
| Spliceosome component analysis | Regulation of Mcl1 splicing | Cancer biology |
In vitro splicing assays
Quantitative high-throughput in vitro splicing assays can measure the second transesterification step and identify inhibitors of spliceosome catalysis. Adenosine phosphorothioates such as ATP alpha S and ATP tau S differentially affect the two steps of mammalian pre-mRNA splicing, allowing step-specific analysis.
Structural biology
Cryo-EM and crystallography have been used to determine the structure of the yeast step II catalytically activated spliceosome, revealing the architecture required for the second transesterification reaction.
Biochemical reconstitution
Biochemical studies of RNA helicase dynamics and factor interactions have defined the roles of Prp16 and Prp22 in the second step. These assays often use purified components or cell extracts to monitor spliceosome rearrangements.
Genetic and mutational analysis
Functional domains of splicing factors such as Prp22 have been mapped by mutagenesis, and ATPase-deficient mutants of Prp16 have been used to trap the step I-II transition.
How CRISPR Can Be Used to Study GO:0000386 second spliceosomal transesterification activity
Knockout
CRISPR knockout of core second-step factors such as PRP8, PRP16 or PRP22 can be used to test their requirement for exon ligation and cell viability. However, because these factors are essential, conditional or inducible knockout systems may be needed.
Point Mutation
Point mutations in catalytic residues or ATPase domains of PRP16 or PRP22 can be introduced to dissect their roles in the second transesterification step. Such mutants can trap specific spliceosomal intermediates.
Knock-in
Tagged knock-in of splicing factors allows localization and interaction studies during step II. Fluorescent or affinity tags can be used to purify step II spliceosomes.
Overexpression
Overexpression of splicing factors or Mcl1 can be used to study how excess protein affects second-step activity and cancer cell survival. Overexpression models can reveal dominant effects on splicing.
How EDITGENE Supports second spliceosomal transesterification activity Research
Researchers studying second spliceosomal transesterification activity-related genes often need to determine whether a candidate gene is causally involved in exon ligation, splicing fidelity or disease-associated splicing changes. EDITGENE provides CRISPR-based cell model services to enable these functional studies.
Contact EDITGENE today to design your custom CRISPR model for second spliceosomal transesterification activity research.
Frequently Asked Questions About second spliceosomal transesterification activity
What is second spliceosomal transesterification activity?
It is the molecular function GO:0000386, which catalyzes the second transesterification reaction of spliceosomal mRNA splicing, joining exons and releasing the intron lariat.
What genes are involved in second spliceosomal transesterification activity?
Key genes include PRP8, PRP16, PRP22, CWC2 and U6 snRNA, as well as MCL1 whose splicing is regulated by spliceosome components.
What is the GO ID for second spliceosomal transesterification activity?
The GO ID is GO:0000386.
How is the second transesterification step different from the first?
The first step uses the branch point adenosine to attack the 5' splice site, while the second step uses the 5' exon 3'-hydroxyl to attack the 3' splice site.
Which proteins regulate the second step of splicing?
Prp16 ATPase drives the step I-II transition, and Prp22 helicase is required for the second step and mRNA release.
Can the second step be inhibited experimentally?
Yes, quantitative in vitro splicing assays have identified inhibitors of spliceosome catalysis, and nucleotide analogs differentially affect the two steps.
What diseases are linked to second-step splicing factors?
Spliceosome components regulate Mcl1 activity in neuroblastoma, linking second-step factors to cancer cell survival.
What methods study second spliceosomal transesterification activity?
In vitro splicing assays, cryo-EM, mutagenesis and ATP analog sensitivity are commonly used.
What is the role of Prp22 in splicing?
Prp22 is an RNA helicase required for the second step and for release of the spliced mRNA.
How can CRISPR help study second-step splicing?
CRISPR knockout, point mutation, knock-in and overexpression models can test the function of splicing factors in exon ligation.
Conclusion
GO:0000386 second spliceosomal transesterification activity is the molecular function that catalyzes exon ligation and intron lariat release during pre-mRNA splicing. It is carried out by the step II catalytically activated spliceosome and regulated by ATP-dependent helicases such as Prp16 and Prp22. Experimental approaches including in vitro splicing assays, structural biology and CRISPR-based models continue to define its mechanism and disease relevance.
References
- 1. Schwer B et al.. 2000. RNA helicase dynamics in pre-mRNA splicing.. EMBO J 19(23):6582-91 PMID: 11101530
- 2. Aupič J et al.. 2023. Monovalent metal ion binding promotes the first transesterification reaction in the spliceosome.. Nat Commun 14(1):8482 PMID: 38123540
- 3. Yan C et al.. 2017. Structure of a yeast step II catalytically activated spliceosome.. Science 355(6321):149-155 PMID: 27980089
- 4. Laetsch TW et al.. 2014. Multiple components of the spliceosome regulate Mcl1 activity in neuroblastoma.. Cell Death Dis 5(2):e1072 PMID: 24556687
- 5. Meissner J et al.. 2024. Characterization of Cwc2, U6 snRNA, and Prp8 interactions destabilized by Prp16 ATPase at the transition between the first and second steps of splicing.. RNA 30(9):1199-1212 PMID: 38876504
- 6. Tazi J et al.. 1992. Adenosine phosphorothioates (ATP alpha S and ATP tau S) differentially affect the two steps of mammalian pre-mRNA splicing.. J Biol Chem 267(7):4322-6 PMID: 1531649
- 7. Schneider S et al.. 2001. Functional domains of the yeast splicing factor Prp22p.. J Biol Chem 276(24):21184-91 PMID: 11283007
- 8. Berg MG et al.. 2012. A quantitative high-throughput in vitro splicing assay identifies inhibitors of spliceosome catalysis.. Mol Cell Biol 32(7):1271-83 PMID: 22252314
