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Cyclin E associates with components of the pre-mRNA splicing machinery in mammalian cells
W Seghezzi1, K Chua, F Shanahan
1Department of Cell Signaling, DNAX Research Institute, Palo Alto, California 94304-1104, USA. seghezzi@dnax.org
Abstract:
Cyclin E-cdk2 is a critical regulator of cell cycle progression from G1 into S phase in mammalian cells. Despite this important function little is known about the downstream targets of this cyclin-kinase complex. Here we have identified components of the pre-mRNA processing machinery as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitated a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B' and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. We demonstrate that SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and that its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.
Insights
Researchers found that cyclin E-cdk2, a key cell cycle regulator, targets pre-mRNA splicing factors. This discovery links cell cycle progression to mRNA processing in mammalian cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cyclin E-cdk2 is a crucial regulator of the G1 to S phase transition in mammalian cell cycles.
- The downstream targets and functions of cyclin E-cdk2 beyond cell cycle control remain largely uncharacterized.
Purpose of the Study:
- To identify novel downstream targets of the cyclin E-cdk2 complex.
- To investigate the potential role of cyclin E-cdk2 in regulating pre-mRNA processing.
Main Methods:
- Immunoprecipitation using cyclin E-specific antibodies to identify associated proteins.
- In vitro kinase assays to assess substrate phosphorylation by cyclin E-cdk2.
- Western blotting and immunoprecipitation of U2 snRNA and spliceosomes.
Main Results:
- Components of the pre-mRNA processing machinery, including spliceosome-associated proteins (SAPs) 114, 145, and 155, were identified as cyclin E-associated proteins.
- SAP 155 was confirmed as a direct in vitro substrate for cyclin E-cdk2, with phosphorylation inhibited by p21.
- Cyclin E antibodies immunoprecipitated U2 snRNA and spliceosomes, indicating a physical association.
Conclusions:
- The findings reveal a novel link between the cell cycle machinery (cyclin E-cdk2) and pre-mRNA splicing.
- SAP 155 phosphorylation by cyclin E-cdk2 prior to splicing step II suggests a regulatory role for cell cycle control over splicing.
- This study provides evidence for cell cycle-dependent regulation of mRNA processing in mammalian cells.