Recombinant Ashbya gossypii Pre-mRNA-splicing factor CWC15 (CWC15)
Description
Overview of Recombinant Ashbya gossypii Pre-mRNA-Splicing Factor CWC15 (CWC15)
Recombinant Ashbya gossypii Pre-mRNA-splicing factor CWC15 (CWC15) is a conserved protein in eukaryotes, initially identified as a spliceosome-associated factor . CWC15 is essential for development in yeast and metazoans; its absence leads to embryo lethality in Arabidopsis . CWC15 is involved in microRNA (miRNA) biogenesis by binding to miRNA promoters and interacting with microprocessor components .
Role in miRNA Biogenesis
CWC15 positively regulates miRNA biogenesis by binding to the promoters of genes encoding miRNAs (MIRs), enhancing their activity, and increasing the occupancy of DNA-dependent RNA polymerases at MIR promoters, which suggests that CWC15 positively regulates the transcription of primary miRNA transcripts (pri-miRNAs) .
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Interaction with Microprocessor Components CWC15 interacts with Serrate (SE) and HYL1, which are key components of the microprocessor complex, and is essential for efficient pri-miRNA processing and HYL1-pri-miRNA interaction .
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Regulation of SE Levels CWC15 interacts with the 20S proteasome and PRP4KA, facilitating SE phosphorylation by PRP4KA and subsequent degradation of non-functional SE by the 20S proteasome . CWC15 ensures optimal miRNA biogenesis by maintaining proper SE levels and modulating pri-miRNA levels .
Experimental Evidence of CWC15 Function
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Knockdown Effects Knockdown of CWC15 using artificial miRNA (amiR CWC15) reduces the accumulation of miRNAs and pri-miRNAs .
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Complementation Studies Expression of a CWC15 open reading frame fused with a GFP tag driven by its native promoter (pCWC15::GFP-CWC15) in amiR CWC15 lines fully rescues the developmental defects caused by CWC15 reduction .
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BiFC Assays Bimolecular fluorescence complementation (BiFC) assays confirm the association of CWC15 with MAC (因子相关复合物) complex components, suggesting its involvement in miRNA biogenesis .
CWC15 and the DCL1 Complex
CWC15 interacts with DDL, HYL1, and SE, but not directly with DCL1 . This interaction enhances pri-miRNA processing by facilitating the degradation of excess SE and promoting the interaction of HYL1 with pri-miRNAs .
CWC15 in Splicing
CWC15 functions in splicing in Arabidopsis, potentially stabilizing the splicing machinery . While CWC15 affects the splicing of some pri-miRNAs, its role in promoting miRNA biogenesis appears independent of its splicing function .
CWC15 and Cancer
CWC15 is associated with somatic mutations in various cancers . The COSMIC database provides detailed information on mutation distribution and variants of CWC15 in different tissue types .
Mutation Distribution in Cancer
| Mutation (Amino Acid) | Mutation ID (COSF) | Count | Mutation Type |
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Product Specs
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Target Background
KEGG: ago:AGOS_AGL168W
STRING: 33169.AAS54323
Q&A
Basic Research Questions
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What is the functional role of CWC15 in Ashbya gossypii?
CWC15 is a pre-mRNA splicing factor involved in intron removal and exon joining during RNA processing. In Ashbya gossypii, it functions as an integral component of the spliceosome, specifically associated with the NineTeen complex (NTC) which is critical for spliceosomal activation . The protein is homologous to the yeast/human splicing factor Cwc15/AD-002 . Its primary function involves facilitating the precise removal of non-coding introns from pre-mRNA transcripts to generate mature mRNA for protein synthesis. As described in structural studies, CWC15 is positioned within the core machinery of the spliceosome, highlighting its fundamental role in RNA processing .
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What is the evolutionary conservation pattern of CWC15 across species?
Phylogenetic analyses reveal that CWC15 is highly conserved across eukaryotes, from fungi to plants and animals, indicating its fundamental role in the splicing machinery . The protein appears to have diverged between plants and animals, with specific amino acid sequences distinguishing the different clades, though the major domains in the N- and C-terminal regions remain conserved across species . This conservation pattern suggests that CWC15 plays an essential role in the splicing process that has been maintained throughout eukaryotic evolution. The high degree of conservation makes A. gossypii CWC15 a valuable model for understanding splicing mechanisms across species .
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What experimental systems are available for studying CWC15 in Ashbya gossypii?
Several experimental approaches can be employed to study CWC15 in A. gossypii:
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Genetic manipulation: A. gossypii is amenable to genetic modifications, allowing for gene disruption, overexpression, or tagging approaches .
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Promoter engineering: New promoters for metabolic engineering in A. gossypii have been developed, which can be utilized for controlled expression of CWC15 .
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Protein expression systems: Recombinant expression systems are available for producing CWC15 protein for biochemical and structural studies .
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Antibody-based detection: Specific antibodies against A. gossypii CWC15 are available for immunological detection methods like Western blotting and immunoprecipitation .
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Genome database resources: The Ashbya Genome Database (AGD) provides comprehensive genomic information that can support CWC15 studies .
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Advanced Research Questions
Data Table: Experimental Evidence for CWC15 Function Across Species
This table summarizes experimental evidence for CWC15 function across different species, highlighting the conservation of its role in RNA processing while noting species-specific aspects of its function and the consequences of its dysfunction.
Research Methodologies for CWC15 Functional Studies
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Genetic Manipulation Approaches
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Protein-Protein Interaction Studies
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Functional Genomics
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Structural Biology
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Biochemical Assays
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In vitro splicing reactions
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Spliceosome assembly assays
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RNA binding assays
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ATPase/helicase activity measurements of associated factors
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