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Bahityar Rahmutulla1, Kazuyuki Matsushita, Mamoru Satoh
1Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba City, Chiba, Japan.
Abstract:
The far-upstream element-binding protein-interacting repressor (FIR) is a c-myc transcriptional suppressor. FIR is alternatively spliced to lack the transcriptional repression domain within exon 2 (FIRΔexon2) in colorectal cancers. FIR and FIRΔexon2 form homo- or heterodimers that complex with SAP155. SAP155, a subunit of the essential splicing factor 3b subcomplex in the spliceosome, is required for proper P27Kip1 pre-mRNA splicing, and P27Kip1 arrests cells at G1. In contrast, FIR was co-immunoprecipitated with Ku86 and DNA-PKcs. siRNA against Ku86/Ku70 decreased FIR and P27Kip1 expression, whereas siRNA against FIR decreased Ku86/XRCC5 and P27Kip1 expression. Thus the mechanical interaction of FIR/FIRΔexon2/SAP155 bridges c-myc and P27Kip1 expression, potentially integrates cell-cycle progression and c-myc transcription in cell. Bleomycin(BLM) is an anticancer agent that introduces DNA breaks. Because DNA breaks generate the recruitment of Ku86/Ku70 to bind to the broken DNA ends, the possible involvement of FIR and Ku86/Ku70 interaction in the BLM-induced DNA damage repair response was investigated in this study. First, BLM treatment reduced SAP155 expression and increased FIR and FIRΔexon2 mRNA expression as well as the ratio of FIRΔexon2:FIR in hepatoblastoma cells (HLE and HLF). Second, FIR or FIRΔexon2 adenovirus vectors (Ad-FIR or Ad-FIRΔexon2) increased Ku86/Ku70 and P27Kip1 expression in vitro. Third, BLM decreased P27Kip1 protein expression, whereas increased P27Kip1 and γH2AX expression with Ad-FIRΔexon2. Together, the interaction of FIR/SAP155 modulates FIR splicing and involves in cell-cycle control or cell fate via P27Kip1 and c-myc in BLM-induced DNA damage pathway. This novel function of FIR splicing will contribute to clinical studies of cancer management through elucidating the mechanical interaction of FIR/FIRΔexon2/SAP155 as a potential target for cancer treatment.
Insights
The far-upstream element-binding protein-interacting repressor (FIR) and its splice variant FIRΔexon2 interact with SAP155 and DNA repair proteins. This interaction influences c-myc and P27Kip1 expression, impacting cell-cycle control and response to DNA damage.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Cycle Regulation
Background:
- The far-upstream element-binding protein-interacting repressor (FIR) is a c-myc transcriptional suppressor.
- FIR undergoes alternative splicing, producing FIRΔexon2, which lacks a transcriptional repression domain and is found in colorectal cancers.
- FIR and FIRΔexon2 interact with SAP155, a splicing factor crucial for P27Kip1 pre-mRNA splicing, which regulates cell-cycle arrest at G1.
Purpose of the Study:
- To investigate the interaction between FIR/FIRΔexon2 and DNA repair proteins, specifically Ku86/Ku70.
- To elucidate the role of FIR splicing in the DNA damage response induced by Bleomycin (BLM).
- To understand how the FIR/SAP155 complex influences cell-cycle control and gene expression in response to DNA damage.
Main Methods:
- Co-immunoprecipitation assays to identify interacting proteins.
- siRNA-mediated knockdown of FIR, Ku86, and Ku70 to assess expression changes.
- Treatment with Bleomycin (BLM) to induce DNA breaks.
- Analysis of mRNA and protein expression levels of FIR, FIRΔexon2, SAP155, P27Kip1, and γH2AX.
- Use of adenovirus vectors (Ad-FIR, Ad-FIRΔexon2) to manipulate FIR expression.
Main Results:
- Bleomycin treatment reduced SAP155 expression and increased FIR and FIRΔexon2 mRNA levels, altering the FIRΔexon2:FIR ratio.
- FIR and FIRΔexon2 overexpression increased Ku86/Ku70 and P27Kip1 expression.
- Bleomycin treatment decreased P27Kip1 protein but increased P27Kip1 and γH2AX expression in cells with Ad-FIRΔexon2.
- siRNA against Ku86/Ku70 decreased FIR and P27Kip1 expression, while siRNA against FIR decreased Ku86/XRCC5 and P27Kip1 expression.
Conclusions:
- The interaction of FIR/FIRΔexon2 with SAP155 modulates FIR splicing and is involved in cell-cycle control via P27Kip1 and c-myc.
- FIR splicing and its interaction with Ku86/Ku70 play a role in the DNA damage response pathway induced by Bleomycin.
- The FIR/FIRΔexon2/SAP155 complex represents a potential therapeutic target for cancer management by influencing DNA damage repair and cell fate.
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