Related Experiment Videos
Initiation of Apaf-1 translation by internal ribosome entry
M J Coldwell1, S A Mitchell, M Stoneley
1Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH.
Abstract:
The apoptotic protease activating factor (Apaf-1) plays a central role in apoptosis: interaction of this protein with procaspase-9 leads to cleavage and activation of this initiator caspase. In common with other mRNAs whose protein products have a major regulatory function, the 5' untranslated region (UTR) of Apaf-1 is long, G-C rich and has the potential to form secondary structure. We have shown that the 5' UTR of Apaf-1 contains an internal ribosome entry segment, located in a 233 nucleotide region towards the 3' end of the leader, and that the translation initiation of this mRNA occurs only by internal ribosome entry. The Apaf-1 IRES is active in almost all human cell types tested, including Human cervical carcinoma (HeLa), Human liver carcinoma (HepG2), Human breast carcinoma (MCF7), Human embryonic kidney (HK293), African Green Monkey kidney (COS7) and Human lung (MRC5). The Apaf-1 IRES initiates translation as efficiently as the HRV IRES, but is less active than the c-myc IRES. We propose that the Apaf-1 IRES ensures that a constant cellular level of Apaf-1 protein is maintained even under conditions where cap-dependent translation is compromised. Oncogene (2000) 19, 899 - 905.
Insights
The apoptotic protease activating factor (Apaf-1) mRNA uses internal ribosome entry sites (IRES) for translation initiation. This mechanism ensures constant Apaf-1 protein levels, even when cap-dependent translation is impaired.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Apoptotic protease activating factor (Apaf-1) is crucial for apoptosis, mediating caspase-9 activation.
- The 5' untranslated region (UTR) of Apaf-1 mRNA is long, G-C rich, and forms secondary structures.
- Translation initiation mechanisms for regulatory proteins are critical for cellular function.
Purpose of the Study:
- To investigate the translation initiation mechanism of Apaf-1 mRNA.
- To identify and characterize the internal ribosome entry segment (IRES) within the Apaf-1 5' UTR.
- To determine the functional significance of Apaf-1 IRES activity in various human cell types.
Main Methods:
- Identification of the internal ribosome entry segment (IRES) within the 233-nucleotide region of the Apaf-1 5' UTR.
- Assays to measure the activity of the Apaf-1 IRES in different human cell lines (HeLa, HepG2, MCF7, HK293, COS7, MRC5).
- Comparative analysis of Apaf-1 IRES activity against known IRES elements (HRV, c-myc).
Main Results:
- The 5' UTR of Apaf-1 contains a functional internal ribosome entry segment (IRES).
- Apaf-1 IRES-mediated translation initiation was observed in multiple human cell types.
- Apaf-1 IRES activity is comparable to HRV IRES but less active than c-myc IRES.
Conclusions:
- Apaf-1 mRNA translation is initiated exclusively via an IRES mechanism.
- The Apaf-1 IRES ensures constitutive expression of Apaf-1 protein.
- This IRES-dependent translation provides a constant Apaf-1 level, vital under conditions inhibiting cap-dependent translation.