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Autoregulation of poly(A)-binding protein synthesis in vitro
O P de Melo Neto1, N Standart, C Martins de Sa
1Department of Biochemistry, University of Cambridge, UK.
Nucleic Acids Research
|June 25, 1995
Summary
Poly(A)-binding protein (PABP) autoregulates its own synthesis. PABP binds to an A-rich sequence in its own mRNA
Area of Science:
- Molecular Biology
- Gene Regulation
- Protein Synthesis
Background:
- Poly(A)-binding protein (PABP) is crucial for mRNA translation and stability.
- PABP mRNA contains a unique 5' untranslated region (UTR) rich in adenylate residues.
- PABP expression is known to be translationally regulated during cellular processes.
Purpose of the Study:
- To investigate the hypothesis that poly(A)-binding protein (PABP) synthesis is autogenously controlled.
- To determine the role of the 5' UTR A-rich sequence in PABP mRNA regulation.
- To elucidate the mechanism of PABP autoregulation.
Main Methods:
- Utilized rabbit reticulocyte cell-free translation systems.
- Performed in vitro transcription of human PABP mRNA.
- Employed deletion and insertion mutagenesis of the 5' UTR A-rich sequence in PABP and reporter mRNAs.
- Conducted UV cross-linking experiments to assess PABP binding.
- Tested the effect of recombinant GST-PABP on mRNA translation.
Main Results:
- Addition of poly(A) stimulated translation of endogenous and in vitro transcribed PABP mRNA.
- The 5' UTR A-rich sequence was found to be necessary and sufficient for poly(A)-mediated translational control.
- UV cross-linking confirmed that PABP binds to the leader adenylate tract.
- Recombinant PABP repressed the translation of mRNAs containing the A-rich sequence in their 5' UTR.
Conclusions:
- PABP binds to an A-rich sequence in the 5' UTR of its own mRNA.
- This binding event leads to the repression of PABP synthesis, demonstrating autogenous control.
- The poly(A)-binding protein (PABP) autoregulates its own translation via its 5' UTR.